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1
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Augustine A, Messaabi A, Fantino E, Merindol N, Meddeb-Mouelhi F, Desgagné-Penix I. Multifactorial interaction and influence of culture conditions on yellow fluorescent protein production in Phaeodactylum tricornutum. BIORESOURCE TECHNOLOGY 2025; 425:132336. [PMID: 40044057 DOI: 10.1016/j.biortech.2025.132336] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/28/2024] [Revised: 03/02/2025] [Accepted: 03/02/2025] [Indexed: 03/16/2025]
Abstract
Phaeodactylum tricornutum is a promising host for light-driven synthesis of heterologous proteins. However, the marine cold-water environment and alkaline-acidic pH shifts in the culture, necessitated by the diatom's growth requirements. In this study, we analyzed the influence of growth condition on maturation and dynamics of the yellow fluorescent protein (YFP) in episomal-transformant P. tricornutum. A mathematical model was developed to detect the parameters that affect biomass and YFP production. Optimized conditions increased YFP mean fluorescence intensity (MFI) per cell by 4.2-fold (3.6 ± 0.6 to 15.4 ± 1.1) and total protein levels in the culture by 1.8-fold (123 ± 4 to 219 ± 9 µg L-1), without affecting biomass. YFP stability studies in P. tricornutum showed that the ubiquitin-proteasome system contributes the degradation of the recombinant protein, whereas newly synthesized YFP remains stable for up to 12 h. This optimization provides insights into the fluorescent protein-based heterologous production in diatoms.
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Affiliation(s)
- Arun Augustine
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boulevard des Forges, Trois-Rivières, QC G9A 5H7, Canada
| | - Anis Messaabi
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boulevard des Forges, Trois-Rivières, QC G9A 5H7, Canada
| | - Elisa Fantino
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boulevard des Forges, Trois-Rivières, QC G9A 5H7, Canada; Plant Biology Research Group, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada
| | - Natacha Merindol
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boulevard des Forges, Trois-Rivières, QC G9A 5H7, Canada
| | - Fatma Meddeb-Mouelhi
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boulevard des Forges, Trois-Rivières, QC G9A 5H7, Canada; Plant Biology Research Group, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada
| | - Isabel Desgagné-Penix
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boulevard des Forges, Trois-Rivières, QC G9A 5H7, Canada; Plant Biology Research Group, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada.
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2
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Islam N, Krishnan HB, Slovin J, Li Z, Fakir T, Luthria D, Natarajan S. High-Resolution Mass Spectrometry Approach for Proteomic and Metabolomic Analyses of High-Protein Soybean Seeds. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2025; 73:6993-7002. [PMID: 40042580 DOI: 10.1021/acs.jafc.5c00375] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/20/2025]
Abstract
Soybeans are a valuable source of vegetable protein and edible oil. Fast neutron (FN) radiation was employed to produce large chromosomal deletions in soybean Glycine max (L.) Merrill. We conducted proteomic and metabolomic profiling of a high-protein soybean mutant (G15FN-12) developed through FN mutagenesis to identify the metabolic pathways that underlie the elevated protein content. A deletion of 137 genes located on chromosome-12 had occurred in G15FN-12. Tandem tag-based protein profiling of the mutant and wild type identified 6098 proteins, of which 175 showed increased abundance and 239 showed decreased abundance in the mutant seeds. Using liquid chromatography-mass spectrometry (LC-MS)-based metabolomic profiling, we identified 610 metabolites, of which 294 metabolites showed increased and 157 showed reduced content in mutant seeds as compared to wild type. Proteomic and metabolomic profiling revealed a decrease in ubiquitin-proteasome-associated proteins and an increase in heat shock proteins in the mutant seed. We hypothesize that decreased protein degradation, together with enhanced refolding of misfolded protein by molecular chaperones, contributes to elevated protein content in the mutant seed. The development of value-added seed traits such as increased protein using advanced metabolic engineering techniques can be achieved by further exploring the metabolic pathways identified in this investigation.
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Affiliation(s)
- Nazrul Islam
- Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, Maryland 20705, United States
| | - Hari B Krishnan
- Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Columbia, Missouri 65201, United States
| | - Janet Slovin
- Genetic Improvement for Fruits & Vegetables Laboratory, USDA-ARS, Beltsville, Maryland 20705, United States
| | - Zenglu Li
- Department of Crop and Soil Sciences and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, Georgia 30602, United States
| | - Tareq Fakir
- Methods and Application of Food Composition Laboratory, NEA, BHNRC, USDA-ARS, Beltsville, Maryland 20705, United States
| | - Devanand Luthria
- Methods and Application of Food Composition Laboratory, NEA, BHNRC, USDA-ARS, Beltsville, Maryland 20705, United States
| | - Savithiry Natarajan
- Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, Maryland 20705, United States
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3
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Abbiati F, Orlandi I, Pagliari S, Campone L, Vai M. Glucosinolates from Seed-Press Cake of Camelina sativa (L.) Crantz Extend Yeast Chronological Lifespan by Modulating Carbon Metabolism and Respiration. Antioxidants (Basel) 2025; 14:80. [PMID: 39857414 PMCID: PMC11759863 DOI: 10.3390/antiox14010080] [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: 12/19/2024] [Revised: 01/08/2025] [Accepted: 01/09/2025] [Indexed: 01/27/2025] Open
Abstract
Glucosinolates (GSLs) are nitrogen/sulfur-containing glycosides widely present in the order of Brassicales, particularly in the Brassicaceae family. Camelina (Camelina sativa (L.) Crantz) is an oilseed plant belonging to this family. Its seeds, in addition to a distinctive fatty acid composition, contain three aliphatic GSLs: glucoarabin, glucocamelinin, and homoglucocamelinin. Our study explored the impact of these GSLs purified from Camelina press cake, a by-product of Camelina oil production, on yeast chronological aging, which is the established model for simulating the aging of post-mitotic quiescent mammalian cells. Supplementing yeast cells with GSLs extends the chronological lifespan (CLS) in a dose-dependent manner. This enhancement relies on an improved mitochondrial respiration efficiency, resulting in a drastic decrease of superoxide anion levels and an increase in ATP production. Furthermore, GSL supplementation affects carbon metabolism. In particular, GSLs support the pro-longevity preservation of TCA cycle enzymatic activities and enhanced glycerol catabolism. These changes contribute positively to the phosphorylating respiration and to an increase in trehalose storage: both of which are longevity-promoting prerequisites.
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Affiliation(s)
- Francesco Abbiati
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (I.O.); (S.P.); (L.C.)
| | - Ivan Orlandi
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (I.O.); (S.P.); (L.C.)
- SYSBIO Centre of Systems Biology, 20126 Milano, Italy
| | - Stefania Pagliari
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (I.O.); (S.P.); (L.C.)
| | - Luca Campone
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (I.O.); (S.P.); (L.C.)
| | - Marina Vai
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (I.O.); (S.P.); (L.C.)
- SYSBIO Centre of Systems Biology, 20126 Milano, Italy
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4
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Hunt LC, Curley M, Nyamkondiwa K, Stephan A, Jiao J, Kavdia K, Pagala VR, Peng J, Demontis F. The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity. PLoS Biol 2025; 23:e3002998. [PMID: 39879147 PMCID: PMC11778781 DOI: 10.1371/journal.pbio.3002998] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Accepted: 12/20/2024] [Indexed: 01/31/2025] Open
Abstract
Ubiquitin-conjugating enzymes (E2s) are key for protein turnover and quality control via ubiquitination. Some E2s also physically interact with the proteasome, but it remains undetermined which E2s maintain proteostasis during aging. Here, we find that E2s have diverse roles in handling a model aggregation-prone protein (huntingtin-polyQ) in the Drosophila retina: while some E2s mediate aggregate assembly, UBE2D/effete (eff) and other E2s are required for huntingtin-polyQ degradation. UBE2D/eff is key for proteostasis also in skeletal muscle: eff protein levels decline with aging, and muscle-specific eff knockdown causes an accelerated buildup in insoluble poly-ubiquitinated proteins (which progressively accumulate with aging) and shortens lifespan. Mechanistically, UBE2D/eff is necessary to maintain optimal proteasome function: UBE2D/eff knockdown reduces the proteolytic activity of the proteasome, and this is rescued by transgenic expression of human UBE2D2, an eff homolog. Likewise, human UBE2D2 partially rescues the lifespan and proteostasis deficits caused by muscle-specific effRNAi and re-establishes the physiological levels of effRNAi-regulated proteins. Interestingly, UBE2D/eff knockdown in young age reproduces part of the proteomic changes that normally occur in old muscles, suggesting that the decrease in UBE2D/eff protein levels that occurs with aging contributes to reshaping the composition of the muscle proteome. However, some of the proteins that are concertedly up-regulated by aging and effRNAi are proteostasis regulators (e.g., chaperones and Pomp) that are transcriptionally induced presumably as part of an adaptive stress response to the loss of proteostasis. Altogether, these findings indicate that UBE2D/eff is a key E2 ubiquitin-conjugating enzyme that ensures protein quality control and helps maintain a youthful proteome composition during aging.
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Affiliation(s)
- Liam C. Hunt
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Michelle Curley
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Kudzai Nyamkondiwa
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Anna Stephan
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Jianqin Jiao
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Kanisha Kavdia
- Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Vishwajeeth R. Pagala
- Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Junmin Peng
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
- Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
| | - Fabio Demontis
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
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5
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Xiao X, Liu Q, Zhang Q, Yan Z, Cai D, Li X. Exogenous Trehalose Assists Zygosaccharomyces rouxii in Resisting High-Temperature Stress Mainly by Activating Genes Rather than Entering Metabolism. J Fungi (Basel) 2024; 10:842. [PMID: 39728338 DOI: 10.3390/jof10120842] [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: 11/07/2024] [Revised: 11/14/2024] [Accepted: 11/15/2024] [Indexed: 12/28/2024] Open
Abstract
Zygosaccharomyces rouxii is a typical aroma-producing yeast in food brewing, but it has low heat resistance and poor proliferation ability at high temperature. Trehalose is generally considered to be a protective agent that helps stable yeast cells resist heat shock stress, but its functional mechanism for yeast cells in the adaptation period under heat stress is unclear. In this study, the physiological metabolism changes, specific gene transcription expression characteristics, and transcriptome differences of Z. rouxii under different carbon sources under high-temperature stress (40 °C) were compared to explore the mechanism of trehalose inducing Z. rouxii to recover and proliferate under high-temperature stress during the adaptation period. The results showed that high concentration of trehalose (20% Tre) could not be used as the main carbon source for the proliferation of Z. rouxii under long-term high-temperature stress, but it helped to maintain the stability of the cell population. The intracellular trehalose of Z. rouxii was mainly derived from the synthesis and metabolism of intracellular glucose, and the extracellular acetic acid concentration showed an upward trend with the improvement of yeast growth. A high concentration of trehalose (20% Tre) can promote the expression of high glucose receptor gene GRT2 (12.0-fold) and induce the up-regulation of HSF1 (27.1-fold), MSN4 (58.9-fold), HXK1 (8.3-fold), and other signal transduction protein genes, and the increase of trehalose concentration will maintain the temporal up-regulation of these genes. Transcriptome analysis showed that trehalose concentration and the presence of glucose had a significant effect on the gene expression of Z. rouxii under high-temperature stress. In summary, trehalose assists Z. rouxii in adapting to high temperature by changing gene expression levels, and assists Z. rouxii in absorbing glucose to achieve cell proliferation.
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Affiliation(s)
- Xiong Xiao
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education and Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China
| | - Quan Liu
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education and Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China
| | - Qian Zhang
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education and Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China
| | - Zhenzhen Yan
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education and Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China
| | - Dongbo Cai
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430068, China
| | - Xin Li
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education and Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China
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6
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Patnaik PK, Nady N, Barlit H, Gülhan A, Labunskyy VM. Lifespan regulation by targeting heme signaling in yeast. GeroScience 2024; 46:5235-5245. [PMID: 38809391 PMCID: PMC11335709 DOI: 10.1007/s11357-024-01218-9] [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: 01/19/2024] [Accepted: 05/21/2024] [Indexed: 05/30/2024] Open
Abstract
Heme is an essential prosthetic group that serves as a co-factor and a signaling molecule. Heme levels decline with age, and its deficiency is associated with multiple hallmarks of aging, including anemia, mitochondrial dysfunction, and oxidative stress. Dysregulation of heme homeostasis has been also implicated in aging in model organisms suggesting that heme may play an evolutionarily conserved role in controlling lifespan. However, the underlying mechanisms and whether heme homeostasis can be targeted to promote healthy aging remain unclear. Here, we used Saccharomyces cerevisiae as a model to investigate the role of heme in aging. For this, we have engineered a heme auxotrophic yeast strain expressing a plasma membrane-bound heme permease from Caenorhabditis elegans (ceHRG-4). This system can be used to control intracellular heme levels independently of the biosynthetic enzymes by manipulating heme concentration in the media. We observed that heme supplementation leads to a significant extension of yeast replicative lifespan. Our findings revealed that the effect of heme on lifespan is independent of the Hap4 transcription factor. Surprisingly, heme-supplemented cells had impaired growth on YPG medium, which requires mitochondrial respiration to be used, suggesting that these cells are respiratory deficient. Together, our results demonstrate that heme homeostasis is fundamentally important for aging biology, and manipulating heme levels can be used as a promising therapeutic target for promoting longevity.
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Affiliation(s)
- Praveen K Patnaik
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, 02118, USA
| | - Nour Nady
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, 02118, USA
| | - Hanna Barlit
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, 02118, USA
| | - Ali Gülhan
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, 02118, USA
| | - Vyacheslav M Labunskyy
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, 02118, USA.
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7
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Patil ND, Bains A, Kaur S, Yadav R, Goksen G, Ali N, AlAsmari AF, Chawla P. Effect of dual modifications with ultrasonication and succinylation on Cicer arietinum protein-iron complexes: Characterization, digestibility, in-vitro cellular mineral uptake and preparation of fortified smoothie. Food Res Int 2024; 186:114344. [PMID: 38729696 DOI: 10.1016/j.foodres.2024.114344] [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: 10/19/2023] [Revised: 04/07/2024] [Accepted: 04/17/2024] [Indexed: 05/12/2024]
Abstract
The research aimed to evaluate the effect of ultrasonication and succinylation on the functional, iron binding, physiochemical, and cellular mineral uptake efficacy of chickpea protein concentrate. Succinylation resulted in significant improvements in the water-holding capacity (WHC) (25.47 %), oil-holding capacity (OHC) (31.38 %), and solubility (5.80 %) of the chickpea protein-iron complex. Mineral bioavailability significantly increased by 4.41 %, and there was a significant increase in cellular mineral uptake (64.64 %), retention (36.68 %), and transport (27.96 %). The ferritin content of the succinylated chickpea protein-iron complex showed a substantial increase of 66.31%. Furthermore, the dual modification approach combining ultrasonication and succinylation reduced the particle size of the protein-iron complex with a substantial reduction of 83.25 %. It also resulted in a significant enhancement of 51.5 % in the SH (sulfhydryl) content and 48.92 % in the surface hydrophobicity. Mineral bioavailability and cellular mineral uptake, retention, and transport were further enhanced through dual modification. In terms of application, the addition of single and dual-modified chickpea protein-iron complex to a fruit-based smoothie demonstrated positive acceptance in sensory attributes. Overall, the combined approach of succinylation and ultrasonication to the chickpea protein-iron complex shows a promising strategy for enhancing the physiochemical and techno-functional characteristics, cellular mineral uptake, and the development of vegan food products.
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Affiliation(s)
- Nikhil Dnyaneshwar Patil
- Department of Food Technology and Nutrition, Lovely Professional University, Phagwara, Punjab 144411, India
| | - Aarti Bains
- Department of Microbiology, Lovely Professional University, Phagwara 144411, Punjab, India
| | - Sawinder Kaur
- Department of Food Technology and Nutrition, Lovely Professional University, Phagwara, Punjab 144411, India
| | - Rahul Yadav
- Shoolini Life Sciences Pvt. Ltd., Shoolini University, Solan 173229, Himachal Pradesh, India
| | - Gulden Goksen
- Department of Food Technology, Vocational School of Technical Sciences at Mersin Tarsus Organized Industrial Zone, Tarsus University, 33100 Mersin, Turkey.
| | - Nemat Ali
- Département of Pharmacology and Toxicology, Collège of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
| | - Abdullah F AlAsmari
- Département of Pharmacology and Toxicology, Collège of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
| | - Prince Chawla
- Department of Food Technology and Nutrition, Lovely Professional University, Phagwara, Punjab 144411, India.
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8
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Gurubaran IS. Mitochondrial damage and clearance in retinal pigment epithelial cells. Acta Ophthalmol 2024; 102 Suppl 282:3-53. [PMID: 38467968 DOI: 10.1111/aos.16661] [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: 01/24/2024] [Accepted: 01/31/2024] [Indexed: 03/13/2024]
Abstract
Age-related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing-associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator-activated receptor gamma coactivator 1-alpha and nuclear factor erythroid 2-related factor 2 (PGC1α/NFE2L2) double-knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell-based model was established to examine the impact of non-functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one-year-old PGC1α/NFE2L2-deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase β. There was an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild-type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C-reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial-mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux-dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin-1β in ARPE-19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD-like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE-19 cells.
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Affiliation(s)
- Iswariyaraja Sridevi Gurubaran
- Department of Medicine, Clinical Medicine Unit, University of Eastern Finland Institute of Clinical Medicine, Kuopio, Northern Savonia, Finland
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9
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Patnaik PK, Nady N, Barlit H, Gülhan A, Labunskyy VM. Lifespan regulation by targeting heme signaling in yeast. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.20.576446. [PMID: 38293148 PMCID: PMC10827197 DOI: 10.1101/2024.01.20.576446] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/01/2024]
Abstract
Heme is an essential prosthetic group that serves as a co-factor and a signaling molecule. Heme levels decline with age, and its deficiency is associated with multiple hallmarks of aging, including anemia, mitochondrial dysfunction, and oxidative stress. Dysregulation of heme homeostasis has been also implicated in aging in model organisms suggesting that heme may play an evolutionarily conserved role in controlling lifespan. However, the underlying mechanisms and whether heme homeostasis can be targeted to promote healthy aging remain unclear. Here we used Saccharomyces cerevisiae as a model to investigate the role of heme in aging. For this, we have engineered a heme auxotrophic yeast strain expressing a plasma membrane-bound heme permease from Caenorhabditis elegans (ceHRG-4). This system can be used to control intracellular heme levels independently of the biosynthetic enzymes by manipulating heme concentration in the media. We observed that heme supplementation leads to significant lifespan extension in yeast. Our findings revealed that the effect of heme on lifespan is independent of the Hap4 transcription factor. Surprisingly, heme-supplemented cells had impaired growth on YPG medium, which requires mitochondrial respiration to be used, suggesting that these cells are respiratory deficient. Together, our results demonstrate that heme homeostasis is fundamentally important for aging biology and manipulating heme levels can be used as a promising therapeutic target for promoting longevity.
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Affiliation(s)
- Praveen K. Patnaik
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
| | - Nour Nady
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
| | - Hanna Barlit
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
| | - Ali Gülhan
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
| | - Vyacheslav M. Labunskyy
- Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
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10
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Abbiati F, Garagnani SA, Orlandi I, Vai M. Sir2 and Glycerol Underlie the Pro-Longevity Effect of Quercetin during Yeast Chronological Aging. Int J Mol Sci 2023; 24:12223. [PMID: 37569599 PMCID: PMC10419316 DOI: 10.3390/ijms241512223] [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: 07/06/2023] [Revised: 07/28/2023] [Accepted: 07/29/2023] [Indexed: 08/13/2023] Open
Abstract
Quercetin (QUER) is a natural polyphenolic compound endowed with beneficial properties for human health, with anti-aging effects. However, although this flavonoid is commercially available as a nutraceutical, target molecules/pathways underlying its pro-longevity potential have yet to be fully clarified. Here, we investigated QUER activity in yeast chronological aging, the established model for simulating the aging of postmitotic quiescent mammalian cells. We found that QUER supplementation at the onset of chronological aging, namely at the diauxic shift, significantly increases chronological lifespan (CLS). Consistent with the antioxidant properties of QUER, this extension takes place in concert with a decrease in oxidative stress. In addition, QUER triggers substantial changes in carbon metabolism. Specifically, it promotes an enhancement of a pro-longevity anabolic metabolism toward gluconeogenesis due to improved catabolism of C2 by-products of yeast fermentation and glycerol. The former is attributable to the Sir2-dependent activity of phosphoenolpyruvate carboxykinase and the latter to the L-glycerol 3-phosphate pathway. Such a combined increased supply of gluconeogenesis leads to an increase in the reserve carbohydrate trehalose, ensuring CLS extension. Moreover, QUER supplementation to chronologically aging cells in water alone amplifies their long-lived phenotype. This is associated with intracellular glycerol catabolism and trehalose increase, further indicating a QUER-specific influence on carbon metabolism that results in CLS extension.
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Affiliation(s)
- Francesco Abbiati
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (S.A.G.); (I.O.)
| | - Stefano Angelo Garagnani
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (S.A.G.); (I.O.)
| | - Ivan Orlandi
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (S.A.G.); (I.O.)
- SYSBIO Centre for Systems Biology, 20126 Milano, Italy
| | - Marina Vai
- Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; (F.A.); (S.A.G.); (I.O.)
- SYSBIO Centre for Systems Biology, 20126 Milano, Italy
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11
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Ma X, Xia Q, Liu K, Wu Z, Li C, Xiao C, Dong N, Hameed M, Anwar MN, Li Z, Shao D, Li B, Qiu Y, Wei J, Ma Z. Palmitoylation at Residue C221 of Japanese Encephalitis Virus NS2A Protein Contributes to Viral Replication Efficiency and Virulence. J Virol 2023; 97:e0038223. [PMID: 37289075 PMCID: PMC10308905 DOI: 10.1128/jvi.00382-23] [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: 03/14/2023] [Accepted: 05/13/2023] [Indexed: 06/09/2023] Open
Abstract
Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.
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Affiliation(s)
- Xiaochun Ma
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
- College of Veterinary Medicine, Shandong Vocational Animal Science and Veterinary College, Weifang, People’s Republic of China
| | - Qiqi Xia
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Ke Liu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Zhuanchang Wu
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, School of Basic Medical Science, Shandong University, Jinan, People’s Republic of China
| | - Chenxi Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Changguang Xiao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Nihua Dong
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Muddassar Hameed
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Muhammad Naveed Anwar
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
- Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan
| | - Zongjie Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Donghua Shao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Beibei Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Yafeng Qiu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Jianchao Wei
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
| | - Zhiyong Ma
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China
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12
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Majtan T, Kožich V, Kruger WD. Recent therapeutic approaches to cystathionine beta-synthase-deficient homocystinuria. Br J Pharmacol 2023; 180:264-278. [PMID: 36417581 PMCID: PMC9822868 DOI: 10.1111/bph.15991] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 11/09/2022] [Accepted: 11/14/2022] [Indexed: 11/27/2022] Open
Abstract
Cystathionine beta-synthase (CBS)-deficient homocystinuria (HCU) is the most common inborn error of sulfur amino acid metabolism. The pyridoxine non-responsive form of the disease manifests itself by massively increasing plasma and tissue concentrations of homocysteine, a toxic intermediate of methionine metabolism that is thought to be the major cause of clinical complications including skeletal deformities, connective tissue defects, thromboembolism and cognitive impairment. The current standard of care involves significant dietary interventions that, despite being effective, often adversely affect quality of life of HCU patients, leading to poor adherence to therapy and inadequate biochemical control with clinical complications. In recent years, the unmet need for better therapeutic options has resulted in development of novel enzyme and gene therapies and exploration of pharmacological approaches to rescue CBS folding defects caused by missense pathogenic mutations. Here, we review scientific evidence and current state of affairs in development of recent approaches to treat HCU.
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Affiliation(s)
- Tomas Majtan
- Department of Pharmacology, University of Fribourg, Faculty of Science and Medicine, Fribourg, 1700, Switzerland
| | - Viktor Kožich
- Department of Pediatrics and Inherited Metabolic Disorders, Charles University-First Faculty of Medicine, Prague, 12808, Czech Republic
- Department of Pediatrics and Inherited Metabolic Disorders, General University Hospital in Prague, Prague, 12808, Czech Republic
| | - Warren D. Kruger
- Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA
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13
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Kandel R, Jung J, Syau D, Kuo T, Songster L, Horn C, Chapman C, Aguayo A, Duttke S, Benner C, Neal SE. Yeast derlin Dfm1 employs a chaperone-like function to resolve misfolded membrane protein stress. PLoS Biol 2023; 21:e3001950. [PMID: 36689475 PMCID: PMC9894555 DOI: 10.1371/journal.pbio.3001950] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 02/02/2023] [Accepted: 12/07/2022] [Indexed: 01/24/2023] Open
Abstract
Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1's previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis.
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Affiliation(s)
- Rachel Kandel
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Jasmine Jung
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Della Syau
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Tiffany Kuo
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Livia Songster
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Casey Horn
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Claire Chapman
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Analine Aguayo
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
| | - Sascha Duttke
- School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America
| | - Christopher Benner
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America
| | - Sonya E. Neal
- Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
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14
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Oroń M, Grochowski M, Jaiswar A, Legierska J, Jastrzębski K, Nowak-Niezgoda M, Kołos M, Kaźmierczak W, Olesiński T, Lenarcik M, Cybulska M, Mikula M, Żylicz A, Miączyńska M, Zettl K, Wiśniewski JR, Walerych D. The molecular network of the proteasome machinery inhibition response is orchestrated by HSP70, revealing vulnerabilities in cancer cells. Cell Rep 2022; 40:111428. [PMID: 36170818 DOI: 10.1016/j.celrep.2022.111428] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Revised: 07/15/2022] [Accepted: 09/08/2022] [Indexed: 11/29/2022] Open
Abstract
Proteasome machinery is a major proteostasis control system in human cells, actively compensated upon its inhibition. To understand this compensation, we compared global protein landscapes upon the proteasome inhibition with carfilzomib, in normal fibroblasts, cells of multiple myeloma, and cancers of lung, colon, and pancreas. Molecular chaperones, autophagy, and endocytosis-related proteins are the most prominent vulnerabilities in combination with carfilzomib, while targeting of the HSP70 family chaperones HSPA1A/B most specifically sensitizes cancer cells to the proteasome inhibition. This suggests a central role of HSP70 in the suppression of the proteasome downregulation, allowing to identify pathways impinging on HSP70 upon the proteasome inhibition. HSPA1A/B indeed controls proteasome-inhibition-induced autophagy, unfolded protein response, and endocytic flux, and directly chaperones the proteasome machinery. However, it does not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, targeting of NRF1 proves effective in decreasing the viability of cancer cells with the inhibited proteasome and HSP70.
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Affiliation(s)
- Magdalena Oroń
- Mossakowski Medical Research Institute PAS, Warsaw, Poland
| | | | | | | | - Kamil Jastrzębski
- International Institute of Molecular and Cell Biology, Warsaw, Poland
| | | | - Małgorzata Kołos
- Central Clinical Hospital of Ministry of Interior and Administration, Warsaw, Poland
| | | | | | | | | | | | - Alicja Żylicz
- International Institute of Molecular and Cell Biology, Warsaw, Poland
| | - Marta Miączyńska
- International Institute of Molecular and Cell Biology, Warsaw, Poland
| | | | | | - Dawid Walerych
- Mossakowski Medical Research Institute PAS, Warsaw, Poland.
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15
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Kwon KM, Lee MJ, Chung HS, Pak JH, Jeon CJ. The Organization of Somatostatin-Immunoreactive Cells in the Visual Cortex of the Gerbil. Biomedicines 2022; 10:biomedicines10010092. [PMID: 35052772 PMCID: PMC8773527 DOI: 10.3390/biomedicines10010092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Revised: 12/29/2021] [Accepted: 12/31/2021] [Indexed: 11/16/2022] Open
Abstract
Somatostatin (SST) is widely expressed in the brain and plays various, vital roles involved in neuromodulation. The purpose of this study is to characterize the organization of SST neurons in the Mongolian gerbil visual cortex (VC) using immunocytochemistry, quantitative analysis, and confocal microscopy. As a diurnal animal, the Mongolian gerbil provides us with a different perspective to other commonly used nocturnal rodent models. In this study, SST neurons were located in all layers of the VC except in layer I; they were most common in layer V. Most SST neurons were multipolar round/oval or stellate cells. No pyramidal neurons were found. Moreover, 2-color immunofluorescence revealed that only 33.50%, 24.05%, 16.73%, 0%, and 64.57% of SST neurons contained gamma-aminobutyric acid, calbindin-D28K, calretinin, parvalbumin, and calcium/calmodulin-dependent protein kinase II, respectively. In contrast, neuropeptide Y and nitric oxide synthase were abundantly expressed, with 80.07% and 75.41% in SST neurons, respectively. Our immunocytochemical analyses of SST with D1 and D2 dopamine receptors and choline acetyltransferase, α7 and β2 nicotinic acetylcholine receptors suggest that dopaminergic and cholinergic fibers contact some SST neurons. The results showed some distinguishable features of SST neurons and provided some insight into their afferent circuitry in the gerbil VC. These findings may support future studies investigating the role of SST neurons in visual processing.
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Affiliation(s)
- Kyung-Min Kwon
- Department of Biology, School of Life Sciences, BK21 FOUR KNU Creative Bio-Research Group, College of Natural Sciences, Brain Science and Engineering Institute, Kyungpook National University, Daegu 41566, Korea; (K.-M.K.); (M.-J.L.)
- Research Institute for Dok-do and Ulleung-do Island, Department of Biology, School of Life Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Korea;
| | - Myung-Jun Lee
- Department of Biology, School of Life Sciences, BK21 FOUR KNU Creative Bio-Research Group, College of Natural Sciences, Brain Science and Engineering Institute, Kyungpook National University, Daegu 41566, Korea; (K.-M.K.); (M.-J.L.)
| | - Han-Saem Chung
- Department of Biology, School of Life Sciences, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea;
| | - Jae-Hong Pak
- Research Institute for Dok-do and Ulleung-do Island, Department of Biology, School of Life Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Korea;
- Department of Biology, School of Life Sciences, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea;
| | - Chang-Jin Jeon
- Department of Biology, School of Life Sciences, BK21 FOUR KNU Creative Bio-Research Group, College of Natural Sciences, Brain Science and Engineering Institute, Kyungpook National University, Daegu 41566, Korea; (K.-M.K.); (M.-J.L.)
- Research Institute for Dok-do and Ulleung-do Island, Department of Biology, School of Life Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Korea;
- Correspondence:
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16
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Baldan S, Meriin AB, Yaglom J, Alexandrov I, Varelas X, Xiao ZXJ, Sherman MY. The Hsp70-Bag3 complex modulates the phosphorylation and nuclear translocation of Hippo pathway protein Yap. J Cell Sci 2021; 134:273417. [PMID: 34761265 DOI: 10.1242/jcs.259107] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Accepted: 10/28/2021] [Indexed: 11/20/2022] Open
Abstract
Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70-Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also controlled YAP nuclear localization in response to cell density, indicating broader roles beyond proteotoxic signaling responses for Bag3 in the regulation of YAP. These data implicate Bag3 as a regulator of Hippo pathway signaling, and suggest mechanisms by which proteotoxic stress signals are propagated.
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Affiliation(s)
- Simone Baldan
- Department of Molecular Biology, Ariel University, Ariel 4077625, Israel
| | - Anatoli B Meriin
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02215, USA
| | - Julia Yaglom
- Department of Molecular Biology, Ariel University, Ariel 4077625, Israel
| | | | - Xaralabos Varelas
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02215, USA
| | | | - Michael Y Sherman
- Department of Molecular Biology, Ariel University, Ariel 4077625, Israel
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17
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Kruger WD. How to fix a broken protein: restoring function to mutant human cystathionine β-synthase. Hum Genet 2021; 141:1299-1308. [PMID: 34636997 DOI: 10.1007/s00439-021-02386-w] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Accepted: 10/05/2021] [Indexed: 12/17/2022]
Abstract
Inborn errors of metabolism (IEM) comprise a large class of recessive genetic diseases involving disorders of cellular metabolism that tend to be caused by missense mutations in which a single incorrect amino acid is substituted in the polypeptide chain. Cystathionine beta-synthase (CBS) deficiency is an example of an IEM that causes large elevations of blood total homocysteine levels, resulting in phenotypes in several tissues. Current treatment strategies involve dietary restriction and vitamin therapy, but these are only partially effective and do not work in all patients. Over 85% of the described mutations in CBS-deficient patients are missense mutations in which the mutant protein fails to fold into an active conformation. The ability of CBS to achieve an active conformation is affected by a variety of intracellular protein networks including the chaperone system and the ubiquitin/proteasome system, collectively referred to as the proteostasis network. Proteostasis modulators are drugs that perturb various aspects of these networks. In this article, we will review the evidence that modulation of the intracellular protein folding environment can be used as a potential therapeutic strategy to treat CBS deficiency and discuss the pros and cons of such a strategy.
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Affiliation(s)
- Warren D Kruger
- Cancer Signaling and Epigenetics Program, Fox Chase Cancer Center, Philadelphia, PA, USA.
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18
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Kang S, Seo H, Lee MG, Yun CW. Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae. J Fungi (Basel) 2021; 7:jof7090756. [PMID: 34575794 PMCID: PMC8469692 DOI: 10.3390/jof7090756] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 09/03/2021] [Accepted: 09/10/2021] [Indexed: 11/17/2022] Open
Abstract
To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.
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Affiliation(s)
| | | | | | - Cheol-Won Yun
- Correspondence: ; Tel.: +82-2-3290-3456; Fax: +82-2-927-9028
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19
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AgDD System: A Chemical Controllable Protein Aggregates in Cells. Methods Mol Biol 2021. [PMID: 34228296 DOI: 10.1007/978-1-0716-1441-9_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.
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20
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Videira NB, Dias MMG, Terra MF, de Oliveira VM, García-Arévalo M, Avelino TM, Torres FR, Batista FAH, Figueira ACM. PPAR Modulation Through Posttranslational Modification Control. NUCLEAR RECEPTORS 2021:537-611. [DOI: 10.1007/978-3-030-78315-0_21] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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21
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Webb SD, Orton LD. Microglial peri-somatic abutments classify two novel types of GABAergic neuron in the inferior colliculus. Eur J Neurosci 2020; 54:5815-5833. [PMID: 33278847 DOI: 10.1111/ejn.15075] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2019] [Revised: 05/14/2020] [Accepted: 05/20/2020] [Indexed: 12/22/2022]
Abstract
Emerging evidence suggests functional roles for microglia in the healthy, mature nervous system. However, we know little of the cellular density and ramified morphology of microglia in sensory systems, and even less of their inter-relationship with inhibitory neurons. We therefore conducted fluorescent multi-channel immunohistochemistry and confocal microscopy in guinea pigs of both sexes for Iba1, GAD67, GFAP, calbindin, and calretinin. We explored these markers in the inferior colliculi (IC), which contain sub-regions specialized for different aspects of auditory processing. First, we found that while the density of Iba1+ somata is similar throughout the IC parenchyma, Iba1+ microglia in dorsal cortex are significantly more ramified than those in the central nucleus or lateral cortex. Conversely, Iba1+ ramifications in ventral central nucleus, a region with the highest density of GAD67+ (putative GABAergic) neurons in IC, are longer with fewer ramifications. Second, we observed extensive abutments of ramified Iba1+ processes onto GAD67+ somata throughout the whole IC and developed novel measures to quantify these. Cluster analyses revealed two novel sub-types of GAD67+ neuron that differ in the quantity of Iba1+ somatic abutments they receive. Unlike previous classification schemes for GAD67+ neurons in IC, these clusters are not related to GAD67+ soma size. Taken together, these data demonstrate that microglial ramifications vary between IC sub-regions in the healthy, adult IC, possibly related to the ongoing demands of their niche. Furthermore, Iba1+ abutments onto neuronal somata are a novel means by which GAD67+ neurons can be classified.
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Affiliation(s)
- Samuel David Webb
- Department of Life Sciences, Manchester Metropolitan University, Manchester, UK
| | - Llwyd David Orton
- Department of Life Sciences, Manchester Metropolitan University, Manchester, UK.,Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK
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22
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Roh H, Kim A, Kim N, Lee Y, Kim DH. Multi-Omics Analysis Provides Novel Insight into Immuno-Physiological Pathways and Development of Thermal Resistance in Rainbow Trout Exposed to Acute Thermal Stress. Int J Mol Sci 2020; 21:E9198. [PMID: 33276666 PMCID: PMC7731343 DOI: 10.3390/ijms21239198] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Revised: 11/23/2020] [Accepted: 11/25/2020] [Indexed: 12/16/2022] Open
Abstract
In recent years, poikilothermic animals such as fish have increasingly been exposed to stressful high-temperature environments due to global warming. However, systemic changes in fish under thermal stress are not fully understood yet at both the transcriptome and proteome level. Therefore, the objective of this study was to investigate the immuno-physiological responses of fish under extreme thermal stress through integrated multi-omics analysis. Trout were exposed to acute thermal stress by raising water temperature from 15 to 25 °C within 30 min. Head-kidney and plasma samples were collected and used for RNA sequencing and two-dimensional gel electrophoresis. Gene enrichment analysis was performed: differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified to interpret the multi-omics results and identify the relevant biological processes through pathway analysis. Thousands of DEGs and 49 DEPs were identified in fish exposed to thermal stress. Most of these genes and proteins were highly linked to DNA replication, protein processing in the endoplasmic reticulum, cell signaling and structure, glycolysis activation, complement-associated hemolysis, processing of released free hemoglobin, and thrombosis and hypertension/vasoconstriction. Notably, we found that immune disorders mediated by the complement system may trigger hemolysis in thermally stressed fish, which could have serious consequences such as ferroptosis and thrombosis. However, antagonistic activities that decrease cell-free hemoglobin, heme, and iron might be involved in alleviating the side effects of thermally induced immuno-physiological disorders. These factors may represent the major thermal resistance traits that allow fish to overcome extreme thermal stress. Our findings, based on integration of multi-omics data from transcriptomics and proteomics analyses, provide novel insight into the pathogenesis of acute thermal stress and temperature-linked epizootics.
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Affiliation(s)
- HyeongJin Roh
- Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan 48513, Korea; (H.R.); (N.K.); (Y.L.)
| | - Ahran Kim
- Pathology Research Division, National Institute of Fisheries Science, Busan 46083, Korea;
| | - Nameun Kim
- Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan 48513, Korea; (H.R.); (N.K.); (Y.L.)
| | - Yoonhang Lee
- Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan 48513, Korea; (H.R.); (N.K.); (Y.L.)
| | - Do-Hyung Kim
- Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan 48513, Korea; (H.R.); (N.K.); (Y.L.)
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BAG3 Proteomic Signature under Proteostasis Stress. Cells 2020; 9:cells9112416. [PMID: 33158300 PMCID: PMC7694386 DOI: 10.3390/cells9112416] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2020] [Revised: 10/28/2020] [Accepted: 10/31/2020] [Indexed: 12/13/2022] Open
Abstract
The multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3) represents a key player in the quality control of the cellular proteostasis network. In response to stress, BAG3 specifically targets aggregation-prone proteins to the perinuclear aggresome and promotes their degradation via BAG3-mediated selective macroautophagy. To adapt cellular homeostasis to stress, BAG3 modulates and functions in various cellular processes and signaling pathways. Noteworthy, dysfunction and deregulation of BAG3 and its pathway are pathophysiologically linked to myopathies, cancer, and neurodegenerative disorders. Here, we report a BAG3 proteomic signature under proteostasis stress. To elucidate the dynamic and multifunctional action of BAG3 in response to stress, we established BAG3 interactomes under basal and proteostasis stress conditions by employing affinity purification combined with quantitative mass spectrometry. In addition to the identification of novel potential BAG3 interactors, we defined proteins whose interaction with BAG3 was altered upon stress. By functional annotation and protein-protein interaction enrichment analysis of the identified potential BAG3 interactors, we confirmed the multifunctionality of BAG3 and highlighted its crucial role in diverse cellular signaling pathways and processes, ensuring cellular proteostasis and cell viability. These include protein folding and degradation, gene expression, cytoskeleton dynamics (including cell cycle and transport), as well as granulostasis, in particular.
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ATP hydrolysis by yeast Hsp104 determines protein aggregate dissolution and size in vivo. Nat Commun 2020; 11:5226. [PMID: 33067463 PMCID: PMC7568574 DOI: 10.1038/s41467-020-19104-1] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Accepted: 09/28/2020] [Indexed: 12/23/2022] Open
Abstract
Signs of proteostasis failure often entwine with those of metabolic stress at the cellular level. Here, we study protein sequestration during glucose deprivation-induced ATP decline in Saccharomyces cerevisiae. Using live-cell imaging, we find that sequestration of misfolded proteins and nascent polypeptides into two distinct compartments, stress granules, and Q-bodies, is triggered by the exhaustion of ATP. Both compartments readily dissolve in a PKA-dependent manner within minutes of glucose reintroduction and ATP level restoration. We identify the ATP hydrolase activity of Hsp104 disaggregase as the critical ATP-consuming process determining compartments abundance and size, even in optimal conditions. Sequestration of proteins into distinct compartments during acute metabolic stress and their retrieval during the recovery phase provide a competitive fitness advantage, likely promoting cell survival during stress. The sequestration of misfolded protein into insoluble aggregates occurs under conditions of proteotoxic stress. Here the authors observe that a reduction in cellular ATP promotes protein sequestration into two separate compartments: Q-bodies and stress granules; and identify Hsp104 as a critical ATP-consuming process that determines those compartments abundance and size.
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25
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Wang X, Wu Z, Li Y, Yang Y, Xiao C, Liu X, Xiang X, Wei J, Shao D, Liu K, Deng X, Wu J, Qiu Y, Li B, Ma Z. p53 promotes ZDHHC1-mediated IFITM3 palmitoylation to inhibit Japanese encephalitis virus replication. PLoS Pathog 2020; 16:e1009035. [PMID: 33108395 PMCID: PMC7647115 DOI: 10.1371/journal.ppat.1009035] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 11/06/2020] [Accepted: 10/06/2020] [Indexed: 01/23/2023] Open
Abstract
The tumor suppressor p53 as an innate antiviral regulator contributes to restricting Japanese encephalitis virus (JEV) replication, but the mechanism is still unclear. The interferon-induced transmembrane protein 3 (IFITM3) is an intrinsic barrier to a range of virus infection, whether IFITM3 is responsible for the p53-mediated anti-JEV response remains elusive. Here, we found that IFITM3 significantly inhibited JEV replication in a protein-palmitoylation-dependent manner and incorporated into JEV virions to diminish the infectivity of progeny viruses. Palmitoylation was also indispensible for keeping IFITM3 from lysosomal degradation to maintain its protein stability. p53 up-regulated IFITM3 expression at the protein level via enhancing IFITM3 palmitoylation. Screening of palmitoyltransferases revealed that zinc finger DHHC domain-containing protein 1 (ZDHHC1) was transcriptionally up-regulated by p53, and consequently ZDHHC1 interacted with IFITM3 to promote its palmitoylation and stability. Knockdown of IFITM3 significantly impaired the inhibitory role of ZDHHC1 on JEV replication. Meanwhile, knockdown of either ZDHHC1 or IFITM3 expression also compromised the p53-mediated anti-JEV effect. Interestingly, JEV reduced p53 expression to impair ZDHHC1 mediated IFITM3 palmitoylation for viral evasion. Our data suggest the existence of a previously unrecognized p53-ZDHHC1-IFITM3 regulatory pathway with an essential role in restricting JEV infection and provide a novel insight into JEV-host interaction.
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Affiliation(s)
- Xin Wang
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
- College of Agriculture and Forestry, Linyi University, Linyi, P.R. China
| | - Zhuanchang Wu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Yuming Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Yifan Yang
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Changguang Xiao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Xiqian Liu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Xiao Xiang
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Jianchao Wei
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Donghua Shao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Ke Liu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Xufang Deng
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Jiaqiang Wu
- Shandong Provincial Animal Disease Control and Breeding, Shandong Academy of Agricultural Sciences, Jinan, P.R. China
| | - Yafeng Qiu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Beibei Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
| | - Zhiyong Ma
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, P.R. China
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The Proteasome Governs Fungal Morphogenesis via Functional Connections with Hsp90 and cAMP-Protein Kinase A Signaling. mBio 2020; 11:mBio.00290-20. [PMID: 32317319 PMCID: PMC7175089 DOI: 10.1128/mbio.00290-20] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Protein homeostasis is critical for proliferation and viability of all organisms. For Candida albicans, protein homeostasis also modulates the transition between yeast and filamentous forms, which is critical for virulence. A key regulator of morphogenesis is the molecular chaperone Hsp90, which mediates proteostasis under physiological and stress conditions. Hsp90 regulates morphogenesis by repressing cyclic AMP-protein kinase A (cAMP-PKA) signaling, such that inhibition of Hsp90 causes filamentation in the absence of an inducing cue. We explored the effect of perturbation of another facet of protein homeostasis and discovered that morphogenesis is also regulated by the proteasome, a large 33-subunit protein complex consisting of a 20S catalytic core and two 19S regulatory particles, which controls degradation of intracellular proteins. We identified a conserved role of the proteasome in morphogenesis as pharmacological inhibition of the proteasome induced filamentation of C. albicans and the related species Candida dubliniensis, Candida tropicalis, Candida krusei, and Candida parapsilosis For C. albicans, genetic depletion of any of 29 subunits of the 19S or 20S particle induced filamentation. Filaments induced by inhibition of either the proteasome or Hsp90 have shared structural characteristics, such as aberrant nuclear content, and shared genetic dependencies, such as intact cAMP-PKA signaling. Consistent with a functional connection between these facets of protein homeostasis that modulate morphogenesis, we observed that proteasome inhibition results in an accumulation of ubiquitinated proteins that overwhelm Hsp90 function, relieving Hsp90-mediated repression of morphogenesis. Together, our findings provide a mechanism whereby interconnected facets of proteostasis regulate C. albicans morphogenesis.IMPORTANCE Fungi cause life-threatening infections and pose a serious threat to human health as there are very few effective antifungal drugs. Candida albicans is a major human fungal pathogen and cause of morbidity and mortality in immunocompromised individuals. A key trait that enables C. albicans virulence is its ability to transition between yeast and filamentous forms. Understanding the mechanisms regulating this virulence trait can facilitate the development of much-needed, novel therapeutic strategies. A key regulator of morphogenesis is the molecular chaperone Hsp90, which is crucial for proteostasis. Here, we expanded our understanding of how proteostasis regulates fungal morphogenesis and identified the proteasome as a repressor of filamentation in C. albicans and related species. Our work suggests that proteasome inhibition overwhelms Hsp90 function, thereby inducing morphogenesis. This work provides a foundation for understanding the role of the proteasome in fungal virulence and offers potential for targeting the proteasome to disarm fungal pathogens.
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Santa Maria SR, Marina DB, Massaro Tieze S, Liddell LC, Bhattacharya S. BioSentinel: Long-Term Saccharomyces cerevisiae Preservation for a Deep Space Biosensor Mission. ASTROBIOLOGY 2020; 23:617-630. [PMID: 31905002 DOI: 10.1089/ast.2019.2073] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2023]
Abstract
The biological risks of the deep space environment must be elucidated to enable a new era of human exploration and scientific discovery beyond low earth orbit (LEO). There is a paucity of deep space biological missions that will inform us of the deleterious biological effects of prolonged exposure to the deep space environment. To safely undertake long-term missions to Mars and space habitation beyond LEO, we must first prove and optimize autonomous biosensors to query the deep space radiation environment. Such biosensors must contain organisms that can survive for extended periods with minimal life support technology and must function reliably with intermittent communication with Earth. NASA's BioSentinel mission, a nanosatellite containing the budding yeast Saccharomyces cerevisiae, is such a biosensor and one of the first biological missions beyond LEO in nearly half a century. It will help fill critical gaps in knowledge about the effects of uniquely composed, chronic, low-flux deep space radiation on biological systems and in particular will provide valuable insight into the DNA damage response to highly ionizing particles. Due to yeast's robustness and desiccation tolerance, it can survive for periods analogous to that of a human Mars mission. In this study, we discuss our optimization of conditions for long-term reagent storage and yeast survival under desiccation in preparation for the BioSentinel mission. We show that long-term yeast cell viability is maximized when cells are air-dried in trehalose solution and stored in a low-relative humidity and low-temperature environment and that dried yeast is sensitive to low doses of deep space-relevant ionizing radiation under these conditions. Our findings will inform the design and development of improved future long-term biological missions into deep space.
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Affiliation(s)
- Sergio R Santa Maria
- COSMIAC Research Center, University of New Mexico, Albuquerque, New Mexico
- Space Biosciences Research, NASA Ames Research Center, Moffett Field, California
| | - Diana B Marina
- Space Biosciences Research, NASA Ames Research Center, Moffett Field, California
- Amyris, Inc., Emeryville, California (present address)
| | - Sofia Massaro Tieze
- Space Biosciences Research, NASA Ames Research Center, Moffett Field, California
- Blue Marble Space Institute of Science, Seattle, Washington
| | - Lauren C Liddell
- Space Biosciences Research, NASA Ames Research Center, Moffett Field, California
- Logyx LLC, Mountain View, California
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28
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Li W, Wu Z, Liang Y. Vrl1 relies on its VPS9-domain to play a role in autophagy in Saccharomyces cerevisiae. Cell Biol Int 2019; 43:875-889. [PMID: 31038239 DOI: 10.1002/cbin.11156] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Accepted: 04/28/2019] [Indexed: 11/10/2022]
Abstract
Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9-domain-containing protein, VARP-like 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9-domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9-domain-containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9-domain-containing proteins, Vps9, Muk1, and Vrl1, all co-localized with Atg8 on autophagosomes in cells blocked in any late step of starvation-induced autophagy, with Vrl1 most often co-localizing with Atg8. A small portion (<25%) of these VPS9-domain-containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy-dependent and -independent routes.
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Affiliation(s)
- Wenjing Li
- Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture and Rural Affairs, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
| | - Zulin Wu
- Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture and Rural Affairs, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
| | - Yongheng Liang
- Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture and Rural Affairs, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
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29
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Liu Z, Dong X, Yi HW, Yang J, Gong Z, Wang Y, Liu K, Zhang WP, Tang C. Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13. Cell Discov 2019; 5:19. [PMID: 30962947 PMCID: PMC6443662 DOI: 10.1038/s41421-019-0089-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Revised: 02/15/2019] [Accepted: 02/19/2019] [Indexed: 01/18/2023] Open
Abstract
The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, while it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have determined the complex structure between Rpn13NTD and K48-diUb. In this structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge-reversal mutation in Rpn13NTD weakens the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, physical blockage of the access of the distal Ub to Rpn13NTD with a proximity-attached Ub monomer can disrupt the interaction between Rpn13 and K48-diUb. Taken together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.
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Affiliation(s)
- Zhu Liu
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China.,2National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070 China
| | - Xu Dong
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China
| | - Hua-Wei Yi
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China.,3University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Ju Yang
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China.,3University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Zhou Gong
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China
| | - Yi Wang
- 4Department of Pharmacology, Institute of Neuroscience, Key Laboratory of Medical Neurobiology of Ministry of Health of China, and Zhejiang Province Key Laboratory of Mental Disorder's Management, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310027 China
| | - Kan Liu
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China
| | - Wei-Ping Zhang
- 4Department of Pharmacology, Institute of Neuroscience, Key Laboratory of Medical Neurobiology of Ministry of Health of China, and Zhejiang Province Key Laboratory of Mental Disorder's Management, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310027 China
| | - Chun Tang
- 1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China.,3University of Chinese Academy of Sciences, Beijing, 100049 China.,5Wuhan National Laboratory for Optoelectronics, Wuhan, Hubei Province 430074 China
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30
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Uddin M, Shibata H. Distribution of calretinin immunopositive somata and fibers in the rabbit midcingulate cortex. J Vet Med Sci 2018; 81:57-65. [PMID: 30473570 PMCID: PMC6361660 DOI: 10.1292/jvms.18-0577] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
The midcingulate cortex (MCC; area 24’) resides in the mid-rostrocaudal part of the cingulate gyrus, and it plays important roles in nociceptive, cognitive and skeletomotor functions. The
MCC has recently been shown to consist of four cortical areas (areas a24a’, a24b’, p24a’ and p24b’) in the rabbit, based on immunohistochemistry. To further characterize the organization of
these areas, here we immunohistochemically identified structures immunopositive (+) for calretinin (CR) as a marker of a subpopulation of inhibitory neurons. CR+ somata were identified as
multipolar and bipolar neurons. The multipolar neurons were predominant throughout the MCC. CR+ somata were present mainly in layer (L) 2/3 and L6, and CR+ fibers occurred mainly in L1, L2/3
and L6. However, there were differences in the distribution of CR+ structures in each area. CR+ somata tended to be most densely distributed in area a24a’, followed by area p24a’, area a24b’
and area p24b’. CR+ fibers were most densely distributed in area p24a’, followed by area p24b’, area a24a’ and area a24b’. In addition, only areas p24a’ and p24b’ enclosed patchy CR+ fibers
and terminals in deep L2/3. These results show the distinct distribution of CR+ structures in each area of the MCC in the rabbit, suggesting that CR+ neurons may contribute to information
processing for cognitive functions in somewhat different manners in each area of the MCC.
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Affiliation(s)
- Mohi Uddin
- Laboratory of Veterinary Anatomy, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.,United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
| | - Hideshi Shibata
- Laboratory of Veterinary Anatomy, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.,United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
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31
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Kim GH, Kim HG, Jeon CJ. Immunocytochemical Localization of Choline Acetyltransferase in the Microbat Visual Cortex. Acta Histochem Cytochem 2018; 51:153-165. [PMID: 30510329 PMCID: PMC6261840 DOI: 10.1267/ahc.18018] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2018] [Accepted: 08/17/2018] [Indexed: 12/23/2022] Open
Abstract
The purpose of the present study was to investigate the organization of choline acetyltransferase (ChAT)-immunoreactive (IR) fibers in the visual cortex of the microbat, using standard immunocytochemistry and confocal microscopy. ChAT-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer III and the lowest density in layer I. However, no ChAT-IR cells were found in the microbat visual cortex. ChAT-IR fibers were classified into two types: small and large varicose fibers. Previously identified sources of cholinergic fibers in the mammalian visual cortex, the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis, all contained strongly labeled ChAT-IR cells in the microbat. The average diameter of ChAT-IR cells in the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis was 16.12 μm, 13.37 μm, and 13.90 μm, respectively. Our double-labeling study with ChAT and gamma-aminobutyric acid (GABA), and triple labeling with ChAT, GABA, and post synaptic density 95 (PSD-95), suggest that some ChAT-IR fibers make contact with GABAergic cells in the microbat visual cortex. Our results should provide a better understanding of the nocturnal bat visual system.
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Affiliation(s)
- Gil-Hyun Kim
- Department of Biology, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, and Brain Science and Engineering Institute, Kyungpook National University
| | - Hang-Gu Kim
- Department of Biology, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, and Brain Science and Engineering Institute, Kyungpook National University
| | - Chang-Jin Jeon
- Department of Biology, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, and Brain Science and Engineering Institute, Kyungpook National University
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32
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Liu L, Levin DE. Intracellular mechanism by which genotoxic stress activates yeast SAPK Mpk1. Mol Biol Cell 2018; 29:2898-2909. [PMID: 30230955 PMCID: PMC6249863 DOI: 10.1091/mbc.e18-07-0441] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Stress-activated MAP kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. The Saccharomyces cerevisiae SAPK Mpk1 (Slt2) is a well-characterized component of the cell-wall integrity (CWI) signaling pathway, which responds to physical and chemical challenges to the cell wall. However, Mpk1 is also activated in response to genotoxic stress through an unknown pathway. We show that, in contrast to cell-wall stress, the pathway for Mpk1 activation by genotoxic stress does not involve the stimulation of the MAP kinase kinases (MEKs) that function immediately upstream of Mpk1. Instead, DNA damage activates Mpk1 through induction of proteasomal degradation of Msg5, the dual-specificity protein phosphatase principally responsible for maintaining Mpk1 in a low-activity state in the absence of stress. Blocking Msg5 degradation in response to genotoxic stress prevented Mpk1 activation. This work raises the possibility that other Mpk1-activating stressors act intracellularly at different points along the canonical Mpk1 activation pathway.
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Affiliation(s)
- Li Liu
- Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118
| | - David E Levin
- Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118.,Department of Microbiology, Boston University School of Medicine, Boston, MA 02118
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33
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Hsp70-Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation. Proc Natl Acad Sci U S A 2018; 115:E7043-E7052. [PMID: 29987014 DOI: 10.1073/pnas.1803130115] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 signaling regulates protein aggregation. We established that the regulated step was the emergence and growth of abnormal protein oligomers containing only a few molecules, indicating that aggregation is regulated at very early stages. The Hsp70-Bag3 complex therefore functions as an important signaling node that senses proteotoxicity and triggers multiple pathways that control cell physiology, including activation of protein aggregation.
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Muñoz-Arellano AJ, Chen X, Molt A, Meza E, Petranovic D. Different Expression Levels of Human Mutant Ubiquitin B +1 (UBB +1) Can Modify Chronological Lifespan or Stress Resistance of Saccharomyces cerevisiae. Front Mol Neurosci 2018; 11:200. [PMID: 29950972 PMCID: PMC6008557 DOI: 10.3389/fnmol.2018.00200] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 05/18/2018] [Indexed: 11/13/2022] Open
Abstract
The ubiquitin-proteasome system (UPS) is the main pathway responsible for the degradation of misfolded proteins, and its dysregulation has been implicated in several neurodegenerative diseases, including Alzheimer's disease (AD). UBB+1, a mutant variant of ubiquitin B, was found to accumulate in neurons of AD patients and it has been linked to UPS dysfunction and neuronal death. Using the yeast Saccharomyces cerevisiae as a model system, we constitutively expressed UBB+1 to evaluate its effects on proteasome function and cell death, particularly under conditions of chronological aging. We showed that the expression of UBB+1 caused inhibition of the three proteasomal proteolytic activities (caspase-like (β1), trypsin-like (β2) and chymotrypsin-like (β5) activities) in yeast. Interestingly, this inhibition did not alter cell viability of growing cells. Moreover, we showed that cells expressing UBB+1 at lower level displayed an increased capacity to degrade induced misfolded proteins. When we evaluated cells during chronological aging, UBB+1 expression at lower level, prevented cells to accumulate reactive oxygen species (ROS) and avert apoptosis, dramatically increasing yeast life span. Since proteasome inhibition by UBB+1 has previously been shown to induce chaperone expression and thus protect against stress, we evaluated our UBB+1 model under heat shock and oxidative stress. Higher expression of UBB+1 caused thermotolerance in yeast due to induction of chaperones, which occurred to a lesser extent at lower expression level of UBB+1 (where we observed the phenotype of extended life span). Altering UPS capacity by differential expression of UBB+1 protects cells against several stresses during chronological aging. This system can be valuable to study the effects of UBB+1 on misfolded proteins involved in neurodegeneration and aging.
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Affiliation(s)
- Ana Joyce Muñoz-Arellano
- Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
| | - Xin Chen
- Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.,Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
| | - Andrea Molt
- Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
| | - Eugenio Meza
- Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
| | - Dina Petranovic
- Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.,Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
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White MC, Schroeder RD, Zhu K, Xiong K, McConkey DJ. HRI-mediated translational repression reduces proteotoxicity and sensitivity to bortezomib in human pancreatic cancer cells. Oncogene 2018; 37:4413-4427. [PMID: 29720726 PMCID: PMC6138554 DOI: 10.1038/s41388-018-0227-y] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Revised: 10/26/2017] [Accepted: 11/03/2017] [Indexed: 12/31/2022]
Abstract
Human cancer cells display extensive heterogeneity in their sensitivities to the proteasome inhibitor bortezomib (Velcade). The molecular mechanisms underlying this heterogeneity remain unclear, and strategies to overcome resistance are limited. Here, we discover that inherent differences in eIF2α phosphorylation among a panel of ten human pancreatic cancer cell lines significantly impacts bortezomib sensitivity, and implicate the HRI (heme-regulated inhibitor) eIF2α kinase as a novel therapeutic target. Within our panel, we identified a subset of cell lines with defective induction of eIF2α phosphorylation, conferring a high degree of sensitivity to bortezomib. These bortezomib-sensitive cells exhibited impaired translation attenuation followed by toxic accumulation of protein aggregates and reactive oxygen species (ROS), whereas the bortezomib-resistant cell lines displayed increased phosphorylation of eIF2α, decreased translation, few protein aggregates, and minimal ROS production. Importantly, we identified HRI as the primary bortezomib-activated eIF2α kinase, and demonstrated that HRI knockdown promoted cell death in the bortezomib-resistant cells. Overall, our data implicate inducible HRI-mediated phosphorylation of eIF2α as a central cytoprotective mechanism following exposure to bortezomib and provide proof-of-concept for the development of HRI inhibitors to overcome proteasome inhibitor resistance.
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Affiliation(s)
- Matthew C White
- Departments of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.,Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.,The Program in Experimental Therapeutics, The University of Texas Graduate School of Biomedical Sciences, Houston, TX, 77030, USA
| | - Rebecca D Schroeder
- The Program in Experimental Therapeutics, The University of Texas Graduate School of Biomedical Sciences, Houston, TX, 77030, USA
| | - Keyi Zhu
- Departments of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.,Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - Katherine Xiong
- Departments of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.,Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - David J McConkey
- Departments of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. .,Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. .,The Program in Experimental Therapeutics, The University of Texas Graduate School of Biomedical Sciences, Houston, TX, 77030, USA. .,Johns Hopkins Greenberg Bladder Cancer Institute, Baltimore, MD, 21287, USA.
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Cui Y, Zhu Y, Lin Y, Chen L, Feng Q, Wang W, Xiang H. New insight into the mechanism underlying the silk gland biological process by knocking out fibroin heavy chain in the silkworm. BMC Genomics 2018; 19:215. [PMID: 29580211 PMCID: PMC5870212 DOI: 10.1186/s12864-018-4602-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2017] [Accepted: 03/13/2018] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Exploring whether and how mutation of silk protein contributes to subsequent re-allocation of nitrogen, and impacts on the timing of silk gland degradation, is important to understand silk gland biology. Rapid development and wide application of genome editing approach in the silkworm provide us an opportunity to address these issues. RESULTS Using CRISPR/Cas9 system, we successfully performed genome editing of Bmfib-H. The loss-of-function mutations caused naked pupa and thin cocoon mutant phenotypes. Compared with the wild type, the posterior silk gland of mutant showed obviously degraded into fragments in advance of programmed cell death of silk gland cells. Comparative transcriptomic analyses of silk gland at the fourth day of the fifth instar larval stage(L5D4)identified 1456 differential expressed genes (DEGs) between posterior silk gland (PSG) and mid silk gland (MSG) and 1388 DEGs between the mutant and the wild type. Hierarchical clustering of all the DEGs indicated a remarkable down-regulated and an up-regulated gene clade in the mutant silk glands, respectively. Down-regulated genes were overrepresented in the pathways involved in cancer, DNA replication and cell proliferation. Intriguingly, up-regulated DEGs are significantly enriched in the proteasome. By further comparison on the transcriptome of MSG and PSG between the wild type and the mutant, we consistently observed that up-regulated DEGs in the mutant PSG were enriched in protein degrading activity and proteasome. Meantime, we observed a series of up-regulated genes involved in autophagy. Since these protein degradation processes would be normally occur after the spinning time, the results suggesting that these progresses were activated remarkably ahead of schedule in the mutant. CONCLUSIONS Accumulation of abnormal fib-H protein might arouse the activation of proteasomes as well as autophagy process, to promote the rapid degradation of such abnormal proteins and the silk gland cells. Our study therefore proposes a subsequent process of protein and partial cellular degradation caused by mutation of silk protein, which might be helpful for understanding its impact of the silk gland biological process, and further exploration the re-allocation of nitrogen in the silkworm.
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Affiliation(s)
- Yong Cui
- Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Yanan Zhu
- Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Yongjian Lin
- Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Lei Chen
- Center for Ecological and Environmental Sciences, Northwestern Polytechnical University, Xi'an, 710129, China
| | - Qili Feng
- Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Wen Wang
- Center for Ecological and Environmental Sciences, Northwestern Polytechnical University, Xi'an, 710129, China.
| | - Hui Xiang
- Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China.
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Kattaia AAAA, Abd El-Baset SA, Mohamed EM. Heat Shock Proteins in Oxidative and Nitrosative Stress. HEAT SHOCK PROTEINS AND STRESS 2018. [DOI: 10.1007/978-3-319-90725-3_7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Norman KL, Chen TC, Zeiner G, Sarnow P. Precursor microRNA-122 inhibits synthesis of Insig1 isoform mRNA by modulating polyadenylation site usage. RNA (NEW YORK, N.Y.) 2017; 23:1886-1893. [PMID: 28928276 PMCID: PMC5689008 DOI: 10.1261/rna.063099.117] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Accepted: 09/09/2017] [Indexed: 06/07/2023]
Abstract
The insulin-induced gene 1 protein (Insig1) inhibits the cholesterol biosynthesis pathway by retaining transcription factor SREBP in the endoplasmic reticulum, and by causing the degradation of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. Liver-specific microRNA miR-122, on the other hand, enhances cholesterol biosynthesis by an unknown mechanism. We have found that Insig1 mRNAs are generated by alternative cleavage and polyadenylation, resulting in specific isoform mRNA species. During high cholesterol abundance, the short 1.4-kb Insig1 mRNA was found to be preferentially translated to yield Insig1 protein. Precursor molecules of miR-122 down-regulated the translation of the 1.4-kb Insig1 isoform mRNA by interfering with the usage of the promoter-proximal cleavage-polyadenylation site that gives rise to the 1.4-kb Insig1 mRNA. These findings argue that precursor miR-122 molecules modulate polyadenylation site usage in Insig1 mRNAs, resulting in down-regulation of Insig1 protein abundance. Thus, precursor microRNAs may have hitherto undetected novel functions in nuclear gene expression.
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Affiliation(s)
- Kara L Norman
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Tzu-Chun Chen
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Gusti Zeiner
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Peter Sarnow
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA
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Gotcha N, Terblanche JS, Nyamukondiwa C. Plasticity and cross-tolerance to heterogeneous environments: divergent stress responses co-evolved in an African fruit fly. J Evol Biol 2017; 31:98-110. [PMID: 29080375 DOI: 10.1111/jeb.13201] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2017] [Revised: 10/10/2017] [Accepted: 10/17/2017] [Indexed: 01/16/2023]
Abstract
Plastic adjustments of physiological tolerance to a particular stressor can result in fitness benefits for resistance that might manifest not only in that same environment but also be advantageous when faced with alternative environmental stressors, a phenomenon termed 'cross-tolerance'. The nature and magnitude of cross-tolerance responses can provide important insights into the underlying genetic architecture, potential constraints on or versatility of an organism's stress responses. In this study, we tested for cross-tolerance to a suite of abiotic factors that likely contribute to setting insect population dynamics and geographic range limits: heat, cold, desiccation and starvation resistance in adult Ceratitis rosa following acclimation to all these isolated individual conditions prior to stress assays. Traits of stress resistance scored included critical thermal (activity) limits, chill coma recovery time (CCRT), heat knockdown time (HKDT), desiccation and starvation resistance. In agreement with other studies, we found that acclimation to one stress typically increased resistance for that same stress experienced later in life. A more novel outcome, however, is that here we also found substantial evidence for cross-tolerance. For example, we found an improvement in heat tolerance (critical thermal maxima, CTmax ) following starvation or desiccation hardening and improved desiccation resistance following cold acclimation, indicating pronounced cross-tolerance to these environmental stressors for the traits examined. We also found that two different traits of the same stress resistance differed in their responsiveness to the same stress conditions (e.g. HKDT was less cross-resistant than CTmax ). The results of this study have two major implications that are of broader importance: (i) that these traits likely co-evolved to cope with diverse or simultaneous stressors, and (ii) that a set of common underlying physiological mechanisms might exist between apparently divergent stress responses in this species. This species may prove to be a valuable model for future work on the evolutionary and mechanistic basis of cross-tolerance.
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Affiliation(s)
- N Gotcha
- Department of Biological Sciences and Biotechnology Sciences, Botswana International University of Science and Technology (BIUST), Palapye, Botswana
| | - J S Terblanche
- Department of Conservation Ecology and Entomology, Centre for Invasion Biology, Stellenbosch University, Stellenbosch, South Africa
| | - C Nyamukondiwa
- Department of Biological Sciences and Biotechnology Sciences, Botswana International University of Science and Technology (BIUST), Palapye, Botswana
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The SAT Protein of Porcine Parvovirus Accelerates Viral Spreading through Induction of Irreversible Endoplasmic Reticulum Stress. J Virol 2017; 91:JVI.00627-17. [PMID: 28566374 DOI: 10.1128/jvi.00627-17] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2017] [Accepted: 05/14/2017] [Indexed: 02/06/2023] Open
Abstract
The SAT protein (SATp) of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER), and SAT deletion induces the slow-spreading phenotype. The in vitro comparison of the wild-type Kresse strain and its SAT knockout (SAT-) mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SAT- virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences in the quantity and the localization of CHOP were detected, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of the induction of irreversible ER stress in porcine testis (PT) cell necrosis and viral egress was confirmed by treatment of infected cells by ER stress-inducing chemicals (MG132, dithiothreitol, and thapsigargin), which accelerated the egress and spreading of both the wild-type and the SAT- viruses. UV stress induction had no beneficial effect on PPV infection, underscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a coexpression experiment.IMPORTANCE SATp is encoded by an alternative open reading frame of the PPV genome. Earlier we showed that SATp of the attenuated PPV NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that slow spreading is a general feature of SAT- PPVs and is the consequence of prolonged cell integrity. PPV infection induced ER stress in infected cells regardless of the presence of SATp, as demonstrated by the morphological changes of the ER and expression of the stress response proteins Xbp1 and CHOP. However, the presence of SATp made the ER stress more severe and accelerated cell death during infection, as shown by the higher rate of expression of CHOP and alteration of the localization of CHOP. The beneficial effect of irreversible ER stress on PPV spread was confirmed by treatment of infected cells with ER stress-inducing chemicals.
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Sap KA, Bezstarosti K, Dekkers DHW, Voets O, Demmers JAA. Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in Drosophila. J Proteome Res 2017; 16:2848-2862. [DOI: 10.1021/acs.jproteome.7b00156] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
- Karen A. Sap
- Proteomics
Center, ‡Netherlands Proteomics Center, and §Department of Biochemistry, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands
| | - Karel Bezstarosti
- Proteomics
Center, ‡Netherlands Proteomics Center, and §Department of Biochemistry, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands
| | - Dick H. W. Dekkers
- Proteomics
Center, ‡Netherlands Proteomics Center, and §Department of Biochemistry, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands
| | - Olaf Voets
- Proteomics
Center, ‡Netherlands Proteomics Center, and §Department of Biochemistry, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands
| | - Jeroen A. A. Demmers
- Proteomics
Center, ‡Netherlands Proteomics Center, and §Department of Biochemistry, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands
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Wang XD, Jiang T, Yu XW, Xu Y. Effects of UPR and ERAD pathway on the prolyl endopeptidase production in Pichia pastoris by controlling of nitrogen source. ACTA ACUST UNITED AC 2017; 44:1053-1063. [DOI: 10.1007/s10295-017-1938-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 02/22/2017] [Indexed: 01/12/2023]
Abstract
Abstract
Prolyl endopeptidase (PEP) is very useful in various industries, while the high cost of enzyme production remains a major obstacle for its industrial applications. Pichia pastoris has been used for the PEP production; however, the fermentation process has not be investigated and little is known about the impact of excessive PEP production on the host cell physiology. Here, we optimized the nitrogen source to improve the PEP expression level and further evaluated the cellular response including UPR and ERAD. During methanol induction phase the PEP activity (1583 U/L) was increased by 1.48-fold under the optimized nitrogen concentration of NH4+ (300 mmol/L) and casamino acids [1.0% (w/v)] in a 3-L bioreactor. Evaluated by RT-PCR the UPR and ERAD pathways were confirmed to be activated. Furthermore, a strong decrease of ERAD-related gene transcription was observed with the addition of nitrogen source, which contributed to a higher PEP expression level.
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Affiliation(s)
- Xiao-Dong Wang
- 0000 0001 0708 1323 grid.258151.a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University 214122 Wuxi People’s Republic of China
- 0000 0001 0708 1323 grid.258151.a State Key Laboratory of Food Science and Technology Jiangnan University 214122 Wuxi People’s Republic of China
| | - Ting Jiang
- 0000 0001 0708 1323 grid.258151.a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University 214122 Wuxi People’s Republic of China
- 0000 0001 0708 1323 grid.258151.a State Key Laboratory of Food Science and Technology Jiangnan University 214122 Wuxi People’s Republic of China
| | - Xiao-Wei Yu
- 0000 0001 0708 1323 grid.258151.a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University 214122 Wuxi People’s Republic of China
- 0000 0001 0708 1323 grid.258151.a State Key Laboratory of Food Science and Technology Jiangnan University 214122 Wuxi People’s Republic of China
| | - Yan Xu
- 0000 0001 0708 1323 grid.258151.a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University 214122 Wuxi People’s Republic of China
- 0000 0001 0708 1323 grid.258151.a State Key Laboratory of Food Science and Technology Jiangnan University 214122 Wuxi People’s Republic of China
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Toh-E A, Ohkusu M, Shimizu K, Yamaguchi M, Ishiwada N, Watanabe A, Kamei K. Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin. Curr Genet 2017; 63:1093-1104. [PMID: 28560585 DOI: 10.1007/s00294-017-0713-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2017] [Revised: 05/18/2017] [Accepted: 05/24/2017] [Indexed: 11/27/2022]
Abstract
We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.
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Affiliation(s)
- Akio Toh-E
- Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan.
| | - Misako Ohkusu
- Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan
| | - Kiminori Shimizu
- Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan
| | - Masashi Yamaguchi
- Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan
| | - Naruhiko Ishiwada
- Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan
| | - Akira Watanabe
- Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan
| | - Katsuhiko Kamei
- Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan
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Kalra B, Tamang AM, Parkash R. Cross-tolerance effects due to adult heat hardening, desiccation and starvation acclimation of tropical drosophilid-Zaprionus indianus. Comp Biochem Physiol A Mol Integr Physiol 2017; 209:65-73. [PMID: 28454925 DOI: 10.1016/j.cbpa.2017.04.014] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2017] [Revised: 04/18/2017] [Accepted: 04/19/2017] [Indexed: 01/02/2023]
Abstract
Some insect taxa from polar or temperate habitats have shown cross-tolerance for multiple stressors but tropical insect taxa have received less attention. Accordingly, we considered adult flies of a tropical drosophilid-Zaprionus indianus for testing direct as well as cross-tolerance effects of rapid heat hardening (HH), desiccation acclimation (DA) and starvation acclimation (SA) after rearing under warmer and drier season specific simulated conditions. We observed significant direct acclimation effects of HH, DA and SA; and four cases of cross-tolerance effects but no cross-tolerance between desiccation and starvation. Cross-tolerance due to heat hardening on desiccation showed 20% higher effect than its reciprocal effect. There is greater reduction of water loss in heat hardened flies (due to increase in amount of cuticular lipids) as compared with desiccation acclimated flies. However, cross-tolerance effect of SA on heat knockdown was two times higher than its reciprocal. Heat hardened and desiccation acclimated adult flies showed substantial increase in the level of trehalose and proline while body lipids increased due to heat hardening or starvation acclimation. However, maximum increase in energy metabolites was stressor specific i.e. trehalose due to DA; proline due to HH and total body lipids due to SA. Rapid changes in energy metabolites due to heat hardening seem compensatory for possible depletion of trehalose and proline due to desiccation stress; and body lipids due to starvation stress. Thus, observed cross-tolerance effects in Z. indianus represent physiological changes to cope with multiple stressors related to warmer and drier subtropical habitats.
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Affiliation(s)
- Bhawna Kalra
- Department of Genetics, M. D. University, Rohtak 124001, India
| | | | - Ravi Parkash
- Department of Genetics, M. D. University, Rohtak 124001, India.
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Orlandi I, Stamerra G, Strippoli M, Vai M. During yeast chronological aging resveratrol supplementation results in a short-lived phenotype Sir2-dependent. Redox Biol 2017; 12:745-754. [PMID: 28412652 PMCID: PMC5397018 DOI: 10.1016/j.redox.2017.04.015] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Revised: 04/05/2017] [Accepted: 04/08/2017] [Indexed: 01/13/2023] Open
Abstract
Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biological properties/functions amongst which are its activity as an antioxidant and as Sirtuin activating compound towards SIRT1 in mammals. Sirtuins comprise a family of NAD+-dependent protein deacetylases that are involved in many physiological and pathological processes including aging and age-related diseases. These enzymes are conserved across species and SIRT1 is the closest mammalian orthologue of Sir2 of Saccharomyces cerevisiae. In the field of aging researches, it is well known that Sir2 is a positive regulator of replicative lifespan and, in this context, the RSV effects have been already examined. Here, we analyzed RSV effects during chronological aging, in which Sir2 acts as a negative regulator of chronological lifespan (CLS). Chronological aging refers to quiescent cells in stationary phase; these cells display a survival-based metabolism characterized by an increase in oxidative stress. We found that RSV supplementation at the onset of chronological aging, namely at the diauxic shift, increases oxidative stress and significantly reduces CLS. CLS reduction is dependent on Sir2 presence both in expired medium and in extreme Calorie Restriction. In addition, all data point to an enhancement of Sir2 activity, in particular Sir2-mediated deacetylation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pck1). This leads to a reduction in the amount of the acetylated active form of Pck1, whose enzymatic activity is essential for gluconeogenesis and CLS extension.
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Affiliation(s)
- Ivan Orlandi
- SYSBIO Centre for Systems Biology Milano, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
| | - Giulia Stamerra
- SYSBIO Centre for Systems Biology Milano, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
| | - Maurizio Strippoli
- SYSBIO Centre for Systems Biology Milano, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
| | - Marina Vai
- SYSBIO Centre for Systems Biology Milano, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
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Sullivan B, Robison G, Osborn J, Kay M, Thompson P, Davis K, Zakharova T, Antipova O, Pushkar Y. On the nature of the Cu-rich aggregates in brain astrocytes. Redox Biol 2017; 11:231-239. [PMID: 28012438 PMCID: PMC5198742 DOI: 10.1016/j.redox.2016.12.007] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2016] [Revised: 11/04/2016] [Accepted: 12/02/2016] [Indexed: 01/15/2023] Open
Abstract
Fulfilling a bevy of biological roles, copper is an essential metal for healthy brain function. Cu dyshomeostasis has been demonstrated to be involved in some neurological conditions including Menkes and Alzheimer's diseases. We have previously reported localized Cu-rich aggregates in astrocytes of the subventricular zone (SVZ) in rodent brains with Cu concentrations in the hundreds of millimolar. Metallothionein, a cysteine-rich protein critical to metal homeostasis and known to participate in a variety of neuroprotective and neuroregenerative processes, was proposed as a binding protein. Here, we present an analysis of metallothionein(1,2) knockout (MTKO) mice and age-matched controls using X-ray fluorescence microscopy. In large structures such as the corpus callosum, cortex, and striatum, there is no significant difference in Cu, Fe, or Zn concentrations in MTKO mice compared to age-matched controls. In the astrocyte-rich subventricular zone where Cu-rich aggregates reside, approximately 1/3 as many Cu-rich aggregates persist in MTKO mice resulting in a decrease in periventricular Cu concentration. Aggregates in both wild-type and MTKO mice show XANES spectra characteristic of CuxSy multimetallic clusters and have similar [S]/[Cu] ratios. Consistent with assignment as a CuxSy multimetallic cluster, the astrocyte-rich SVZ of both MTKO and wild-type mice exhibit autofluorescent bodies, though MTKO mice exhibit fewer. Furthermore, XRF imaging of Au-labeled lysosomes and ubiquitin demonstrates a lack of co-localization with Cu-rich aggregates suggesting they are not involved in a degradation pathway. Overall, these data suggest that Cu in aggregates is bound by either metallothionein-3 or a yet unknown protein similar to metallothionein.
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Affiliation(s)
- Brendan Sullivan
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Gregory Robison
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Jenna Osborn
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Martin Kay
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Peter Thompson
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Katherine Davis
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Taisiya Zakharova
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States
| | - Olga Antipova
- BioCAT, Advanced Photon Source, Argonne National Laboratory, 9700 S. Cass Ave., Argonne, IL 60439, United States; XSD, Argonne National Laboratory, 9700 S. Cass Ave., Argonne, IL 60439, United States
| | - Yulia Pushkar
- Department of Physics and Astronomy, Purdue University, 525 Northwestern Ave., West Lafayette, IN 47907, United States.
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Kim JE, Hyun HW, Min SJ, Kang TC. Sustained HSP25 Expression Induces Clasmatodendrosis via ER Stress in the Rat Hippocampus. Front Cell Neurosci 2017; 11:47. [PMID: 28275338 PMCID: PMC5319974 DOI: 10.3389/fncel.2017.00047] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2016] [Accepted: 02/13/2017] [Indexed: 12/16/2022] Open
Abstract
Heat shock protein (HSP) 25 (murine/rodent 25 kDa, human 27 kDa) is one of the major astroglial HSP families, which has a potent anti-apoptotic factor contributing to a higher resistance of astrocytes to the stressful condition. However, impaired removals of HSP25 decrease astroglial viability. In the present study, we investigated whether HSP25 is involved in astroglial apoptosis or clasmatodendrosis (autophagic astroglial death) in the rat hippocampus induced by status epilepticus (SE). Following SE, HSP25 expression was transiently increased in astrocytes within the dentate gyrus (DG), while it was sustained in CA1 astrocytes until 4 weeks after SE. HSP25 knockdown exacerbated SE-induced apoptotic astroglial degeneration, but mitigated clasmatodendrosis accompanied by abrogation of endoplasmic reticulum (ER) stress without changed seizure susceptibility or severity. These findings suggest that sustained HSP25 induction itself may result in clasmatodendrosis via prolonged ER stress. To the best of our knowledge, the present study demonstrates for the first time the double-edge properties of HSP25 in astroglial death induced by SE.
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Affiliation(s)
- Ji-Eun Kim
- Department of Anatomy and Neurobiology, Institute of Epilepsy Research, College of Medicine, Hallym University Chuncheon, South Korea
| | - Hye-Won Hyun
- Department of Anatomy and Neurobiology, Institute of Epilepsy Research, College of Medicine, Hallym University Chuncheon, South Korea
| | - Su-Ji Min
- Department of Anatomy and Neurobiology, Institute of Epilepsy Research, College of Medicine, Hallym University Chuncheon, South Korea
| | - Tae-Cheon Kang
- Department of Anatomy and Neurobiology, Institute of Epilepsy Research, College of Medicine, Hallym University Chuncheon, South Korea
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Nishimoto T, Watanabe T, Furuta M, Kataoka M, Kishida M. Roles of Catalase and Trehalose in the Protection from Hydrogen Peroxide Toxicity in Saccharomyces cerevisiae. Biocontrol Sci 2017; 21:179-82. [PMID: 27667523 DOI: 10.4265/bio.21.179] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022]
Abstract
The roles of catalase and trehalose in Saccharomyces cerevisiae subject to hydrogen peroxide (H2O2) treatment were examined by measuring the catalase activity and intracellular trehalose levels in mutants lacking catalase or trehalose synthetase. Intracellular trehalose was elevated but the survival rate after H2O2 treatment remained low in mutants with deletion of the Catalase T gene. On the other hand, deletion of the trehalose synthetase gene increased the catalase activity in mutated yeast to levels higher than those in the wild-type strain, and these mutants exhibited some degree of tolerance to H2O2 treatment. These results suggest that Catalase T is critical in the yeast response to oxidative damage caused by H2O2 treatment, but trehalose also plays a role in protection against H2O2 treatment.
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Affiliation(s)
- Takuto Nishimoto
- Division of Applied Life Science, Graduate School of Life and Environmental Sciences
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Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells. Biochem Biophys Res Commun 2016; 479:690-696. [DOI: 10.1016/j.bbrc.2016.09.151] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2016] [Accepted: 09/28/2016] [Indexed: 01/01/2023]
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50
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Wike CL, Graves HK, Wason A, Hawkins R, Gopalakrishnan J, Schumacher J, Tyler JK. Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans. Cell Cycle 2016; 15:2216-2225. [PMID: 27248858 PMCID: PMC4993543 DOI: 10.1080/15384101.2016.1192728] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Revised: 04/20/2016] [Accepted: 05/17/2016] [Indexed: 02/04/2023] Open
Abstract
The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.
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Affiliation(s)
- Candice L. Wike
- Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Hillary K. Graves
- Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Arpit Wason
- Institute for Biochemistry and Center for Molecular Medicine, University of Cologne, Cologne, Germany
| | - Reva Hawkins
- Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jay Gopalakrishnan
- Institute for Biochemistry and Center for Molecular Medicine, University of Cologne, Cologne, Germany
| | - Jill Schumacher
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jessica K. Tyler
- Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
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