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Ge C, Zhao X, Zhang J, Li P, Hao J, Lu M, Li C, Ge L, Tu L, Zhang Q. Role of DLAT in cuproptosis and autophagy in hippocampal tissue of PTSD rats with high-voltage electrical burns. Burns 2025; 51:107519. [PMID: 40319822 DOI: 10.1016/j.burns.2025.107519] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2024] [Revised: 01/21/2025] [Accepted: 04/19/2025] [Indexed: 05/07/2025]
Abstract
BACKGROUND The mechanisms behind high-voltage electrical burn (HVEB)-induced post-traumatic stress disorder (PTSD) remain unclear. Accordingly, we aimed to identify the molecular changes in hippocampal tissue following HVEB-induced PTSD. METHODS The GSE60303 dataset was used to identify differentially expressed cuproptosis-related genes (DE-CRGs) and to perform weighted gene co-expression network analysis. A core gene and associated genes were identified, followed by enrichment analysis. Additionally, a rat model of HVEB PTSD was established, and behavioral tests were conducted. Histological assessments and the evaluation of related protein and gene expression levels were performed on hippocampal tissue. RESULTS Twelve DE-CRGs were identified in the hippocampal tissue of PTSD rats, with DLAT identified as the core gene. Analysis of DLAT-associated genes revealed enrichment in the cAMP signaling pathway and autophagy. Behavioral tests confirmed that HVEB induced PTSD-like behavior in rats. DLAT expression was decreased in the hippocampal tissue of HVEB PTSD rats, accompanied by changes in the expression of cuproptosis, cAMP pathway, and autophagy-related genes. CONCLUSION DLAT is reduced in the hippocampal tissue of HVEB PTSD rats. The downregulation of DLAT may contribute to the development of PTSD-like behaviors in HVEB rats by promoting cuproptosis, activating the cAMP pathway, and enhancing autophagy.
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Affiliation(s)
- Chenyang Ge
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Xuegang Zhao
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Jing Zhang
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Peixuan Li
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Jiawen Hao
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Mengyuan Lu
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Congying Li
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Lili Ge
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Lihong Tu
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China
| | - Qingfu Zhang
- Burn and Wound Repair Center, Hebei Medical University Third Hospital, Shijiazhuang, Hebei Province 050035, China.
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Makarchikov AF, Wins P, Bettendorff L. Biochemical and medical aspects of vitamin B 1 research. Neurochem Int 2025; 185:105962. [PMID: 40058602 DOI: 10.1016/j.neuint.2025.105962] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2024] [Revised: 03/04/2025] [Accepted: 03/06/2025] [Indexed: 03/15/2025]
Abstract
Vitamin B1 is an indispensable food factor for the human and animal body. In animals, vitamin B1 is found in the form of thiamine and its phosphate esters - thiamine mono-, di- and triphosphate, as well as an adenylated derivative - adenosine thiamine triphosphate. At present, the only vitamin B1 form with biochemical functions being elucidated is thiamine diphosphate, which serves as a coenzyme for several important enzymes involved in carbohydrate, amino acid, fatty acid and energy metabolism. Here we review the latest developments in the field of vitamin B1 research in animals. Transport, metabolism and biological role of thiamine and its derivatives are considered as well as the involvement of vitamin B1-dependent processes in human diseases and its therapeutic issues, a field that has gained momentum with several important recent developments.
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Affiliation(s)
- Alexander F Makarchikov
- Grodno State Agrarian University, 28 Tereshkova St., 230005, Grodno, Belarus; Institute of Biochemistry of Biologically Active Compounds of NAS of Belarus, 7 Antoni Tyzenhauz Square, 230023, Grodno, Belarus
| | - Pierre Wins
- Laboratory of Neurophysiology, GIGA Institute, University of Liège, Avenue Hippocrate 15, B-4000, Liege, Belgium
| | - Lucien Bettendorff
- Laboratory of Neurophysiology, GIGA Institute, University of Liège, Avenue Hippocrate 15, B-4000, Liege, Belgium.
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3
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Wang C, Ma C, Xu Y, Chang S, Wu H, Yan C, Chen J, Wu Y, An S, Xu J, Han Q, Jiang Y, Jiang Z, Chu X, Gao H, Zhang X, Chang Y. Dynamics of the mammalian pyruvate dehydrogenase complex revealed by in-situ structural analysis. Nat Commun 2025; 16:917. [PMID: 39843418 PMCID: PMC11754459 DOI: 10.1038/s41467-025-56171-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 01/10/2025] [Indexed: 01/24/2025] Open
Abstract
The multi-enzyme pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle and plays vital roles in metabolism, energy production, and cellular signaling. Although all components have been individually characterized, the intact PDHc structure remains unclear, hampering our understanding of its composition and dynamical catalytic mechanisms. Here, we report the in-situ architecture of intact mammalian PDHc by cryo-electron tomography. The organization of peripheral E1 and E3 components varies substantially among the observed PDHcs, with an average of 21 E1 surrounding each PDHc core, and up to 12 E3 locating primarily along the pentagonal openings. In addition, we observed dynamic interactions of the substrate translocating lipoyl domains (LDs) with both E1 and E2, and the interaction interfaces were further analyzed by molecular dynamics simulations. By revealing intrinsic dynamics of PDHc peripheral compositions, our findings indicate a distinctive activity regulation mechanism, through which the number of E1, E3 and functional LDs may be coordinated to meet constantly changing demands of metabolism.
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Affiliation(s)
- Chen Wang
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Cheng Ma
- Protein Facility, Core Facilities, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Yuanyou Xu
- Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China
| | - Shenghai Chang
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Hangjun Wu
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Chunlan Yan
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Jinghua Chen
- Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China
| | - Yongping Wu
- College of Veterinary Medicine, College of Animal Science and Technology, Zhejiang A&F University, Hangzhou, Zhejiang, China
| | - Shaoya An
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Jiaqi Xu
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Qin Han
- Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Yujie Jiang
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Zhinong Jiang
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Xiakun Chu
- Advanced Materials Thrust, The Hong Kong University of Science and Technology (Guangzhou), Guangzhou, Guangdong, China
| | - Haichun Gao
- Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
| | - Xing Zhang
- Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
- Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
| | - Yunjie Chang
- Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
- Department of Infectious Diseases of Sir Run Run Shaw Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
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Jiang Z, Xiong N, Yan R, Li ST, Liu H, Mao Q, Sun Y, Shen S, Ye L, Gao P, Zhang P, Jia W, Zhang H. PDHX acetylation facilitates tumor progression by disrupting PDC assembly and activating lactylation-mediated gene expression. Protein Cell 2025; 16:49-63. [PMID: 39311688 DOI: 10.1093/procel/pwae052] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2024] [Accepted: 08/29/2024] [Indexed: 01/07/2025] Open
Abstract
Deactivation of the mitochondrial pyruvate dehydrogenase complex (PDC) is important for the metabolic switching of cancer cell from oxidative phosphorylation to aerobic glycolysis. Studies examining PDC activity regulation have mainly focused on the phosphorylation of pyruvate dehydrogenase (E1), leaving other post-translational modifications largely unexplored. Here, we demonstrate that the acetylation of Lys 488 of pyruvate dehydrogenase complex component X (PDHX) commonly occurs in hepatocellular carcinoma, disrupting PDC assembly and contributing to lactate-driven epigenetic control of gene expression. PDHX, an E3-binding protein in the PDC, is acetylated by the p300 at Lys 488, impeding the interaction between PDHX and dihydrolipoyl transacetylase (E2), thereby disrupting PDC assembly to inhibit its activation. PDC disruption results in the conversion of most glucose to lactate, contributing to the aerobic glycolysis and H3K56 lactylation-mediated gene expression, facilitating tumor progression. These findings highlight a previously unrecognized role of PDHX acetylation in regulating PDC assembly and activity, linking PDHX Lys 488 acetylation and histone lactylation during hepatocellular carcinoma progression and providing a potential biomarker and therapeutic target for further development.
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Affiliation(s)
- Zetan Jiang
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Nanchi Xiong
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- Insitute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei 230601, China
| | - Ronghui Yan
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Shi-Ting Li
- Medical Research Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou 510080, China
| | - Haiying Liu
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Qiankun Mao
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Yuchen Sun
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Shengqi Shen
- Medical Research Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou 510080, China
| | - Ling Ye
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Ping Gao
- Medical Research Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou 510080, China
| | - Pinggen Zhang
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Weidong Jia
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Huafeng Zhang
- Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230027, China
- Insitute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei 230601, China
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Bell MB, Kane MS, Ouyang X, Young ME, Jegga AG, Chatham JC, Darley‐Usmar V, Zhang J. Brain Transcriptome Changes Associated With an Acute Increase of Protein O-GlcNAcylation and Implications for Neurodegenerative Disease. J Neurochem 2025; 169:e16302. [PMID: 39823370 PMCID: PMC11741514 DOI: 10.1111/jnc.16302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Revised: 12/16/2024] [Accepted: 12/24/2024] [Indexed: 01/19/2025]
Abstract
Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) has been considered as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer's disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the potential of using OGA inhibition to treat neurodegenerative diseases. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using the OGA inhibitor Thiamet G (TG), in normal mouse brains. We hypothesized that the transcriptome signature in response to a 3 h TG treatment (50 mg/kg) provides a comprehensive view of the effect of OGA inhibition. We then performed mRNA sequencing of the brain using NovaSeq PE 150 (n = 5 each group). We identified 1234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. AMPK subunit Prkab2 was one gene in the kinase activity pathway, and the increase after acute and chronic treatment was confirmed using qPCR. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the impact of OGA inhibition in the treatment of AD. In particular, OGA inhibitors appear to have downstream effects related to bioenergetics which may limit their therapeutic benefits.
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Affiliation(s)
- Margaret B. Bell
- Department of PathologyUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Mariame S. Kane
- Department of PathologyUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Xiaosen Ouyang
- Department of PathologyUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Martin E. Young
- Department of MedicineUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Anil G. Jegga
- Division of Biomedical Informatics, Department of Pediatrics, Cincinnati Children's Hospital Medical CenterUniversity of Cincinnati College of MedicineCincinnatiOhioUSA
| | - John C. Chatham
- Department of PathologyUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Victor Darley‐Usmar
- Department of PathologyUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Jianhua Zhang
- Department of PathologyUniversity of Alabama at BirminghamBirminghamAlabamaUSA
- Birmingham VA Medical CenterUniversity of Alabama at BirminghamBirminghamAlabamaUSA
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Bell M, Kane MS, Ouyang X, Young ME, Jegga AG, Chatham JC, Darley-Usmar V, Zhang J. Acute increase of protein O-GlcNAcylation in mice leads to transcriptome changes in the brain opposite to what is observed in Alzheimer's Disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.19.613769. [PMID: 39345543 PMCID: PMC11429956 DOI: 10.1101/2024.09.19.613769] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/01/2024]
Abstract
Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) is explored as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer's disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the mechanistic path of using OGA inhibition to treat AD. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using OGA inhibitor Thiamet G (TG), on normal mouse brains. We hypothesized that the transcritome signature in respones to TG treatment provides a comprehensive view of the effect of OGA inhibition. We sacrificed the mice and dissected their brains after 3 hours of saline or 50 mg/kg TG treatment, and then performed mRNA sequencing using NovaSeq PE 150 (n=5 each group). We identified 1,234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the potential benefits of OGA inhibition in the treatment of AD. In particular, although OGA inhibitors are promising to treat AD, their downstream chronic effects related to bioenergetics may be a limiting factor. Abstract Figure
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Gómez-Fernández D, Romero-González A, Suárez-Rivero JM, Cilleros-Holgado P, Álvarez-Córdoba M, Piñero-Pérez R, Romero-Domínguez JM, Reche-López D, López-Cabrera A, Ibáñez-Mico S, Castro de Oliveira M, Rodríguez-Sacristán A, González-Granero S, García-Verdugo JM, Sánchez-Alcázar JA. A Multi-Target Pharmacological Correction of a Lipoyltransferase LIPT1 Gene Mutation in Patient-Derived Cellular Models. Antioxidants (Basel) 2024; 13:1023. [PMID: 39199267 PMCID: PMC11351668 DOI: 10.3390/antiox13081023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Revised: 08/12/2024] [Accepted: 08/20/2024] [Indexed: 09/01/2024] Open
Abstract
Mutations in the lipoyltransferase 1 (LIPT1) gene are rare inborn errors of metabolism leading to a fatal condition characterized by lipoylation defects of the 2-ketoacid dehydrogenase complexes causing early-onset seizures, psychomotor retardation, abnormal muscle tone, severe lactic acidosis, and increased urine lactate, ketoglutarate, and 2-oxoacid levels. In this article, we characterized the disease pathophysiology using fibroblasts and induced neurons derived from a patient bearing a compound heterozygous mutation in LIPT1. A Western blot analysis revealed a reduced expression of LIPT1 and absent expression of lipoylated pyruvate dehydrogenase E2 (PDH E2) and alpha-ketoglutarate dehydrogenase E2 (α-KGDH E2) subunits. Accordingly, activities of PDH and α-KGDH were markedly reduced, associated with cell bioenergetics failure, iron accumulation, and lipid peroxidation. In addition, using a pharmacological screening, we identified a cocktail of antioxidants and mitochondrial boosting agents consisting of pantothenate, nicotinamide, vitamin E, thiamine, biotin, and α-lipoic acid, which is capable of rescuing LIPT1 pathophysiology, increasing the LIPT1 expression and lipoylation of mitochondrial proteins, improving cell bioenergetics, and eliminating iron overload and lipid peroxidation. Furthermore, our data suggest that the beneficial effect of the treatment is mainly mediated by SIRT3 activation. In conclusion, we have identified a promising therapeutic approach for correcting LIPT1 mutations.
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Affiliation(s)
- David Gómez-Fernández
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Ana Romero-González
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Juan M. Suárez-Rivero
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Paula Cilleros-Holgado
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Mónica Álvarez-Córdoba
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Rocío Piñero-Pérez
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - José Manuel Romero-Domínguez
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Diana Reche-López
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Alejandra López-Cabrera
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
| | - Salvador Ibáñez-Mico
- Hospital Clínico Universitario Virgen de la Arrixaca, Servicio de Neuropediatría, 30120 Murcia, Spain;
| | - Marta Castro de Oliveira
- Neuropediatria, Neurolinkia, C. Jardín de la Isla, 8, Local 4 y 5, 41014 Sevilla, Spain;
- FEA Pediatría, Centro Universitario Hospitalar de Faro, R. Leão Penedo, 8000-386 Faro, Portugal
- Neuropediatria, Servicio de Pediatría, Hospital Universitario Virgen Macarena, 41009 Sevilla, Spain;
| | - Andrés Rodríguez-Sacristán
- Neuropediatria, Servicio de Pediatría, Hospital Universitario Virgen Macarena, 41009 Sevilla, Spain;
- Departamento de Farmacología, Radiología y Pediatría de la Facultad de Medicina de la Universidad de Sevilla, 41009 Sevilla, Spain
| | - Susana González-Granero
- Laboratory of Comparative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia and CIBERNED-ISCIII, 46980 Valencia, Spain; (S.G.-G.); (J.M.G.-V.)
| | - José Manuel García-Verdugo
- Laboratory of Comparative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia and CIBERNED-ISCIII, 46980 Valencia, Spain; (S.G.-G.); (J.M.G.-V.)
| | - José A. Sánchez-Alcázar
- Centro Andaluz de Biología del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), 41013 Sevilla, Spain; (D.G.-F.); (A.R.-G.); (J.M.S.-R.); (P.C.-H.); (M.Á.-C.); (R.P.-P.); (J.M.R.-D.); (D.R.-L.); (A.L.-C.)
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Zdanowicz R, Afanasyev P, Pruška A, Harrison JA, Giese C, Boehringer D, Leitner A, Zenobi R, Glockshuber R. Stoichiometry and architecture of the human pyruvate dehydrogenase complex. SCIENCE ADVANCES 2024; 10:eadn4582. [PMID: 39018392 PMCID: PMC466950 DOI: 10.1126/sciadv.adn4582] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Accepted: 06/05/2024] [Indexed: 07/19/2024]
Abstract
The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.
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Affiliation(s)
- Rafal Zdanowicz
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, 8093 Zürich, Switzerland
| | - Pavel Afanasyev
- Cryo-EM Knowledge Hub, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
| | - Adam Pruška
- Department of Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland
| | - Julian A. Harrison
- Department of Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland
| | - Christoph Giese
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, 8093 Zürich, Switzerland
| | - Daniel Boehringer
- Cryo-EM Knowledge Hub, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
| | - Alexander Leitner
- Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
| | - Renato Zenobi
- Department of Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland
| | - Rudi Glockshuber
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, 8093 Zürich, Switzerland
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9
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Miquel E, Villarino R, Martínez-Palma L, Cassina A, Cassina P. Pyruvate dehydrogenase kinase 2 knockdown restores the ability of amyotrophic lateral sclerosis-linked SOD1G93A rat astrocytes to support motor neuron survival by increasing mitochondrial respiration. Glia 2024; 72:999-1011. [PMID: 38372421 DOI: 10.1002/glia.24516] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Revised: 12/28/2023] [Accepted: 02/05/2024] [Indexed: 02/20/2024]
Abstract
Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration. Various studies using cellular and animal models of ALS indicate that there is a complex interplay between MN and neighboring non-neuronal cells, such as astrocytes, resulting in noncell autonomous neurodegeneration. Astrocytes in ALS exhibit a lower ability to support MN survival than nondisease-associated ones, which is strongly correlated with low-mitochondrial respiratory activity. Indeed, pharmacological inhibition of pyruvate dehydrogenase kinase (PDK) led to an increase in the mitochondrial oxidative phosphorylation pathway as the primary source of cell energy in SOD1G93A astrocytes and restored the survival of MN. Among the four PDK isoforms, PDK2 is ubiquitously expressed in astrocytes and presents low expression levels in neurons. Herein, we hypothesize whether selective knockdown of PDK2 in astrocytes may increase mitochondrial activity and, in turn, reduce SOD1G93A-associated toxicity. To assess this, cultured neonatal SOD1G93A rat astrocytes were incubated with specific PDK2 siRNA. This treatment resulted in a reduction of the enzyme expression with a concomitant decrease in the phosphorylation rate of the pyruvate dehydrogenase complex. In addition, PDK2-silenced SOD1G93A astrocytes exhibited restored mitochondrial bioenergetics parameters, adopting a more complex mitochondrial network. This treatment also decreased lipid droplet content in SOD1G93A astrocytes, suggesting a switch in energetic metabolism. Significantly, PDK2 knockdown increased the ability of SOD1G93A astrocytes to support MN survival, further supporting the major role of astrocyte mitochondrial respiratory activity in astrocyte-MN interactions. These results suggest that PDK2 silencing could be a cell-specific therapeutic tool to slow the progression of ALS.
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Affiliation(s)
- Ernesto Miquel
- Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Rosalía Villarino
- Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Laura Martínez-Palma
- Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Adriana Cassina
- Departamento de Bioquímica, Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Patricia Cassina
- Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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10
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Szabo E, Nagy B, Czajlik A, Komlodi T, Ozohanics O, Tretter L, Ambrus A. Mitochondrial Alpha-Keto Acid Dehydrogenase Complexes: Recent Developments on Structure and Function in Health and Disease. Subcell Biochem 2024; 104:295-381. [PMID: 38963492 DOI: 10.1007/978-3-031-58843-3_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/05/2024]
Abstract
The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons. Our chapter offers a wealth of up-to-date information on these multienzyme complexes for a comprehensive understanding of their significance in health and disease.
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Affiliation(s)
- Eszter Szabo
- Department of Biochemistry, Semmelweis University, Budapest, Hungary
| | - Balint Nagy
- Department of Biochemistry, Semmelweis University, Budapest, Hungary
| | - Andras Czajlik
- Department of Biochemistry, Semmelweis University, Budapest, Hungary
| | - Timea Komlodi
- Department of Biochemistry, Semmelweis University, Budapest, Hungary
| | - Oliver Ozohanics
- Department of Biochemistry, Semmelweis University, Budapest, Hungary
| | - Laszlo Tretter
- Department of Biochemistry, Semmelweis University, Budapest, Hungary
| | - Attila Ambrus
- Department of Biochemistry, Semmelweis University, Budapest, Hungary.
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11
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Zlatic SA, Werner E, Surapaneni V, Lee CE, Gokhale A, Singleton K, Duong D, Crocker A, Gentile K, Middleton F, Dalloul JM, Liu WLY, Patgiri A, Tarquinio D, Carpenter R, Faundez V. Systemic proteome phenotypes reveal defective metabolic flexibility in Mecp2 mutants. Hum Mol Genet 2023; 33:12-32. [PMID: 37712894 PMCID: PMC10729867 DOI: 10.1093/hmg/ddad154] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Revised: 09/01/2023] [Accepted: 09/11/2023] [Indexed: 09/16/2023] Open
Abstract
Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.
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Affiliation(s)
- Stephanie A Zlatic
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
| | - Erica Werner
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
| | - Veda Surapaneni
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
| | - Chelsea E Lee
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
| | - Avanti Gokhale
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
| | - Kaela Singleton
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
| | - Duc Duong
- Department of Biochemistry, Emory University, 1510 Clifton Rd NE, Atlanta, GA 30322, United States
| | - Amanda Crocker
- Program in Neuroscience, Middlebury College, Bicentennial Way, Middlebury, VT 05753, United States
| | - Karen Gentile
- Department of Neuroscience and Physiology, SUNY Upstate Medical University, 505 Irving Avenue, Syracuse, NY 13210, United States
| | - Frank Middleton
- Department of Neuroscience and Physiology, SUNY Upstate Medical University, 505 Irving Avenue, Syracuse, NY 13210, United States
| | - Joseph Martin Dalloul
- Pharmacology and Chemical Biology, Emory University, 1510 Clifton Rd NE, Atlanta, GA 30322, United States
| | - William Li-Yun Liu
- Pharmacology and Chemical Biology, Emory University, 1510 Clifton Rd NE, Atlanta, GA 30322, United States
| | - Anupam Patgiri
- Pharmacology and Chemical Biology, Emory University, 1510 Clifton Rd NE, Atlanta, GA 30322, United States
| | - Daniel Tarquinio
- Center for Rare Neurological Diseases, 5600 Oakbrook Pkwy, Norcross, GA 30093, United States
| | - Randall Carpenter
- Rett Syndrome Research Trust, 67 Under Cliff Rd, Trumbull, CT 06611, United States
| | - Victor Faundez
- Department of Cell Biology, Emory University, 615 Michael Steet, Atlanta, GA 30322, United States
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12
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D’Angelo D, Vecellio Reane D, Raffaello A. Neither too much nor too little: mitochondrial calcium concentration as a balance between physiological and pathological conditions. Front Mol Biosci 2023; 10:1336416. [PMID: 38148906 PMCID: PMC10749936 DOI: 10.3389/fmolb.2023.1336416] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Accepted: 12/04/2023] [Indexed: 12/28/2023] Open
Abstract
Ca2+ ions serve as pleiotropic second messengers in the cell, regulating several cellular processes. Mitochondria play a fundamental role in Ca2+ homeostasis since mitochondrial Ca2+ (mitCa2+) is a key regulator of oxidative metabolism and cell death. MitCa2+ uptake is mediated by the mitochondrial Ca2+ uniporter complex (MCUc) localized in the inner mitochondrial membrane (IMM). MitCa2+ uptake stimulates the activity of three key enzymes of the Krebs cycle, thereby modulating ATP production and promoting oxidative metabolism. As Paracelsus stated, "Dosis sola facit venenum,"in pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP), enabling the release of apoptotic factors and ultimately leading to cell death. Excessive mitCa2+ accumulation is also associated with a pathological increase of reactive oxygen species (ROS). In this article, we review the precise regulation and the effectors of mitCa2+ in physiopathological processes.
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Affiliation(s)
- Donato D’Angelo
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | - Denis Vecellio Reane
- Department of Biomedical Sciences, University of Padua, Padua, Italy
- Institute for Diabetes and Obesity, Helmholtz Zentrum München, Munich, Germany
| | - Anna Raffaello
- Department of Biomedical Sciences, Myology Center (CIR-Myo), University of Padua, Padua, Italy
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13
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Cimmino TP, Pagano E, Stornaiuolo M, Esposito G, Ammendola R, Cattaneo F. Formyl-peptide receptor 2 signalling triggers aerobic metabolism of glucose through Nox2-dependent modulation of pyruvate dehydrogenase activity. Open Biol 2023; 13:230336. [PMID: 37875162 PMCID: PMC10597678 DOI: 10.1098/rsob.230336] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Accepted: 09/20/2023] [Indexed: 10/26/2023] Open
Abstract
The human formyl-peptide receptor 2 (FPR2) is activated by an array of ligands. By phospho-proteomic analysis we proved that FPR2 stimulation induces redox-regulated phosphorylation of many proteins involved in cellular metabolic processes. In this study, we investigated metabolic pathways activated in FPR2-stimulated CaLu-6 cells. The results showed an increased concentration of metabolites involved in glucose metabolism, and an enhanced uptake of glucose mediated by GLUT4, the insulin-regulated member of GLUT family. Accordingly, we observed that FPR2 transactivated IGF-IRβ/IRβ through a molecular mechanism that requires Nox2 activity. Since cancer cells support their metabolism via glycolysis, we analysed glucose oxidation and proved that FPR2 signalling promoted kinase activity of the bifunctional enzyme PFKFB2 through FGFR1/FRS2- and Akt-dependent phosphorylation. Furthermore, FPR2 stimulation induced IGF-IRβ/IRβ-, PI3K/Akt- and Nox-dependent inhibition of pyruvate dehydrogenase activity, thus preventing the entry of pyruvate in the tricarboxylic acid cycle. Consequently, we observed an enhanced FGFR-dependent lactate dehydrogenase (LDH) activity and lactate production in FPR2-stimulated cells. As LDH expression is transcriptionally regulated by c-Myc and HIF-1, we demonstrated that FPR2 signalling promoted c-Myc phosphorylation and Nox-dependent HIF-1α stabilization. These results strongly indicate that FPR2-dependent signalling can be explored as a new therapeutic target in treatment of human cancers.
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Affiliation(s)
- Tiziana Pecchillo Cimmino
- Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, 80131 Naples, Italy
| | - Ester Pagano
- Department of Pharmacy, School of Medicine, University of Naples Federico II, 80131 Naples, Italy
| | - Mariano Stornaiuolo
- Department of Pharmacy, School of Medicine, University of Naples Federico II, 80131 Naples, Italy
| | - Gabriella Esposito
- Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, 80131 Naples, Italy
| | - Rosario Ammendola
- Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, 80131 Naples, Italy
| | - Fabio Cattaneo
- Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, 80131 Naples, Italy
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14
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Zlatic SA, Werner E, Surapaneni V, Lee CE, Gokhale A, Singleton K, Duong D, Crocker A, Gentile K, Middleton F, Dalloul JM, Liu WLY, Patgiri A, Tarquinio D, Carpenter R, Faundez V. Systemic Proteome Phenotypes Reveal Defective Metabolic Flexibility in Mecp2 Mutants. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.03.535431. [PMID: 37066332 PMCID: PMC10103972 DOI: 10.1101/2023.04.03.535431] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 04/24/2023]
Abstract
Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.
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Affiliation(s)
| | - Erica Werner
- Department of Cell Biology, Emory University, Atlanta, GA, USA, 30322
| | - Veda Surapaneni
- Department of Cell Biology, Emory University, Atlanta, GA, USA, 30322
| | - Chelsea E. Lee
- Department of Cell Biology, Emory University, Atlanta, GA, USA, 30322
| | - Avanti Gokhale
- Department of Cell Biology, Emory University, Atlanta, GA, USA, 30322
| | - Kaela Singleton
- Department of Cell Biology, Emory University, Atlanta, GA, USA, 30322
| | - Duc Duong
- Department of Biochemistry, Emory University, Atlanta, GA, USA, 30322
| | - Amanda Crocker
- Program in Neuroscience, Middlebury College, Middlebury, Vermont 05753
| | - Karen Gentile
- Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
| | - Frank Middleton
- Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
| | - Joseph Martin Dalloul
- Department of Pharmacology & Chemical Biology, Emory University, Atlanta, GA, USA, 30322
| | - William Li-Yun Liu
- Department of Pharmacology & Chemical Biology, Emory University, Atlanta, GA, USA, 30322
| | - Anupam Patgiri
- Department of Pharmacology & Chemical Biology, Emory University, Atlanta, GA, USA, 30322
| | | | | | - Victor Faundez
- Department of Cell Biology, Emory University, Atlanta, GA, USA, 30322
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15
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Salinas ND, Ma R, Dickey TH, McAleese H, Ouahes T, Long CA, Miura K, Lambert LE, Tolia NH. A potent and durable malaria transmission-blocking vaccine designed from a single-component 60-copy Pfs230D1 nanoparticle. NPJ Vaccines 2023; 8:124. [PMID: 37596283 PMCID: PMC10439124 DOI: 10.1038/s41541-023-00709-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Accepted: 07/12/2023] [Indexed: 08/20/2023] Open
Abstract
Malaria transmission-blocking vaccines (TBVs) reduce disease transmission by breaking the continuous cycle of infection between the human host and the mosquito vector. Domain 1 (D1) of Pfs230 is a leading TBV candidate and comprises the majority of transmission-reducing activity (TRA) elicited by Pfs230. Here we show that the fusion of Pfs230D1 to a 60-copy multimer of the catalytic domain of dihydrolipoyl acetyltransferase protein (E2p) results in a single-component nanoparticle composed of 60 copies of the fusion protein with high stability, homogeneity, and production yields. The nanoparticle presents a potent human transmission-blocking epitope within Pfs230D1, indicating the antigen is correctly oriented on the surface of the nanoparticle. Two vaccinations of New Zealand White rabbits with the Pfs230D1 nanoparticle elicited a potent and durable antibody response with high TRA when formulated in two distinct adjuvants suitable for translation to human use. This single-component nanoparticle vaccine may play a key role in malaria control and has the potential to improve production pipelines and the cost of manufacturing of a potent and durable TBV.
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Affiliation(s)
- Nichole D Salinas
- Host-Pathogen Interactions and Structural Vaccinology Section, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Rui Ma
- Host-Pathogen Interactions and Structural Vaccinology Section, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Thayne H Dickey
- Host-Pathogen Interactions and Structural Vaccinology Section, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Holly McAleese
- Vaccine Development Unit, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Tarik Ouahes
- Vaccine Development Unit, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Carole A Long
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA
| | - Kazutoyo Miura
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA
| | - Lynn E Lambert
- Vaccine Development Unit, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Niraj H Tolia
- Host-Pathogen Interactions and Structural Vaccinology Section, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
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16
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Wang Z, Wang Y, Yan J, Wei Y, Zhang Y, Wang X, Leng X. Analysis of cuproptosis-related genes in Ulcerative colitis and immunological characterization based on machine learning. Front Med (Lausanne) 2023; 10:1115500. [PMID: 37529244 PMCID: PMC10389668 DOI: 10.3389/fmed.2023.1115500] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2022] [Accepted: 07/03/2023] [Indexed: 08/03/2023] Open
Abstract
Cuproptosis is a novel form of cell death, mediated by protein lipid acylation and highly associated with mitochondrial metabolism, which is regulated in the cell. Ulcerative colitis (UC) is a chronic inflammatory bowel disease that recurs frequently, and its incidence is increasing worldwide every year. Currently, a growing number of studies have shown that cuproptosis-related genes (CRGs) play a crucial role in the development and progression of a variety of tumors. However, the regulatory role of CRGs in UC has not been fully elucidated. Firstly, we identified differentially expressed genes in UC, Likewise, CRGs expression profiles and immunological profiles were evaluated. Using 75 UC samples, we typed UC based on the expression profiles of CRGs, followed by correlative immune cell infiltration analysis. Using the weighted gene co-expression network analysis (WGCNA) methodology, the cluster's differentially expressed genes (DEGs) were produced. Then, the performances of extreme gradient boosting models (XGB), support vector machine models (SVM), random forest models (RF), and generalized linear models (GLM) were constructed and predicted. Finally, the effectiveness of the best machine learning model was evaluated using five external datasets, receiver operating characteristic curve (ROC), the area under the curve of ROC (AUC), a calibration curve, a nomogram, and a decision curve analysis (DCA). A total of 13 CRGs were identified as significantly different in UC and control samples. Two subtypes were identified in UC based on CRGs expression profiles. Immune cell infiltration analysis of subtypes showed significant differences between immune cells of different subtypes. WGCNA results showed a total of 8 modules with significant differences between subtypes, with the turquoise module being the most specific. The machine learning results showed satisfactory performance of the XGB model (AUC = 0.981). Finally, the construction of the final 5-gene-based XGB model, validated by the calibration curve, nomogram, decision curve analysis, and five external datasets (GSE11223: AUC = 0.987; GSE38713: AUC = 0.815; GSE53306: AUC = 0.946; GSE94648: AUC = 0.809; GSE87466: AUC = 0.981), also proved to predict subtypes of UC with accuracy. Our research presents a trustworthy model that can predict the likelihood of developing UC and methodically outlines the complex relationship between CRGs and UC.
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Affiliation(s)
- Zhengyan Wang
- Changchun University of Chinese Medicine, Changchun, China
| | - Ying Wang
- The Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, China
| | - Jing Yan
- Changchun University of Chinese Medicine, Changchun, China
| | - Yuchi Wei
- Changchun University of Chinese Medicine, Changchun, China
| | - Yinzhen Zhang
- Changchun University of Chinese Medicine, Changchun, China
| | - Xukai Wang
- Department of Orthopedics, The Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, China
| | - Xiangyang Leng
- Changchun University of Chinese Medicine, Changchun, China
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17
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Moon DO. Calcium's Role in Orchestrating Cancer Apoptosis: Mitochondrial-Centric Perspective. Int J Mol Sci 2023; 24:ijms24108982. [PMID: 37240331 DOI: 10.3390/ijms24108982] [Citation(s) in RCA: 38] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2023] [Revised: 05/16/2023] [Accepted: 05/17/2023] [Indexed: 05/28/2023] Open
Abstract
Calcium is an essential intracellular messenger that plays a vital role in controlling a broad range of cellular processes, including apoptosis. This review offers an in-depth analysis of calcium's multifaceted role in apoptosis regulation, focusing on the associated signaling pathways and molecular mechanisms. We will explore calcium's impact on apoptosis through its effects on different cellular compartments, such as the mitochondria and endoplasmic reticulum (ER), and discuss the connection between calcium homeostasis and ER stress. Additionally, we will highlight the interplay between calcium and various proteins, including calpains, calmodulin, and Bcl-2 family members, and the role of calcium in regulating caspase activation and pro-apoptotic factor release. By investigating the complex relationship between calcium and apoptosis, this review aims to deepen our comprehension of the fundamental processes, and pinpointing possible treatment options for illnesses associated with imbalanced cell death is crucial.
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Affiliation(s)
- Dong-Oh Moon
- Department of Biology Education, Daegu University, 201, Daegudae-ro, Gyeongsan-si 38453, Gyeongsangbuk-do, Republic of Korea
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18
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Forsberg BO. The structure and evolutionary diversity of the fungal E3-binding protein. Commun Biol 2023; 6:480. [PMID: 37137945 PMCID: PMC10156792 DOI: 10.1038/s42003-023-04854-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 04/20/2023] [Indexed: 05/05/2023] Open
Abstract
The pyruvate dehydrogenase complex (PDC) is a central metabolic enzyme in all living cells composed majorly of E1, E2, and E3. Tight coupling of their reactions makes each component essential, so that any loss impacts oxidative metabolism pathologically. E3 retention is mediated by the E3-binding protein (E3BP), which is here resolved within the PDC core from N.crassa, resolved to 3.2Å. Fungal and mammalian E3BP are shown to be orthologs, arguing E3BP as a broadly eukaryotic gene. Fungal E3BP architectures predicted from sequence data and computational models further bridge the evolutionary distance between N.crassa and humans, and suggest discriminants for E3-specificity. This is confirmed by similarities in their respective E3-binding domains, where an interaction previously not described is also predicted. This provides evolutionary parallels for a crucial interaction human metabolism, an interaction specific to fungi that can be targeted, and an example of protein evolution following gene neofunctionalization.
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Affiliation(s)
- Bjoern O Forsberg
- Department of Physiology and Pharmacology, Karolinska Institutet, Biomedicum, Solnavägen 9, 171 77, Stockholm, Sweden.
- Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, OX3 7BN, Oxford, UK.
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de Ridder I, Kerkhofs M, Lemos FO, Loncke J, Bultynck G, Parys JB. The ER-mitochondria interface, where Ca 2+ and cell death meet. Cell Calcium 2023; 112:102743. [PMID: 37126911 DOI: 10.1016/j.ceca.2023.102743] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2023] [Revised: 04/20/2023] [Accepted: 04/24/2023] [Indexed: 05/03/2023]
Abstract
Endoplasmic reticulum (ER)-mitochondria contact sites are crucial to allow Ca2+ flux between them and a plethora of proteins participate in tethering both organelles together. Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a pivotal role at such contact sites, participating in both ER-mitochondria tethering and as Ca2+-transport system that delivers Ca2+ from the ER towards mitochondria. At the ER-mitochondria contact sites, the IP3Rs function as a multi-protein complex linked to the voltage-dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane, via the chaperone glucose-regulated protein 75 (GRP75). This IP3R-GRP75-VDAC1 complex supports the efficient transfer of Ca2+ from the ER into the mitochondrial intermembrane space, from which the Ca2+ ions can reach the mitochondrial matrix through the mitochondrial calcium uniporter. Under physiological conditions, basal Ca2+ oscillations deliver Ca2+ to the mitochondrial matrix, thereby stimulating mitochondrial oxidative metabolism. However, when mitochondrial Ca2+ overload occurs, the increase in [Ca2+] will induce the opening of the mitochondrial permeability transition pore, thereby provoking cell death. The IP3R-GRP75-VDAC1 complex forms a hub for several other proteins that stabilize the complex and/or regulate the complex's ability to channel Ca2+ into the mitochondria. These proteins and their mechanisms of action are discussed in the present review with special attention for their role in pathological conditions and potential implication for therapeutic strategies.
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Affiliation(s)
- Ian de Ridder
- KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, Leuven BE-3000, Belgium
| | - Martijn Kerkhofs
- KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, Leuven BE-3000, Belgium
| | - Fernanda O Lemos
- KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, Leuven BE-3000, Belgium
| | - Jens Loncke
- KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, Leuven BE-3000, Belgium
| | - Geert Bultynck
- KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, Leuven BE-3000, Belgium.
| | - Jan B Parys
- KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, Leuven BE-3000, Belgium.
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20
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Lipoamide dehydrogenase (LADH) deficiency: medical perspectives of the structural and functional characterization of LADH and its pathogenic variants. Biol Futur 2023:10.1007/s42977-023-00155-6. [PMID: 36842090 DOI: 10.1007/s42977-023-00155-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Accepted: 02/08/2023] [Indexed: 02/27/2023]
Abstract
(Dihydro)lipoamide dehydrogenase (LADH) deficiency is an autosomal recessive genetic metabolic disorder. It generally presents with an onset in the neonatal age and premature death. The clinical picture usually involves metabolic decompensation and lactic acidosis that lead to neurological, cardiological, and/or hepatological outcomes. Severity of the disease is due to the fact that LADH is a common E3 subunit to the pyruvate, alpha-ketoglutarate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes and is also part of the glycine cleavage system; hence, a loss in LADH activity adversely affects several central metabolic pathways simultaneously. The severe clinical manifestations, however, often do not parallel the LADH activity loss, which implies the existence of auxiliary pathological pathways; stimulated reactive oxygen species (ROS) production as well as dissociation from the relevant multienzyme complexes proved to be auxiliary exacerbating pathomechanisms for selected disease-causing LADH mutations. This review provides an overview on the therapeutic challenges of inherited metabolic diseases, structural and functional characteristics of the mitochondrial alpha-keto acid dehydrogenase complexes, molecular pathogenesis and structural basis of LADH deficiency, and relevant potential future medical perspectives.
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21
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Liu S, Xia X, Zhen J, Li Z, Zhou ZH. Structures and comparison of endogenous 2-oxoglutarate and pyruvate dehydrogenase complexes from bovine kidney. Cell Discov 2022; 8:126. [PMID: 36414632 PMCID: PMC9681731 DOI: 10.1038/s41421-022-00487-y] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Accepted: 10/20/2022] [Indexed: 11/23/2022] Open
Abstract
The α-keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of α-keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of α-keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in native states remain unknown. Here, we report the cryo-electron microscopy structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at resolutions of 3.5 Å and 3.8 Å, respectively. The structures of multiple proteins were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, the native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. In addition, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the twofold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides a direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among α-keto acid dehydrogenase complexes.
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Affiliation(s)
- Shiheng Liu
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA, USA
| | - Xian Xia
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA, USA
| | - James Zhen
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA, USA
- Molecular Biology Institute, UCLA, Los Angeles, CA, USA
| | - Zihang Li
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA, USA
| | - Z Hong Zhou
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.
- California NanoSystems Institute, UCLA, Los Angeles, CA, USA.
- Molecular Biology Institute, UCLA, Los Angeles, CA, USA.
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22
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Ouyang S, Zhang Q, Lou L, Zhu K, Li Z, Liu P, Zhang X. The Double-Edged Sword of SIRT3 in Cancer and Its Therapeutic Applications. Front Pharmacol 2022; 13:871560. [PMID: 35571098 PMCID: PMC9092499 DOI: 10.3389/fphar.2022.871560] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Accepted: 03/31/2022] [Indexed: 11/13/2022] Open
Abstract
Reprogramming of cellular energy metabolism is considered an emerging feature of cancer. Mitochondrial metabolism plays a crucial role in cancer cell proliferation, survival, and metastasis. As a major mitochondrial NAD+-dependent deacetylase, sirtuin3 (SIRT3) deacetylates and regulates the enzymes involved in regulating mitochondrial energy metabolism, including fatty acid oxidation, the Krebs cycle, and the respiratory chain to maintain metabolic homeostasis. In this article, we review the multiple roles of SIRT3 in various cancers, and systematically summarize the recent advances in the discovery of its activators and inhibitors. The roles of SIRT3 vary in different cancers and have cell- and tumor-type specificity. SIRT3 plays a unique function by mediating interactions between mitochondria and intracellular signaling. The critical functions of SIRT3 have renewed interest in the development of small molecule modulators that regulate its activity. Delineation of the underlying mechanism of SIRT3 as a critical regulator of cell metabolism and further characterization of the mitochondrial substrates of SIRT3 will deepen our understanding of the role of SIRT3 in tumorigenesis and progression and may provide novel therapeutic strategies for cancer targeting SIRT3.
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Affiliation(s)
- Shumin Ouyang
- National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Qiyi Zhang
- National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Linlin Lou
- National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Kai Zhu
- Innovation Practice Center, Changchun University of Chinese Medicine, Changchun, China
| | - Zeyu Li
- National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Peiqing Liu
- National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Xiaolei Zhang
- National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China
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23
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Tüting C, Kyrilis FL, Müller J, Sorokina M, Skalidis I, Hamdi F, Sadian Y, Kastritis PL. Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction. Nat Commun 2021; 12:6933. [PMID: 34836937 PMCID: PMC8626477 DOI: 10.1038/s41467-021-27287-4] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Accepted: 11/08/2021] [Indexed: 11/23/2022] Open
Abstract
Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.
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Affiliation(s)
- Christian Tüting
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
| | - Fotis L Kyrilis
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
- Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany
| | - Johannes Müller
- Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany
| | - Marija Sorokina
- Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany
- RGCC International GmbH, Baarerstrasse 95, Zug, 6300, Switzerland
- BioSolutions GmbH Weinbergweg 22, 06120, Halle/Saale, Germany
| | - Ioannis Skalidis
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
- Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany
| | - Farzad Hamdi
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
| | - Yashar Sadian
- Bioimaging Center (cryoGEnic), Université de Genève, Sciences II, 1211, Genève 4, Switzerland
| | - Panagiotis L Kastritis
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany.
- Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany.
- Biozentrum, Martin Luther University Halle-Wittenberg, Weinbergweg 22, Halle/Saale, Germany.
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24
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Zhang L, Zhang J, Liu Y, Zhang P, Nie J, Zhao R, Shi Q, Sun H, Jiao D, Chen Y, Zhao X, Huang Y, Li Y, Zhao JY, Xu W, Zhao SM, Wang C. Mitochondrial STAT5A promotes metabolic remodeling and the Warburg effect by inactivating the pyruvate dehydrogenase complex. Cell Death Dis 2021; 12:634. [PMID: 34148062 PMCID: PMC8214628 DOI: 10.1038/s41419-021-03908-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Revised: 05/31/2021] [Accepted: 06/07/2021] [Indexed: 12/12/2022]
Abstract
Signal transducer and activator 5a (STAT5A) is a classical transcription factor that plays pivotal roles in various biological processes, including tumor initiation and progression. A fraction of STAT5A is localized in the mitochondria, but the biological functions of mitochondrial STAT5A remain obscure. Here, we show that STAT5A interacts with pyruvate dehydrogenase complex (PDC), a mitochondrial gatekeeper enzyme connecting two key metabolic pathways, glycolysis and the tricarboxylic acid cycle. Mitochondrial STAT5A disrupts PDC integrity, thereby inhibiting PDC activity and remodeling cellular glycolysis and oxidative phosphorylation. Mitochondrial translocation of STAT5A is increased under hypoxic conditions. This strengthens the Warburg effect in cancer cells and promotes in vitro cell growth under hypoxia and in vivo tumor growth. Our findings indicate distinct pro-oncogenic roles of STAT5A in energy metabolism, which is different from its classical function as a transcription factor.
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Affiliation(s)
- Liang Zhang
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Jianong Zhang
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Yan Liu
- Institute of metabolism and integrative biology (IMIB), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Pingzhao Zhang
- Fudan University Shanghai Cancer Center and Department of Pathology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, 200032, Shanghai, China
| | - Ji Nie
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Rui Zhao
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Qin Shi
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Huiru Sun
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Dongyue Jiao
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Yingji Chen
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Xiaying Zhao
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Yan Huang
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Yao Li
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Jian-Yuan Zhao
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 200438, Shanghai, China
| | - Wei Xu
- Shanghai Fifth People's Hospital and Institutes of Biomedical Sciences, Fudan University, 20032, Shanghai, China
| | - Shi-Min Zhao
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China.
| | - Chenji Wang
- Obstetrics & Gynecology Hospital of Fudan University, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Key Laboratory of Reproduction Regulation of NPFPC (SIPPR, IRD), School of Life Sciences, Fudan University, 200438, Shanghai, China.
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Nemeria NS, Zhang X, Leandro J, Zhou J, Yang L, Houten SM, Jordan F. Toward an Understanding of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxoacid Dehydrogenase Complexes. Life (Basel) 2021; 11:407. [PMID: 33946784 PMCID: PMC8146983 DOI: 10.3390/life11050407] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2021] [Revised: 04/21/2021] [Accepted: 04/22/2021] [Indexed: 12/24/2022] Open
Abstract
The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.
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Affiliation(s)
- Natalia S. Nemeria
- Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA; (J.Z.); (L.Y.)
| | - Xu Zhang
- Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA; (J.Z.); (L.Y.)
| | - Joao Leandro
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; (J.L.); (S.M.H.)
| | - Jieyu Zhou
- Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA; (J.Z.); (L.Y.)
| | - Luying Yang
- Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA; (J.Z.); (L.Y.)
| | - Sander M. Houten
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; (J.L.); (S.M.H.)
| | - Frank Jordan
- Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA; (J.Z.); (L.Y.)
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26
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Nagy B, Polak M, Ozohanics O, Zambo Z, Szabo E, Hubert A, Jordan F, Novaček J, Adam-Vizi V, Ambrus A. Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure. Biochim Biophys Acta Gen Subj 2021; 1865:129889. [PMID: 33684457 DOI: 10.1016/j.bbagen.2021.129889] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Revised: 02/05/2021] [Accepted: 03/02/2021] [Indexed: 12/19/2022]
Abstract
BACKGROUND The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers. METHODS We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling. RESULTS We report the 2.9 Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1-150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc. CONCLUSIONS The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly. GENERAL SIGNIFICANCE The revealed structural information will support the future pharmacologically targeting of the hKGDHc.
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Affiliation(s)
- Balint Nagy
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Martin Polak
- Central European Institute of Technology, Masaryk University, Brno, Czech Republic
| | - Oliver Ozohanics
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Zsofia Zambo
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Eszter Szabo
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Agnes Hubert
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Frank Jordan
- Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ, USA
| | - Jiří Novaček
- Central European Institute of Technology, Masaryk University, Brno, Czech Republic
| | - Vera Adam-Vizi
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
| | - Attila Ambrus
- Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary.
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Chen S, Liu X, Peng C, Tan C, Sun H, Liu H, Zhang Y, Wu P, Cui C, Liu C, Yang D, Li Z, Lu J, Guan J, Ke X, Wang R, Bo X, Xu X, Han J, Liu J. The phytochemical hyperforin triggers thermogenesis in adipose tissue via a Dlat-AMPK signaling axis to curb obesity. Cell Metab 2021; 33:565-580.e7. [PMID: 33657393 DOI: 10.1016/j.cmet.2021.02.007] [Citation(s) in RCA: 131] [Impact Index Per Article: 32.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 10/22/2020] [Accepted: 02/08/2021] [Indexed: 12/15/2022]
Abstract
Stimulation of adipose tissue thermogenesis is regarded as a promising avenue in the treatment of obesity. However, pharmacologic engagement of this process has proven difficult. Using the Connectivity Map (CMap) approach, we identified the phytochemical hyperforin (HPF) as an anti-obesity agent. We found that HPF efficiently promoted thermogenesis by stimulating AMPK and PGC-1α via a Ucp1-dependent pathway. Using LiP-SMap (limited proteolysis-mass spectrometry) combined with a microscale thermophoresis assay and molecular docking analysis, we confirmed dihydrolipoamide S-acetyltransferase (Dlat) as a direct molecular target of HPF. Ablation of Dlat significantly attenuated HPF-mediated adipose tissue browning both in vitro and in vivo. Furthermore, genome-wide association study analysis indicated that a variation in DLAT is significantly associated with obesity in humans. These findings suggest that HPF is a promising lead compound in the pursuit of a pharmacological approach to promote energy expenditure in the treatment of obesity.
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Affiliation(s)
- Suzhen Chen
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China; Department of Otolaryngology Head and Neck Surgery & Center of Sleep Medicine, Shanghai Key Laboratory of Sleep Disordered Breathing, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China.
| | - Xiaoxiao Liu
- State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
| | - Chao Peng
- National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai 201210, China
| | - Chang Tan
- Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China
| | - Honglin Sun
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - He Liu
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - Yao Zhang
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - Ping Wu
- National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai 201210, China
| | - Can Cui
- Center for Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Chuchu Liu
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - Di Yang
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - Zhiqiang Li
- Affiliated Hospital of Qingdao University and Biomedical Sciences Institute of Qingdao University, Qingdao University, Qingdao, China
| | - Junxi Lu
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - Jian Guan
- Department of Otolaryngology Head and Neck Surgery & Center of Sleep Medicine, Shanghai Key Laboratory of Sleep Disordered Breathing, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China
| | - Xisong Ke
- Center for Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Renxiao Wang
- State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
| | - Xiaohai Bo
- Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China
| | - Xiaojun Xu
- State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China.
| | - Junfeng Han
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China.
| | - Junli Liu
- Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China.
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Kyrilis FL, Semchonok DA, Skalidis I, Tüting C, Hamdi F, O'Reilly FJ, Rappsilber J, Kastritis PL. Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts. Cell Rep 2021; 34:108727. [PMID: 33567276 DOI: 10.1016/j.celrep.2021.108727] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Revised: 12/02/2020] [Accepted: 01/14/2021] [Indexed: 12/29/2022] Open
Abstract
The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.
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Affiliation(s)
- Fotis L Kyrilis
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany; Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany
| | - Dmitry A Semchonok
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
| | - Ioannis Skalidis
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany; Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany
| | - Christian Tüting
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
| | - Farzad Hamdi
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany
| | - Francis J O'Reilly
- Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany
| | - Juri Rappsilber
- Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany; Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom
| | - Panagiotis L Kastritis
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany; Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany; Biozentrum, Martin Luther University Halle-Wittenberg, Weinbergweg 22, Halle/Saale, Germany.
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29
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Gherardi G, Monticelli H, Rizzuto R, Mammucari C. The Mitochondrial Ca 2+ Uptake and the Fine-Tuning of Aerobic Metabolism. Front Physiol 2020; 11:554904. [PMID: 33117189 PMCID: PMC7575740 DOI: 10.3389/fphys.2020.554904] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Accepted: 09/14/2020] [Indexed: 12/13/2022] Open
Abstract
Recently, the role of mitochondrial activity in high-energy demand organs and in the orchestration of whole-body metabolism has received renewed attention. In mitochondria, pyruvate oxidation, ensured by efficient mitochondrial pyruvate entry and matrix dehydrogenases activity, generates acetyl CoA that enters the TCA cycle. TCA cycle activity, in turn, provides reducing equivalents and electrons that feed the electron transport chain eventually producing ATP. Mitochondrial Ca2+ uptake plays an essential role in the control of aerobic metabolism. Mitochondrial Ca2+ accumulation stimulates aerobic metabolism by inducing the activity of three TCA cycle dehydrogenases. In detail, matrix Ca2+ indirectly modulates pyruvate dehydrogenase via pyruvate dehydrogenase phosphatase 1, and directly activates isocitrate and α-ketoglutarate dehydrogenases. Here, we will discuss the contribution of mitochondrial Ca2+ uptake to the metabolic homeostasis of organs involved in systemic metabolism, including liver, skeletal muscle, and adipose tissue. We will also tackle the role of mitochondrial Ca2+ uptake in the heart, a high-energy consuming organ whose function strictly depends on appropriate Ca2+ signaling.
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Affiliation(s)
- Gaia Gherardi
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | | | - Rosario Rizzuto
- Department of Biomedical Sciences, University of Padua, Padua, Italy
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30
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Forsberg BO, Aibara S, Howard RJ, Mortezaei N, Lindahl E. Arrangement and symmetry of the fungal E3BP-containing core of the pyruvate dehydrogenase complex. Nat Commun 2020; 11:4667. [PMID: 32938938 PMCID: PMC7494870 DOI: 10.1038/s41467-020-18401-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Accepted: 08/20/2020] [Indexed: 11/21/2022] Open
Abstract
The pyruvate dehydrogenase complex (PDC) is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Similar to the E3-binding protein (E3BP) of mammalian PDC, PX selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. Here, we report reconstructions of PDC from the filamentous fungus Neurospora crassa by cryo-electron microscopy, where we find protein X (PX) interior to the PDC core as opposed to substituting E2 core subunits as in mammals. Steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, explaining previous observations in Saccharomyces cerevisiae. The PX-binding site is conserved in (and specific to) fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC. The pyruvate dehydrogenase complex (PDC) is a multienzyme complex connecting glycolysis to mitochondrial oxidation of pyruvate. Cryo-EM analysis of PDC from Neurospora crassa reveals localization of fungi-specific protein X (PX) and confirms that it functions like the mammalian E3BP, recruiting the E3 component of PDC.
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Affiliation(s)
- B O Forsberg
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 17165, Solna, Sweden
| | - S Aibara
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 17165, Solna, Sweden.,Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany
| | - R J Howard
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 17165, Solna, Sweden
| | - N Mortezaei
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 17165, Solna, Sweden.,Vironova AB, 11330, Stockholm, Sweden
| | - E Lindahl
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 17165, Solna, Sweden. .,Department of Applied Physics, Swedish eScience Research Center, KTH Royal Institute of Technology, 17168, Solna, Sweden.
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31
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Guo Y, Qiu W, Roche TE, Hackert ML. Crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 2020; 76:292-301. [PMID: 32627744 DOI: 10.1107/s2053230x20007943] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Accepted: 06/11/2020] [Indexed: 11/11/2022]
Abstract
Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (Rfree = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.
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Affiliation(s)
- Youzhong Guo
- Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Weihua Qiu
- Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Thomas E Roche
- Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA
| | - Marvin L Hackert
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA
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32
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Abstract
A striking change has happened in the field of immunology whereby specific metabolic processes have been shown to be a critical determinant of immune cell activation. Multiple immune receptor types rewire metabolic pathways as a key part of how they promote effector functions. Perhaps surprisingly for immunologists, the Krebs cycle has emerged as the central immunometabolic hub of the macrophage. During proinflammatory macrophage activation, there is an accumulation of the Krebs cycle intermediates succinate and citrate, and the Krebs cycle–derived metabolite itaconate. These metabolites have distinct nonmetabolic signaling roles that influence inflammatory gene expression. A key bioenergetic target for the Krebs cycle, the electron transport chain, also becomes altered, generating reactive oxygen species from Complexes I and III. Similarly, alternatively activated macrophages require α-ketoglutarate-dependent epigenetic reprogramming to elicit anti-inflammatory gene expression. In this review, we discuss these advances and speculate on the possibility of targeting these events therapeutically for inflammatory diseases.
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Affiliation(s)
- Dylan G. Ryan
- School of Biochemistry and Immunology and Trinity Biomedical Sciences Institute, Trinity College, Dublin 2, Ireland
| | - Luke A.J. O'Neill
- School of Biochemistry and Immunology and Trinity Biomedical Sciences Institute, Trinity College, Dublin 2, Ireland
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33
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Meng H, Yan WY, Lei YH, Wan Z, Hou YY, Sun LK, Zhou JP. SIRT3 Regulation of Mitochondrial Quality Control in Neurodegenerative Diseases. Front Aging Neurosci 2019; 11:313. [PMID: 31780922 PMCID: PMC6861177 DOI: 10.3389/fnagi.2019.00313] [Citation(s) in RCA: 82] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2019] [Accepted: 10/29/2019] [Indexed: 12/21/2022] Open
Abstract
Neurodegenerative diseases are disorders that are characterized by a progressive decline of motor and/or cognitive functions caused by the selective degeneration and loss of neurons within the central nervous system. The most common neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Neurons have high energy demands, and dysregulation of mitochondrial quality and function is an important cause of neuronal degeneration. Mitochondrial quality control plays an important role in maintaining mitochondrial integrity and ensuring normal mitochondrial function; thus, defects in mitochondrial quality control are also significant causes of neurodegenerative diseases. The mitochondrial deacetylase SIRT3 has been found to have a large effect on mitochondrial function. Recent studies have also shown that SIRT3 has a role in mitochondrial quality control, including in the refolding or degradation of misfolded/unfolded proteins, mitochondrial dynamics, mitophagy, and mitochondrial biogenesis, all of which are affected in neurodegenerative diseases.
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Affiliation(s)
- Hao Meng
- Department of Pathophysiology, College of Basic Medical Sciences, Jilin University, Changchun, China
- Department of Neurosurgery, The First Hospital of Jilin University, Changchun, China
| | - Wan-Yu Yan
- Department of Pathophysiology, College of Basic Medical Sciences, Jilin University, Changchun, China
| | - Yu-Hong Lei
- Department of Neurosurgery, The First Hospital of Jilin University, Changchun, China
| | - Zheng Wan
- Department of Neurosurgery, The First Hospital of Jilin University, Changchun, China
| | - Ye-Ye Hou
- Department of Neurosurgery, The First Hospital of Jilin University, Changchun, China
| | - Lian-Kun Sun
- Department of Pathophysiology, College of Basic Medical Sciences, Jilin University, Changchun, China
| | - Jue-Pu Zhou
- Department of Neurosurgery, The First Hospital of Jilin University, Changchun, China
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34
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Investigation of inhibitory potential of quercetin to the pyruvate dehydrogenase kinase 3: Towards implications in anticancer therapy. Int J Biol Macromol 2019; 136:1076-1085. [DOI: 10.1016/j.ijbiomac.2019.06.158] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2019] [Revised: 06/19/2019] [Accepted: 06/21/2019] [Indexed: 12/27/2022]
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35
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Dahiya R, Mohammad T, Gupta P, Haque A, Alajmi MF, Hussain A, Hassan MI. Molecular interaction studies on ellagic acid for its anticancer potential targeting pyruvate dehydrogenase kinase 3. RSC Adv 2019; 9:23302-23315. [PMID: 35514501 PMCID: PMC9067284 DOI: 10.1039/c9ra02864a] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Accepted: 07/15/2019] [Indexed: 11/21/2022] Open
Abstract
Pyruvate dehydrogenase kinase 3 (PDK3) plays a central role in the cancer metabolic switch through the reversible phosphorylation of pyruvate dehydrogenase complex thereby blocking the entry of pyruvate for its catabolism into the TCA cycle, and thus it is considered as an important drug target for various types of cancers. We have successfully expressed full length human PDK3 and investigated its interaction mechanism with dietary polyphenols in the search for potential inhibitors. Molecular docking analysis revealed that the selected compounds preferentially bind to the ATP-binding pocket of PDK3 and interact with functionally important residues. In silico observations were further complemented by experimental measurements of the fluorescence quenching of PDK3 and confirmed with the isothermal titration calorimetry measurements. Ellagic acid (EA) significantly binds and inhibits the kinase activity of PDK3. In vitro cytotoxicity and the anti-proliferative properties of EA were evaluated by MTT assay. Conformational dynamics of the EA-PDK3 complex during molecular dynamics simulation revealed that a stable complex was maintained by a significant number of hydrogen bonds throughout the 100 ns trajectories. In conclusion, EA may be considered as a promising molecule for PDK3 inhibition and could be exploited as a lead molecule against PDK3 associated diseases.
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Affiliation(s)
- Rashmi Dahiya
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia Jamia Nagar New Delhi 110025 India
| | - Taj Mohammad
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia Jamia Nagar New Delhi 110025 India
| | - Preeti Gupta
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia Jamia Nagar New Delhi 110025 India
| | - Anzarul Haque
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia Jamia Nagar New Delhi 110025 India
| | - Mohamed F Alajmi
- Department of Pharmacognosy College of Pharmacy, King Saud University Riyadh 11451 Kingdom of Saudi Arabia
| | - Afzal Hussain
- Department of Pharmacognosy College of Pharmacy, King Saud University Riyadh 11451 Kingdom of Saudi Arabia
| | - Md Imtaiyaz Hassan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia Jamia Nagar New Delhi 110025 India
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Prajapati S, Haselbach D, Wittig S, Patel MS, Chari A, Schmidt C, Stark H, Tittmann K. Structural and Functional Analyses of the Human PDH Complex Suggest a "Division-of-Labor" Mechanism by Local E1 and E3 Clusters. Structure 2019; 27:1124-1136.e4. [PMID: 31130485 DOI: 10.1016/j.str.2019.04.009] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2018] [Revised: 01/17/2019] [Accepted: 04/11/2019] [Indexed: 11/27/2022]
Abstract
The pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-Å resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes. Such uneven, clustered organization translates into specific duties for E1-E2 clusters (oxidative decarboxylation and acetyl transfer) and E3BP-E3 clusters (regeneration of reduced lipoamide) corresponding to half-reactions of the PDHc catalytic cycle. The addition of substrate coenzyme A modulates the conformational landscape of PDHc, in particular of the lipoyl domains, extending the postulated multiple random coupling mechanism. The conformational and associated chemical landscapes of PDHc are thus not determined entirely stochastically, but are restrained and channeled through an asymmetric architecture and further modulated by substrate binding.
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Affiliation(s)
- Sabin Prajapati
- Department of Molecular Enzymology, Göttingen Centre for Molecular Biosciences and Albrecht-von-Haller Institute, Georg-August University Göttingen, Julia-Lermontowa-Weg 3, 37077 Göttingen, Germany; Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Am Fassberg 11, 37077 Göttingen, Germany
| | - David Haselbach
- Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Am Fassberg 11, 37077 Göttingen, Germany
| | - Sabine Wittig
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3a, 06120 Halle/Saale, Germany
| | - Mulchand S Patel
- Jacobs School of Medicine and Biomedical Sciences, Department of Biochemistry, University at Buffalo, 955 Main Street, Buffalo, NY 14203, USA
| | - Ashwin Chari
- Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Am Fassberg 11, 37077 Göttingen, Germany
| | - Carla Schmidt
- Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3a, 06120 Halle/Saale, Germany.
| | - Holger Stark
- Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Am Fassberg 11, 37077 Göttingen, Germany.
| | - Kai Tittmann
- Department of Molecular Enzymology, Göttingen Centre for Molecular Biosciences and Albrecht-von-Haller Institute, Georg-August University Göttingen, Julia-Lermontowa-Weg 3, 37077 Göttingen, Germany; Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Am Fassberg 11, 37077 Göttingen, Germany.
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37
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Hong SM, Lee YK, Park I, Kwon SM, Min S, Yoon G. Lactic acidosis caused by repressed lactate dehydrogenase subunit B expression down-regulates mitochondrial oxidative phosphorylation via the pyruvate dehydrogenase (PDH)-PDH kinase axis. J Biol Chem 2019; 294:7810-7820. [PMID: 30923124 DOI: 10.1074/jbc.ra118.006095] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2018] [Revised: 03/21/2019] [Indexed: 01/12/2023] Open
Abstract
Aerobic glycolysis and mitochondrial dysfunction are key metabolic features of cancer cells, but their interplay during cancer development remains unclear. We previously reported that human hepatoma cells with mitochondrial defects exhibit down-regulated lactate dehydrogenase subunit B (LDHB) expression. Here, using several molecular and biochemical assays and informatics analyses, we investigated how LDHB suppression regulates mitochondrial respiratory activity and contributes to liver cancer progression. We found that transcriptional LDHB down-regulation is an upstream event during suppressed oxidative phosphorylation. We also observed that LDHB knockdown increases inhibitory phosphorylation of pyruvate dehydrogenase (PDH) via lactate-mediated PDH kinase (PDK) activation and thereby attenuates oxidative phosphorylation activity. Interestingly, monocarboxylate transporter 1 was the major lactate transporter in hepatoma cells, and its expression was essential for PDH phosphorylation by modulating intracellular lactate levels. Finally, bioinformatics analysis of the hepatocellular carcinoma cohort from The Cancer Genome Atlas revealed that a low LDHB/LDHA ratio is statistically significantly associated with poor prognostic outcomes. A low ratio was also associated with a significant enrichment in glycolysis genes and negatively correlated with PDK1 and 2 expression, supporting a close link between LDHB suppression and the PDK-PDH axis. These results suggest that LDHB suppression is a key mechanism that enhances glycolysis and is critically involved in the maintenance and propagation of mitochondrial dysfunction via lactate release in liver cancer progression.
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Affiliation(s)
- Sun Mi Hong
- From the Departments of Biochemistry and.,Biomedical Sciences (BK21 Plus), Ajou University School of Medicine, Suwon 16499, Korea
| | | | - Imkyong Park
- From the Departments of Biochemistry and.,Biomedical Sciences (BK21 Plus), Ajou University School of Medicine, Suwon 16499, Korea
| | | | - Seongki Min
- From the Departments of Biochemistry and.,Biomedical Sciences (BK21 Plus), Ajou University School of Medicine, Suwon 16499, Korea
| | - Gyesoon Yoon
- From the Departments of Biochemistry and .,Biomedical Sciences (BK21 Plus), Ajou University School of Medicine, Suwon 16499, Korea
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38
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Luo F, Li Y, Yuan F, Zuo J. Hexokinase II promotes the Warburg effect by phosphorylating alpha subunit of pyruvate dehydrogenase. Chin J Cancer Res 2019; 31:521-532. [PMID: 31354221 PMCID: PMC6613503 DOI: 10.21147/j.issn.1000-9604.2019.03.14] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Objective Tumor cells rely heavily on glycolysis regardless of oxygen tension, a phenomenon called the Warburg effect. Hexokinase II (HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumor cells. Mitochondrial HKII couples glycolysis and oxidative phosphorylation while maintaining mitochondrial membrane integrity. In this study, we investigated the role of HKII in promoting the Warburg effect in cancer cells. Methods HKII-mediated phosphorylation of the alpha subunit of pyruvate dehydrogenase (PDHA1) was tested in HEK293T cells and clear cell renal cell carcinoma (ccRCC) specimens using gene knockdown, western blotting, immunohistochemistry, and immunofluorescence. Results It was determined that HKII could not only transform glucose into glucose-6-phosphate, but also transfer the phosphate group of ATP onto PDHA1. In addition, it was found that HKII increased the phosphorylation of Ser293 on PDHA1, decreasing pyruvate dehydrogenase (PDH) complex activity and thus rerouting the metabolic pathway and promoting the Warburg effect. The overexpression of HKII correlated with the phosphorylation of PDHA1 and disease progression in ccRCC. Conclusions The data presented here suggest that HKII is an important biomarker in the evaluation and treatment of cancer.
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Affiliation(s)
- Fangxiu Luo
- Department of Pathology, Ruijin Hospital North, Shanghai Jiaotong University School of Medicine, Shanghai 201801, China
| | - You Li
- Department of General Surgery, Ruijin Hospital North, Shanghai Jiaotong University School of Medicine, Shanghai 201801, China
| | - Fei Yuan
- Department of Pathology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
| | - Junli Zuo
- Department of Geriatrics, Ruijin Hospital North, Shanghai Jiaotong University School of Medicine, Shanghai 201801, China
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39
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Zhou J, Yang L, Ozohanics O, Zhang X, Wang J, Ambrus A, Arjunan P, Brukh R, Nemeria NS, Furey W, Jordan F. A multipronged approach unravels unprecedented protein-protein interactions in the human 2-oxoglutarate dehydrogenase multienzyme complex. J Biol Chem 2018; 293:19213-19227. [PMID: 30323066 DOI: 10.1074/jbc.ra118.005432] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2018] [Revised: 09/17/2018] [Indexed: 10/28/2022] Open
Abstract
The human 2-oxoglutaric acid dehydrogenase complex (hOGDHc) plays a pivotal role in the tricarboxylic acid (TCA) cycle, and its diminished activity is associated with neurodegenerative diseases. The hOGDHc comprises three components, hE1o, hE2o, and hE3, and we recently reported functionally active E1o and E2o components, enabling studies on their assembly. No atomic-resolution structure for the hE2o component is currently available, so here we first studied the interactions in the binary subcomplexes (hE1o-hE2o, hE1o-hE3, and hE2o-hE3) to gain insight into the strength of their interactions and to identify the interaction loci in them. We carried out multiple physico-chemical studies, including fluorescence, hydrogen-deuterium exchange MS (HDX-MS), and chemical cross-linking MS (CL-MS). Our fluorescence studies suggested a strong interaction for the hE1o-hE2o subcomplex, but a much weaker interaction in the hE1o-hE3 subcomplex, and failed to identify any interaction in the hE2o-hE3 subcomplex. The HDX-MS studies gave evidence for interactions in the hE1o-hE2o and hE1o-hE3 subcomplexes comprising full-length components, identifying: (i) the N-terminal region of hE1o, in particular the two peptides 18YVEEM22 and 27ENPKSVHKSWDIF39 as constituting the binding region responsible for the assembly of the hE1o with both the hE2o and hE3 components into hOGDHc, an hE1 region absent in available X-ray structures; and (ii) a novel hE2o region comprising residues from both a linker region and from the catalytic domain as being a critical region interacting with hE1o. The CL-MS identified the loci in the hE1o and hE2o components interacting with each other.
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Affiliation(s)
- Jieyu Zhou
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
| | - Luying Yang
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
| | - Oliver Ozohanics
- the Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, 27-29 Tuzolto Utca, Budapest H-1094, Hungary
| | - Xu Zhang
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
| | - Junjie Wang
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
| | - Attila Ambrus
- the Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, 27-29 Tuzolto Utca, Budapest H-1094, Hungary
| | - Palaniappa Arjunan
- the Biocrystallography Laboratory, Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240.,the Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and
| | - Roman Brukh
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
| | - Natalia S Nemeria
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102,
| | - William Furey
- the Biocrystallography Laboratory, Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240.,the Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and
| | - Frank Jordan
- From the Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102,
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40
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Drakulic S, Rai J, Petersen SV, Golas MM, Sander B. Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate dehydrogenase complex. Cell Mol Life Sci 2018; 75:3009-3026. [PMID: 29445841 PMCID: PMC11105750 DOI: 10.1007/s00018-018-2775-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2017] [Revised: 01/31/2018] [Accepted: 02/06/2018] [Indexed: 12/18/2022]
Abstract
The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.
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Affiliation(s)
- Srdja Drakulic
- Department of Biomedicine, Aarhus University, 8000, Aarhus C, Denmark
| | - Jay Rai
- Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, 8000, Aarhus C, Denmark
| | | | - Monika M Golas
- Department of Biomedicine, Aarhus University, 8000, Aarhus C, Denmark.
- Department of Human Genetics, Hannover Medical School, 30625 Hannover, Germany.
| | - Bjoern Sander
- Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, 8000, Aarhus C, Denmark.
- Institute of Pathology, Hannover Medical School, 30625 Hannover, Germany.
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41
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Chakraborty J, Nemeria NS, Farinas E, Jordan F. Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2-oxo acid dehydrogenase complexes. FEBS Open Bio 2018; 8:880-896. [PMID: 29928569 PMCID: PMC5986005 DOI: 10.1002/2211-5463.12431] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Revised: 03/29/2018] [Accepted: 04/09/2018] [Indexed: 11/25/2022] Open
Abstract
The Escherichia coli 2‐oxoglutarate dehydrogenase complex (OGDHc) comprises multiple copies of three enzymes—E1o, E2o, and E3—and transthioesterification takes place within the catalytic domain of E2o. The succinyl group from the thiol ester of S8‐succinyldihydrolipoyl‐E2o is transferred to the thiol group of coenzyme A (CoA), forming the all‐important succinyl‐CoA. Here, we report mechanistic studies of enzymatic transthioesterification on OGDHc. Evidence is provided for the importance of His375 and Asp374 in E2o for the succinyl transfer reaction. The magnitude of the rate acceleration provided by these residues (54‐fold from each with alanine substitution) suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid–base catalysis. Further evidence ruling out a role in acid–base catalysis is provided by site‐saturation mutagenesis studies at His375 (His375Trp substitution with little penalty) and substitutions to other potential hydrogen bond participants at Asp374. Taking into account that the rate constant for reductive succinylation of the E2o lipoyl domain (LDo) by E1o and 2‐oxoglutarate (99 s−1) was approximately twofold larger than the rate constant for kcat of 48 s−1 for the overall reaction (NADH production), it could be concluded that succinyl transfer to CoA and release of succinyl‐CoA, rather than reductive succinylation, is the rate‐limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2‐oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl‐CoA formation, so important for several biological processes including post‐translational succinylation of protein lysines. Enzymes 2‐oxoglutarate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html); dihydrolipoamide succinyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html); dihydrolipoamide dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html); pyruvate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html); dihydrolipoamide acetyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html).
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Affiliation(s)
- Joydeep Chakraborty
- Department of Chemistry and Environmental Science New Jersey Institute of Technology Newark NJ USA
| | | | - Edgardo Farinas
- Department of Chemistry and Environmental Science New Jersey Institute of Technology Newark NJ USA
| | - Frank Jordan
- Department of Chemistry Rutgers University Newark NJ USA
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42
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Jiang J, Baiesc FL, Hiromasa Y, Yu X, Hui WH, Dai X, Roche TE, Zhou ZH. Atomic Structure of the E2 Inner Core of Human Pyruvate Dehydrogenase Complex. Biochemistry 2018; 57:2325-2334. [PMID: 29608861 DOI: 10.1021/acs.biochem.8b00357] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Pyruvate dehydrogenase complex (PDC) is a large multienzyme complex that catalyzes the irreversible conversion of pyruvate to acetyl-coenzyme A with reduction of NAD+. Distinctive from PDCs in lower forms of life, in mammalian PDC, dihydrolipoyl acetyltransferase (E2; E2p in PDC) and dihydrolipoamide dehydrogenase binding protein (E3BP) combine to form a complex that plays a central role in the organization, regulation, and integration of catalytic reactions of PDC. However, the atomic structure and organization of the mammalian E2p/E3BP heterocomplex are unknown. Here, we report the structure of the recombinant dodecahedral core formed by the C-terminal inner-core/catalytic (IC) domain of human E2p determined at 3.1 Å resolution by cryo electron microscopy (cryoEM). The structure of the N-terminal fragment and four other surface areas of the human E2p IC domain exhibit significant differences from those of the other E2 crystal structures, which may have implications for the integration of E3BP in mammals. This structure also allowed us to obtain a homology model for the highly homologous IC domain of E3BP. Analysis of the interactions of human E2p or E3BP with their adjacent IC domains in the dodecahedron provides new insights into the organization of the E2p/E3BP heterocomplex and suggests a potential contribution by E3BP to catalysis in mammalian PDC.
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Affiliation(s)
- Jiansen Jiang
- Department of Microbiology, Immunology and Molecular Genetics , University of California, Los Angeles , Los Angeles , California 90095 , United States.,California Nanosystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States
| | - Flavius L Baiesc
- Department of Microbiology, Immunology and Molecular Genetics , University of California, Los Angeles , Los Angeles , California 90095 , United States
| | - Yasuaki Hiromasa
- Faculty of Agriculture, Attached Promotive Center for International Education and Research of Agriculture , Kyushu University , Fukuoka 812-8581 , Japan.,Department of Biochemistry and Molecular Biophysics , Kansas State University , Manhattan , Kansas 66506 , United States
| | - Xuekui Yu
- Department of Microbiology, Immunology and Molecular Genetics , University of California, Los Angeles , Los Angeles , California 90095 , United States.,California Nanosystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States
| | - Wong Hoi Hui
- California Nanosystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States
| | - Xinghong Dai
- Department of Microbiology, Immunology and Molecular Genetics , University of California, Los Angeles , Los Angeles , California 90095 , United States.,California Nanosystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States
| | - Thomas E Roche
- Department of Biochemistry and Molecular Biophysics , Kansas State University , Manhattan , Kansas 66506 , United States
| | - Z Hong Zhou
- Department of Microbiology, Immunology and Molecular Genetics , University of California, Los Angeles , Los Angeles , California 90095 , United States.,California Nanosystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States
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43
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Yang Z, Wang Y, Zhang Y, He X, Zhong CQ, Ni H, Chen X, Liang Y, Wu J, Zhao S, Zhou D, Han J. RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis. Nat Cell Biol 2018; 20:186-197. [PMID: 29358703 DOI: 10.1038/s41556-017-0022-y] [Citation(s) in RCA: 219] [Impact Index Per Article: 31.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Accepted: 12/05/2017] [Indexed: 01/22/2023]
Abstract
Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.
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Affiliation(s)
- Zhentao Yang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Yan Wang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Yingying Zhang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Xiadi He
- State Key Laboratory of Genetic Engineering, School of Life Science and Institute of Biomedical Sciences, The Obstetrics & Gynecology Hospital of Fudan University, Shanghai, China
| | - Chuan-Qi Zhong
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Hengxiao Ni
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Xin Chen
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Yaoji Liang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Jianfeng Wu
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Shimin Zhao
- State Key Laboratory of Genetic Engineering, School of Life Science and Institute of Biomedical Sciences, The Obstetrics & Gynecology Hospital of Fudan University, Shanghai, China
| | - Dawang Zhou
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Jiahuai Han
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
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44
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Hezaveh S, Zeng AP, Jandt U. Full Enzyme Complex Simulation: Interactions in Human Pyruvate Dehydrogenase Complex. J Chem Inf Model 2018; 58:362-369. [PMID: 29298056 DOI: 10.1021/acs.jcim.7b00557] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The pyruvate dehydrogenase complex (PDC) is a large macromolecular machine consisting of dozens of interacting enzymes that are connected and regulated by highly flexible domains, also called swinging arms. The overall structure and function of these domains and how they organize the complex function have not been elucidated in detail to date. This lack of structural and dynamic understanding is frequently observed in multidomain enzymatic complexes. Here we present the first full and dynamic structural model of full human PDC (hPDC), including binding of the linking arms to the surrounding E1 and E3 enzymes via their binding domains with variable stoichiometries. All of the linking domains were modeled at atomistic and coarse-grained levels, and the latter was parametrized to reproduce the same properties of those from the atomistic model. The radii of gyration of the wild-type full complex and functional trimeric subunits were in agreement with available experimental data. Furthermore, the E1 and E3 population effect on the overall structure of the full complex was studied. The results indicated that decreasing the number of E1s increases the flexibility of the now nonoccupied arms. Furthermore, their flexibility depends on the presence of other E1s and E3s in the vicinity, even if they are associated with other arms. As one consequence, the radius of gyration decreases with decreasing number of E1s. This effect also provides an indication of the optimal configuration of E1 and E3 on the basis of the assumption that a certain stability of the enymatic cloud is necessary to avoid free metabolic diffusion of intermediates (metabolic channeling). Our approach and results open a window for future enzyme engineering in a more effective way by evaluating the effect of different linker arm lengths, flexibilities, and combinations of mutations on the activity of other complex enzymes that involve flexible domains, including for example processive enzymes.
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Affiliation(s)
- Samira Hezaveh
- Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology , Denickestr. 15, 21071 Hamburg, Germany
| | - An-Ping Zeng
- Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology , Denickestr. 15, 21071 Hamburg, Germany
| | - Uwe Jandt
- Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology , Denickestr. 15, 21071 Hamburg, Germany
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45
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Richard AJ, Hang H, Stephens JM. Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue. J Biol Chem 2017; 292:19733-19742. [PMID: 28982698 PMCID: PMC5712614 DOI: 10.1074/jbc.m117.811794] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2017] [Revised: 10/03/2017] [Indexed: 12/19/2022] Open
Abstract
STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene cytokine-inducible SH2-containing protein (cish). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation.
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Affiliation(s)
- Allison J Richard
- From the Adipocyte Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808 and
| | - Hardy Hang
- From the Adipocyte Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808 and
| | - Jacqueline M Stephens
- From the Adipocyte Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808 and
- the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
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46
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Fagernes CE, Stensløkken KO, Røhr ÅK, Berenbrink M, Ellefsen S, Nilsson GE. Extreme anoxia tolerance in crucian carp and goldfish through neofunctionalization of duplicated genes creating a new ethanol-producing pyruvate decarboxylase pathway. Sci Rep 2017; 7:7884. [PMID: 28801642 PMCID: PMC5554223 DOI: 10.1038/s41598-017-07385-4] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2017] [Accepted: 06/23/2017] [Indexed: 11/09/2022] Open
Abstract
Without oxygen, most vertebrates die within minutes as they cannot meet cellular energy demands with anaerobic metabolism. However, fish of the genus Carassius (crucian carp and goldfish) have evolved a specialized metabolic system that allows them to survive prolonged periods without oxygen by producing ethanol as their metabolic end-product. Here we show that this has been made possible by the evolution of a pyruvate decarboxylase, analogous to that in brewer's yeast and the first described in vertebrates, in addition to a specialized alcohol dehydrogenase. Whole-genome duplication events have provided additional gene copies of the pyruvate dehydrogenase multienzyme complex that have evolved into a pyruvate decarboxylase, while other copies retained the essential function of the parent enzymes. We reveal the key molecular substitution in duplicated pyruvate dehydrogenase genes that underpins one of the most extreme hypoxic survival strategies among vertebrates and that is highly deleterious in humans.
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Affiliation(s)
| | - Kåre-Olav Stensløkken
- Institute of Basic Medical Sciences, University of Oslo, N-0372, Oslo, Norway.,Center for Heart Failure Research, University of Oslo, N-0317, Oslo, Norway
| | - Åsmund K Røhr
- Department of Biosciences, University of Oslo, N-0316, Oslo, Norway
| | - Michael Berenbrink
- Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, United Kingdom
| | - Stian Ellefsen
- The Lillehammer Research Center for Medicine and Exercise Physiology, Inland Norway University of Applied Sciences, N-2604, Lillehammer, Norway
| | - Göran E Nilsson
- Department of Biosciences, University of Oslo, N-0316, Oslo, Norway.
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47
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Global view of cognate kinase activation by the human pyruvate dehydrogenase complex. Sci Rep 2017; 7:42760. [PMID: 28230160 PMCID: PMC5322387 DOI: 10.1038/srep42760] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2016] [Accepted: 01/13/2017] [Indexed: 12/11/2022] Open
Abstract
The human pyruvate dehydrogenase complex (PDC) comprises four multidomain components, E1, E3, E2 and an E3-binding protein (E3BP), the latter two forming the core as E2·E3BP sub-complex. Pyruvate flux through PDC is regulated via phosphorylation (inactivation) at E1 by four PDC kinases (PDKs), and reactivation by two PDC phosphatases. Up-regulation of PDK isoform gene expression is reported in several forms of cancer, while PDKs may be further activated by PDC by binding to the E2·E3BP core. Hence, the PDK: E2·E3BP interaction provides new therapeutic targets. We carried out both functional kinetic and thermodynamic studies to demonstrate significant differences in the activation of PDK isoforms by binding to the E2·E3BP core: (i) PDK2 needs no activation by E2·E3BP for efficient functioning, while PDK4 was the least effective of the four isoforms, and could not be activated by E2·E3BP. Hence, development of inhibitors to the interaction of PDK2 and PDK4 with E2·E3BP is not promising; (ii) Design of inhibitors to interfere with interaction of E2·E3BP with PDK1 and PDK3 is promising. PDK3 needs E2·E3BP core for activation, an activation best achieved by synergistic combination of E2-derived catalytic domain and tridomain.
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48
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Reengineering of the human pyruvate dehydrogenase complex: from disintegration to highly active agglomerates. Biochem J 2017; 474:865-875. [PMID: 27986918 DOI: 10.1042/bcj20160916] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Revised: 12/13/2016] [Accepted: 12/15/2016] [Indexed: 11/17/2022]
Abstract
The pyruvate dehydrogenase complex (PDC) plays a central role in cellular metabolism and regulation. As a metabolite-channeling multi-enzyme complex it acts as a complete nanomachine due to its unique geometry and by coupling a cascade of catalytic reactions using 'swinging arms'. Mammalian and specifically human PDC (hPDC) is assembled from multiple copies of E1 and E3 bound to a large E2/E3BP 60-meric core. A less restrictive and smaller catalytic core, which is still active, is highly desired for both fundamental research on channeling mechanisms and also to create a basis for further modification and engineering of new enzyme cascades. Here, we present the first experimental results of the successful disintegration of the E2/E3BP core while retaining its activity. This was achieved by C-terminal α-helixes double truncations (eight residues from E2 and seven residues from E3BP). Disintegration of the hPDC core via double truncations led to the formation of highly active (approximately 70% of wildtype) apparently unordered clusters or agglomerates and inactive non-agglomerated species (hexamer/trimer). After additional deletion of N-terminal 'swinging arms', the aforementioned C-terminal truncations also caused the formation of agglomerates of minimized E2/E3BP complexes. It is likely that these 'swinging arm' regions are not solely responsible for the formation of the large agglomerates.
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49
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Kupynyak NI, Ikkert OV, Shlykov SG, Babich LG, Manko VV. Mitochondrial ryanodine-sensitive Ca 2+ channels of rat liver. Cell Biochem Funct 2017; 35:42-49. [PMID: 28052355 DOI: 10.1002/cbf.3243] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2016] [Revised: 10/26/2016] [Accepted: 11/21/2016] [Indexed: 11/06/2022]
Abstract
To examine ryanodine-sensitive Ca2+ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca2+ concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca2+ concentration and affecting the energy state of mictochondria in hepatocytes.
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Affiliation(s)
- N I Kupynyak
- Ivan Franko National University of Lviv, Lviv, Ukraine.,Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
| | - O V Ikkert
- Ivan Franko National University of Lviv, Lviv, Ukraine
| | - S G Shlykov
- Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine
| | - L G Babich
- Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine
| | - V V Manko
- Ivan Franko National University of Lviv, Lviv, Ukraine
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Abstract
The family of 2-oxoacid dehydrogenase complexes (2-OADC), typified by the pyruvate dehydrogenase multi-enzyme complex (PDC) as its most prominent member, are massive molecular machines (Mr, 4-10 million) controlling key steps in glucose homeostasis (PDC), citric acid cycle flux (OGDC, 2-oxoglutarate dehydrogenase) and the metabolism of the branched-chain amino acids, leucine, isoleucine and valine (BCOADC, branched-chain 2-OADC). These highly organised mitochondrial arrays, composed of multiple copies of three separate enzymes, have been widely studied as paradigms for the analysis of enzyme cooperativity, substrate channelling, protein-protein interactions and the regulation of activity by phosphorylation . This chapter will highlight recent advances in our understanding of the structure-function relationships, the overall organisation and the transport and assembly of PDC in particular, focussing on both native and recombinant forms of the complex and their individual components or constituent domains. Biophysical approaches, including X-ray crystallography (MX), nuclear magnetic resonance spectroscopy (NMR), cryo-EM imaging, analytical ultracentrifugation (AUC) and small angle X-ray and neutron scattering (SAXS and SANS), have all contributed significant new information on PDC subunit organisation, stoichiometry, regulatory mechanisms and mode of assembly. Moreover, the recognition of specific genetic defects linked to PDC deficiency, in combination with the ability to analyse recombinant PDCs housing both novel naturally-occurring and engineered mutations, have all stimulated renewed interest in these classical metabolic assemblies. In addition, the role played by PDC, and its constituent proteins, in certain disease states will be briefly reviewed, focussing on the development of new and exciting areas of medical and immunological research.
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Affiliation(s)
- Olwyn Byron
- School of Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
| | - John Gordon Lindsay
- Institute of Molecular, Cell and Systems Biology, Davidson Building, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
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