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Nakagawa T, Hata K, Izumi Y, Nakashima H, Katada S, Matsuda T, Bamba T, Nakashima K. E3 ubiquitin ligase RMND5A maintains the self-renewal state of human neural stem/precursor cells by regulating Wnt and mTOR signaling pathways. FEBS Lett 2025. [PMID: 40377017 DOI: 10.1002/1873-3468.70067] [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: 01/22/2025] [Revised: 04/11/2025] [Accepted: 04/18/2025] [Indexed: 05/18/2025]
Abstract
During cortical development, neural stem/precursor cells (NS/PCs) sequentially produce neurons, astrocytes, and oligodendrocytes. Before producing these cells, human (h) NS/PCs undergo prolonged self-renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. Here, we performed a gene knockout screen using the CRISPR/Cas9 system to search for genes involved in hNS/PC self-renewal. We identified RMND5A, encoding an E3 ubiquitin ligase, among the candidate genes. We further demonstrated that knockdown of RMND5A decreased proliferation and promoted neuronal differentiation of hNS/PCs through the activation and suppression of the Wnt and mTOR signaling pathways, respectively. Taken together, our findings suggest that RMND5A participates in the maintenance of hNS/PC self-renewal by modulating the Wnt and mTOR signaling pathways. Impact statement During cortical development, human neural stem/precursor cells (hNS/PCs) undergo prolonged self-renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. We identified RMND5A, an E3 ubiquitin ligase, as essential for maintaining self-renewal of hNS/PCs, providing valuable insights into the evolutionary expansion of the human brain.
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Affiliation(s)
- Takumi Nakagawa
- Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Kosuke Hata
- Division of Metabolomics, Medical Research Center for High Depth Omics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Yoshihiro Izumi
- Division of Metabolomics, Medical Research Center for High Depth Omics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Hideyuki Nakashima
- Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Sayako Katada
- Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Taito Matsuda
- Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
- Laboratory of Neural Regeneration and Brain Repair, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology (NAIST), Ikoma, Japan
- Life Science Collaboration Center (LiSCo), Nara Institute of Science and Technology (NAIST), Ikoma, Japan
| | - Takeshi Bamba
- Division of Metabolomics, Medical Research Center for High Depth Omics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Kinichi Nakashima
- Stem Cell Biology and Medicine, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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2
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Chen D, Liang X, Zhang L, Zhang J, Gao L, Yan D, Zuo K, Guo H, Du S, Liu J. E3 Ubiquitin Ligase FBXO32 Promotes LPS-Induced Cardiac Injury by Regulating ANXA1/PI3K/AKT Signaling. Inflammation 2025:10.1007/s10753-025-02273-w. [PMID: 40126756 DOI: 10.1007/s10753-025-02273-w] [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: 08/07/2024] [Revised: 02/10/2025] [Accepted: 02/14/2025] [Indexed: 03/26/2025]
Abstract
Sepsis-induced cardiomyopathy (SIC) is a severe complication of sepsis. Therefore, understanding SIC pathogenesis and developing new therapeutic targets are of great significance. This study investigated the role of F-box-only protein 32 (FBXO32) in SIC pathogenesis. LPS-induced cardiac injury models were established in rats and H9c2 cells using lipopolysaccharide. The effects of FBXO32 on myocardial apoptosis and mitochondrial structure and function were determined using electron microscopy, reactive oxygen species detection, and JC-1 staining. The molecular mechanism was elucidated using western blotting and co-immunoprecipitation. The results showed elevated FBXO32 expression in both in vivo and in vitro LPS-induced cardiac injury models. Fbxo32 knockdown alleviated apoptosis and mitochondrial and cardiac dysfunction. Mechanistic analysis revealed that FBXO32 promoted ubiquitination and degradation of annexin A1 (ANXA1), inhibiting the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) pathways. Rescue experiments demonstrated that Anxa1 knockdown reversed the effects of Fbxo32 knockdown. This study suggests that FBXO32 exacerbates LPS-induced cardiac injury progression by mediating ANXA1 ubiquitination and inhibiting the PI3K/AKT signaling pathway.
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Affiliation(s)
- De Chen
- The First Clinical Medical College of Lanzhou University, Lanzhou, Gansu Province, China
| | - Xuan Liang
- Department of Allergy, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China
| | - Lei Zhang
- Department of Intensive Care Unit, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China
| | - Jingjing Zhang
- Medical Genetics Center, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China
| | - Lina Gao
- The First Clinical Medical College of Lanzhou University, Lanzhou, Gansu Province, China
| | - Dong Yan
- The First Clinical Medical College of Lanzhou University, Lanzhou, Gansu Province, China
| | - Kun Zuo
- Department of Intensive Care Unit, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China
| | - Hong Guo
- Department of Intensive Care Unit, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China
| | - Song Du
- Department of Intensive Care Unit, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China
| | - Jian Liu
- The First Clinical Medical College of Lanzhou University, Lanzhou, Gansu Province, China.
- Department of Intensive Care Unit, Gansu Provincial Maternity and Child-Care Hospital/Gansu Provincial Central Hospital, Lanzhou, Gansu Province, China.
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3
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Kumar P, Kinger S, Dubey AR, Jagtap YA, Choudhary A, Karmakar S, Lal G, Kumar A, Bhattacharyya S, Poluri KM, Mishra A. Ketorolac disturbs proteasome functions and induces mitochondrial abnormality-associated apoptosis. IUBMB Life 2025; 77:e2937. [PMID: 39723629 DOI: 10.1002/iub.2937] [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: 11/04/2024] [Accepted: 12/11/2024] [Indexed: 12/28/2024]
Abstract
Non-steroidal anti-inflammatory drugs (NSAIDs) are recommended to treat moderate-to-severe pain. Previous studies suggest that NSAIDs can suppress cellular proliferation and elevate apoptosis in different cancer cells. Ketorolac is an NSAID and can reduce the cancer cells' viability. However, molecular mechanisms by which Ketorolac can induce apoptosis and be helpful as an anti-tumor agent against carcinogenesis are unclear. Here, we observed treatment with Ketorolac disturbs proteasome functions, which induces aggregation of aberrant ubiquitinated proteins. Ketorolac exposure also induced the aggregation of expanded polyglutamine proteins, results cellular proteostasis disturbance. We found that the treatment of Ketorolac aggravates the accumulation of various cell cycle-linked proteins, which results in pro-apoptotic induction in cells. Ketorolac-mediated proteasome disturbance leads to mitochondrial abnormalities. Finally, we have observed that Ketorolac treatment depolarized mitochondrial membrane potential, released cytochrome c into cytoplasm, and induced apoptosis in cells, which could be due to proteasome functional depletion. Perhaps more in-depth research is required to understand the details of NSAID-based anti-proliferative molecular mechanisms that can elevate apoptosis in cancer cells and generate anti-tumor potential with the combination of putative cancer drugs.
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Affiliation(s)
- Prashant Kumar
- Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
| | - Sumit Kinger
- Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
| | - Ankur Rakesh Dubey
- Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
| | - Yuvraj Anandrao Jagtap
- Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
| | - Akash Choudhary
- Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
| | - Surojit Karmakar
- National Centre for Cell Science (NCCS), Pune, Maharashtra, India
| | - Girdhari Lal
- National Centre for Cell Science (NCCS), Pune, Maharashtra, India
| | - Amit Kumar
- Department of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, Madhya Pradesh, India
| | - Sudipta Bhattacharyya
- Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
| | - Krishna Mohan Poluri
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
| | - Amit Mishra
- Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India
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4
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Liao Y, Zhang W, Zhou M, Zhu C, Zou Z. Ubiquitination in pyroptosis pathway: A potential therapeutic target for sepsis. Cytokine Growth Factor Rev 2024; 80:72-86. [PMID: 39294049 DOI: 10.1016/j.cytogfr.2024.09.001] [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: 08/28/2024] [Accepted: 09/03/2024] [Indexed: 09/20/2024]
Abstract
Sepsis remains a significant clinical challenge, causing numerous deaths annually and representing a major global health burden. Pyroptosis, a unique form of programmed cell death characterized by cell lysis and the release of inflammatory mediators, is a crucial factor in the pathogenesis and progression of sepsis, septic shock, and organ dysfunction. Ubiquitination, a key post-translational modification influencing protein fate, has emerged as a promising target for managing various inflammatory conditions, including sepsis. This review integrates the current knowledge on sepsis, pyroptosis, and the ubiquitin system, focusing on the molecular mechanisms of ubiquitination within pyroptotic pathways activated during sepsis. By exploring how modulating ubiquitination can regulate pyroptosis and its associated inflammatory signaling pathways, this review provides insights into potential therapeutic strategies for sepsis, highlighting the need for further research into these complex molecular networks.
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Affiliation(s)
- Yan Liao
- School of Anesthesiology, Naval Medical University, Shanghai 200433, China
| | - Wangzheqi Zhang
- School of Anesthesiology, Naval Medical University, Shanghai 200433, China
| | - Miao Zhou
- Department of Anesthesiology, the Affiliated Cancer Hospital of Nanjing Medical University, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University, Nanjing, Jiangsu 210009, China
| | - Chenglong Zhu
- School of Anesthesiology, Naval Medical University, Shanghai 200433, China.
| | - Zui Zou
- School of Anesthesiology, Naval Medical University, Shanghai 200433, China.
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5
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Tyrna P, Procyk G, Szeleszczuk Ł, Młynarczuk-Biały I. Different Strategies to Overcome Resistance to Proteasome Inhibitors-A Summary 20 Years after Their Introduction. Int J Mol Sci 2024; 25:8949. [PMID: 39201634 PMCID: PMC11354503 DOI: 10.3390/ijms25168949] [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: 07/02/2024] [Revised: 08/09/2024] [Accepted: 08/14/2024] [Indexed: 09/02/2024] Open
Abstract
Proteasome inhibitors (PIs), bortezomib, carfilzomib, and ixazomib, are the first-line treatment for multiple myeloma (MM). They inhibit cytosolic protein degradation in cells, which leads to the accumulation of misfolded and malfunctioned proteins in the cytosol and endoplasmic reticulum, resulting in cell death. Despite being a breakthrough in MM therapy, malignant cells develop resistance to PIs via different mechanisms. Understanding these mechanisms drives research toward new anticancer agents to overcome PI resistance. In this review, we summarize the mechanism of action of PIs and how MM cells adapt to these drugs to develop resistance. Finally, we explore these mechanisms to present strategies to interfere with PI resistance. The strategies include new inhibitors of the ubiquitin-proteasome system, drug efflux inhibitors, autophagy disruption, targeting stress response mechanisms, affecting survival and cell cycle regulators, bone marrow microenvironment modulation, and immunotherapy. We list potential pharmacological targets examined in in vitro, in vivo, and clinical studies. Some of these strategies have already provided clinicians with new anti-MM medications, such as panobinostat and selinexor. We hope that further exploration of the subject will broaden the range of therapeutic options and improve patient outcomes.
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Affiliation(s)
- Paweł Tyrna
- Histology and Embryology Students’ Science Association, Department of Histology and Embryology, Faculty of Medicine, Warsaw Medical University, Chalubinskiego 5, 02-004 Warsaw, Poland;
| | - Grzegorz Procyk
- 1st Chair and Department of Cardiology, Medical University of Warsaw, Banacha 1A, 02-097 Warsaw, Poland;
| | - Łukasz Szeleszczuk
- Department of Organic and Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1 Str., 02-093 Warsaw, Poland;
| | - Izabela Młynarczuk-Biały
- Department of Histology and Embryology, Faculty of Medicine, Warsaw Medical University, Chalubinskiego 5, 02-004 Warsaw, Poland
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6
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Zhou X, Xu R, Wu Y, Zhou L, Xiang T. The role of proteasomes in tumorigenesis. Genes Dis 2024; 11:101070. [PMID: 38523673 PMCID: PMC10958230 DOI: 10.1016/j.gendis.2023.06.037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Revised: 05/10/2023] [Accepted: 06/27/2023] [Indexed: 03/26/2024] Open
Abstract
Protein homeostasis is the basis of normal life activities, and the proteasome family plays an extremely important function in this process. The proteasome 20S is a concentric circle structure with two α rings and two β rings overlapped. The proteasome 20S can perform both ATP-dependent and non-ATP-dependent ubiquitination proteasome degradation by binding to various subunits (such as 19S, 11S, and 200 PA), which is performed by its active subunit β1, β2, and β5. The proteasome can degrade misfolded, excess proteins to maintain homeostasis. At the same time, it can be utilized by tumors to degrade over-proliferate and unwanted proteins to support their growth. Proteasomes can affect the development of tumors from several aspects including tumor signaling pathways such as NF-κB and p53, cell cycle, immune regulation, and drug resistance. Proteasome-encoding genes have been found to be overexpressed in a variety of tumors, providing a potential novel target for cancer therapy. In addition, proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib have been put into clinical application as the first-line treatment of multiple myeloma. More and more studies have shown that it also has different therapeutic effects in other tumors such as hepatocellular carcinoma, non-small cell lung cancer, glioblastoma, and neuroblastoma. However, proteasome inhibitors are not much effective due to their tolerance and singleness in other tumors. Therefore, further studies on their mechanisms of action and drug interactions are needed to investigate their therapeutic potential.
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Affiliation(s)
- Xiangyi Zhou
- Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing 400030, China
| | - Ruqing Xu
- Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
| | - Yue Wu
- Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
| | - Li Zhou
- Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing 400030, China
| | - Tingxiu Xiang
- Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing 400030, China
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7
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Lobas AA, Saei AA, Lyu H, Zubarev RA, Gorshkov MV. Chemical Proteomics Reveals that the Anticancer Drug Everolimus Affects the Ubiquitin-Proteasome System. ACS Pharmacol Transl Sci 2024; 7:787-796. [PMID: 38481686 PMCID: PMC10928898 DOI: 10.1021/acsptsci.3c00316] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Revised: 01/15/2024] [Accepted: 01/19/2024] [Indexed: 02/09/2025]
Abstract
Rapamycin is a natural antifungal, immunosuppressive, and antiproliferative compound that allosterically inhibits mTOR complex 1. The ubiquitin-proteasome system (UPS) responsible for protein turnover is usually not listed among the pathways affected by mTOR signaling. However, some previous studies have indicated the interplay between the UPS and mTOR. It has also been reported that rapamycin and its analogs can allosterically inhibit the proteasome itself. In this work, we studied the molecular effect of rapamycin and its analogs (rapalogs), everolimus and temsirolimus, on the A549 cell line by expression proteomics. The analysis of differentially expressed proteins showed that the cellular response to everolimus treatment is strikingly different from that to rapamycin and temsirolimus. In the cluster analysis, the effect of everolimus was similar to that of bortezomib, a well-established proteasome inhibitor. UPS-related pathways were enriched in the cluster of proteins specifically upregulated upon everolimus and bortezomib treatments, suggesting that both compounds have similar proteasome inhibition effects. In particular, the total amount of ubiquitin was significantly elevated in the samples treated with everolimus and bortezomib, and analysis of the polyubiquitination patterns revealed elevated intensities of the ubiquitin peptide with a GG modification at the K48 residue, consistent with a bottleneck in proteasomal protein degradation. Moreover, the everolimus treatment resulted in both ubiquitin phosphorylation and generation of a significant amount of semitryptic peptides, illustrating the increase in the protease activity. These observations suggest that everolimus affects the UPS in a unique way, and its mechanism of action is different from that of its close chemical analogs, rapamycin and temsirolimus.
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Affiliation(s)
- Anna A. Lobas
- V.
L. Talrose Institute for Energy Problems of Chemical Physics, Federal
Research Center for Chemical Physics, Russian
Academy of Sciences, 119334 Moscow, Russia
| | - Amir Ata Saei
- Division
of Physiological Chemistry I, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden
- Biozentrum, University of Basel, 4056 Basel, Switzerland
- Center
for Translational Microbiome Research, Department of Microbiology,
Tumor and Cell Biology, Karolinska Institutet, SE-17 177 Stockholm, Sweden
| | - Hezheng Lyu
- Division
of Physiological Chemistry I, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden
| | - Roman A. Zubarev
- Division
of Physiological Chemistry I, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden
- The
National Medical Research Center for Endocrinology, 115478 Moscow, Russia
| | - Mikhail V. Gorshkov
- V.
L. Talrose Institute for Energy Problems of Chemical Physics, Federal
Research Center for Chemical Physics, Russian
Academy of Sciences, 119334 Moscow, Russia
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Beller P, Fink P, Wolf F, Männle D, Helmle I, Kuttenlochner W, Unterfrauner D, Engelbrecht A, Staudt ND, Kulik A, Groll M, Gross H, Kaysser L. Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG. J Biol Chem 2024; 300:105507. [PMID: 38029966 PMCID: PMC10776993 DOI: 10.1016/j.jbc.2023.105507] [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: 07/24/2023] [Revised: 11/17/2023] [Accepted: 11/21/2023] [Indexed: 12/01/2023] Open
Abstract
Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a β-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin β-lactone warheads.
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Affiliation(s)
- Patrick Beller
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Phillipp Fink
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Felix Wolf
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Daniel Männle
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Irina Helmle
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Wolfgang Kuttenlochner
- Department of Bioscience, Center for Protein Assemblies (CPA), TUM School of Natural Sciences, Technical University of Munich, Garching, Germany
| | - Daniel Unterfrauner
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Alicia Engelbrecht
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Nicole D Staudt
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Andreas Kulik
- Interfaculty Institute for Microbiology and Infection Medicine Tübingen (IMIT), Microbial Bioactive Compounds, University of Tübingen, Tübingen, Germany
| | - Michael Groll
- Department of Bioscience, Center for Protein Assemblies (CPA), TUM School of Natural Sciences, Technical University of Munich, Garching, Germany
| | - Harald Gross
- Department of Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Tübingen, Germany
| | - Leonard Kaysser
- Department of Pharmaceutical Biology, Institute for Drug Discovery, University of Leipzig, Leipzig, Germany.
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9
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Thomas T, Salcedo-Tacuma D, Smith DM. Structure, Function, and Allosteric Regulation of the 20S Proteasome by the 11S/PA28 Family of Proteasome Activators. Biomolecules 2023; 13:1326. [PMID: 37759726 PMCID: PMC10526260 DOI: 10.3390/biom13091326] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 08/24/2023] [Accepted: 08/24/2023] [Indexed: 09/29/2023] Open
Abstract
The proteasome, a complex multi-catalytic protease machinery, orchestrates the protein degradation essential for maintaining cellular homeostasis, and its dysregulation also underlies many different types of diseases. Its function is regulated by many different mechanisms that encompass various factors such as proteasome activators (PAs), adaptor proteins, and post-translational modifications. This review highlights the unique characteristics of proteasomal regulation through the lens of a distinct family of regulators, the 11S, REGs, or PA26/PA28. This ATP-independent family, spanning from amoebas to mammals, exhibits a common architectural structure; yet, their cellular biology and criteria for protein degradation remain mostly elusive. We delve into their evolution and cellular biology, and contrast their structure and function comprehensively, emphasizing the unanswered questions regarding their regulatory mechanisms and broader roles in proteostasis. A deeper understanding of these processes will illuminate the roles of this regulatory family in biology and disease, thus contributing to the advancement of therapeutic strategies.
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Affiliation(s)
- Taylor Thomas
- Department of Biochemistry and Molecular Medicine, School of Medicine, West Virginia University, 64 Medical Center Drive, Morgantown, WV 26506, USA
| | - David Salcedo-Tacuma
- Department of Biochemistry and Molecular Medicine, School of Medicine, West Virginia University, 64 Medical Center Drive, Morgantown, WV 26506, USA
| | - David M. Smith
- Department of Biochemistry and Molecular Medicine, School of Medicine, West Virginia University, 64 Medical Center Drive, Morgantown, WV 26506, USA
- Department of Neuroscience, Rockefeller Neuroscience Institute, West Virginia University, Morgantown, WV 26506, USA
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10
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Saha A, Suga H, Brik A. Combining Chemical Protein Synthesis and Random Nonstandard Peptides Integrated Discovery for Modulating Biological Processes. Acc Chem Res 2023; 56:1953-1965. [PMID: 37312234 PMCID: PMC10357587 DOI: 10.1021/acs.accounts.3c00178] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Indexed: 06/15/2023]
Abstract
Chemical manipulation of naturally occurring peptides offers a convenient route for generating analogs to screen against different therapeutic targets. However, the limited success of the conventional chemical libraries has urged chemical biologists to adopt alternative methods such as phage and mRNA displays and create libraries of a large number of variants for the screening and selection of novel peptides. Messenger RNA (mRNA) display provides great advantages in terms of the library size and the straightforward recovery of the selected polypeptide sequences. Importantly, the integration of the flexible in vitro translation (FIT) system with the mRNA display provides the basis of the random nonstandard peptides integrated discovery (RaPID) approach for the introduction of diverse nonstandard motifs, such as unnatural side chains and backbone modifications. This platform allows the discovery of functionalized peptides with tight binding against virtually any protein of interest (POI) and therefore shows great potential in the pharmaceutical industry. However, this method has been limited to targets generated by recombinant expression, excluding its applications to uniquely modified proteins, particularly those with post-translational modifications.Chemical protein synthesis allows a wide range of changes to the protein's chemical composition to be performed, including side chain and backbone modifications and access to post-translationally modified proteins, which are often inaccessible or difficult to achieve via recombinant expression methods. Notably, d-proteins can be prepared via chemical synthesis, which has been used in mirror image phase display for the discovery of nonproteolytic d-peptide binders.Combining chemical protein synthesis with the RaPID system allows the production of a library of trillions of cyclic peptides and subsequent selection for novel cyclic peptide binders targeting a uniquely modified protein to assist in studying its unexplored biology and possibly the discovery of new drug candidates.Interestingly, the small post-translational modifier protein ubiquitin (Ub), with its various polymeric forms, regulates directly or indirectly many biochemical processes, e.g., proteasomal degradation, DNA damage repair, cell cycle regulation, etc. In this Account, we discuss combining the RaPID approach against various synthetic Ub chains for selecting effective and specific macrocyclic peptide binders. This offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling. We highlight experimental approaches and conceptual adaptations required to design and modulate the activity of Lys48- and Lys63-linked Ub chains by macrocyclic peptides. We also present the applications of these approaches to shed light on related biological activities and ultimately their activity against cancer. Finally, we contemplate future developments still pending in this exciting multidisciplinary field.
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Affiliation(s)
- Abhishek Saha
- Schulich
Faculty of Chemistry, Technion-Israel Institute
of Technology, Haifa 3200008, Israel
| | - Hiroaki Suga
- Department
of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
| | - Ashraf Brik
- Schulich
Faculty of Chemistry, Technion-Israel Institute
of Technology, Haifa 3200008, Israel
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11
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Gulyaeva NV. Glucocorticoids Orchestrate Adult Hippocampal Plasticity: Growth Points and Translational Aspects. BIOCHEMISTRY. BIOKHIMIIA 2023; 88:565-589. [PMID: 37331704 DOI: 10.1134/s0006297923050012] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 04/10/2023] [Accepted: 04/10/2023] [Indexed: 06/20/2023]
Abstract
The review analyzes modern concepts about the control of various mechanisms of the hippocampal neuroplasticity in adult mammals and humans by glucocorticoids. Glucocorticoid hormones ensure the coordinated functioning of key components and mechanisms of hippocampal plasticity: neurogenesis, glutamatergic neurotransmission, microglia and astrocytes, systems of neurotrophic factors, neuroinflammation, proteases, metabolic hormones, neurosteroids. Regulatory mechanisms are diverse; along with the direct action of glucocorticoids through their receptors, there are conciliated glucocorticoid-dependent effects, as well as numerous interactions between various systems and components. Despite the fact that many connections in this complex regulatory scheme have not yet been established, the study of the factors and mechanisms considered in the work forms growth points in the field of glucocorticoid-regulated processes in the brain and primarily in the hippocampus. These studies are fundamentally important for the translation into the clinic and the potential treatment/prevention of common diseases of the emotional and cognitive spheres and respective comorbid conditions.
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Affiliation(s)
- Natalia V Gulyaeva
- Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, 117485, Russia.
- Research and Clinical Center for Neuropsychiatry of Moscow Healthcare Department, Moscow, 115419, Russia
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12
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Imran MAS, Carrera M, Pérez-Polo S, Pérez J, Barros L, Dios S, Gestal C. Insights into Common Octopus (Octopus vulgaris) Ink Proteome and Bioactive Peptides Using Proteomic Approaches. Mar Drugs 2023; 21:md21040206. [PMID: 37103345 PMCID: PMC10142993 DOI: 10.3390/md21040206] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2023] [Revised: 03/21/2023] [Accepted: 03/21/2023] [Indexed: 03/29/2023] Open
Abstract
The common octopus (Octopus vulgaris) is nowadays the most demanded cephalopod species for human consumption. This species was also postulated for aquaculture diversification to supply its increasing demand in the market worldwide, which only relies on continuously declining field captures. In addition, they serve as model species for biomedical and behavioral studies. Body parts of marine species are usually removed before reaching the final consumer as by-products in order to improve preservation, reduce shipping weight, and increase product quality. These by-products have recently attracted increasing attention due to the discovery of several relevant bioactive compounds. Particularly, the common octopus ink has been described as having antimicrobial and antioxidant properties, among others. In this study, the advanced proteomics discipline was applied to generate a common octopus reference proteome to screen potential bioactive peptides from fishing discards and by-products such as ink. A shotgun proteomics approach by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using an Orbitrap Elite instrument was used to create a reference dataset from octopus ink. A total of 1432 different peptides belonging to 361 non-redundant annotated proteins were identified. The final proteome compilation was investigated by integrated in silico studies, including gene ontology (GO) term enrichment, pathways, and network studies. Different immune functioning proteins involved in the innate immune system, such as ferritin, catalase, proteasome, Cu/Zn superoxide dismutase, calreticulin, disulfide isomerase, heat shock protein, etc., were found in ink protein networks. Additionally, the potential of bioactive peptides from octopus ink was addressed. These bioactive peptides can exert beneficial health properties such as antimicrobial, antioxidant, antihypertensive, and antitumoral properties and are therefore considered lead compounds for developing pharmacological, functional foods or nutraceuticals.
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13
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Lu C, Lu P, Gong L, Zhu LJ, An Y, Wang Y. Rational design and development of novel NAE inhibitors for the treatment of pancreatic cancer. Med Chem Res 2023. [DOI: 10.1007/s00044-022-02979-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
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14
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Cecarini V, Selmi S, Cuccioloni M, Gong C, Bonfili L, Zheng Y, Cortese M, Angeletti M, Kilani S, Eleuteri AM. Targeting Proteolysis with Cyanogenic Glycoside Amygdalin Induces Apoptosis in Breast Cancer Cells. Molecules 2022; 27:7591. [PMID: 36364419 PMCID: PMC9657530 DOI: 10.3390/molecules27217591] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2022] [Revised: 10/24/2022] [Accepted: 11/02/2022] [Indexed: 09/29/2023] Open
Abstract
BACKGROUND Breast cancer is the most diagnosed cancer among women, and its incidence and mortality are rapidly growing worldwide. In this regard, plant-derived natural compounds have been shown to be effective as chemotherapeutic and preventative agents. Apricot kernels are a rich source of nutrients including proteins, lipids, fibers, and phenolic compounds and contain the aromatic cyanogenic glycoside amygdalin that has been shown to exert a cytotoxic effect on cancer cells by affecting the cell cycle, inducing apoptosis, and regulating the immune function. METHODS Here, we describe a previously unexplored proapoptotic mechanism of action of amygdalin in breast cancer (MCF7) cells that involves the modulation of intracellular proteolysis. For comparative purposes, the same investigations were also conducted upon cell treatment with two apricot kernel aqueous extracts from Prunus armeniaca L. RESULTS We observed that both the 20S and 26S proteasome activities were downregulated in the MCF7 cells upon 24 h treatments. Simultaneously, the autophagy cascade resulted in being impaired due to cathepsin B and L inhibition that also contributed to a reduction in cancer cell migration. The inhibition of these proteolytic systems finally promoted the activation of apoptotic events in the MCF7 cells. CONCLUSION Collectively, our data unveil a novel mechanism of the anticancer activity of amygdalin, prompting further investigations for potential application in cancer preventative strategies.
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Affiliation(s)
- Valentina Cecarini
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
| | - Salima Selmi
- Research Unit for Bioactive Natural Products and Biotechnology UR17ES49, Faculty of Dental Medicine of Monastir, University of Monastir, Avicenne Street, Monastir 5000, Tunisia
- Department of Pharmaceutical Sciences A, Faculty of Pharmacy of Monastir, University of Monastir, Avicenne Street, Monastir 5019, Tunisia
| | - Massimiliano Cuccioloni
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
| | - Chunmei Gong
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
| | - Laura Bonfili
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
| | - Yadong Zheng
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
| | - Manuela Cortese
- CHiP Research Center, School of Pharmacy, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy
| | - Mauro Angeletti
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
| | - Soumaya Kilani
- Research Unit for Bioactive Natural Products and Biotechnology UR17ES49, Faculty of Dental Medicine of Monastir, University of Monastir, Avicenne Street, Monastir 5000, Tunisia
- Department of Pharmaceutical Sciences A, Faculty of Pharmacy of Monastir, University of Monastir, Avicenne Street, Monastir 5019, Tunisia
| | - Anna Maria Eleuteri
- School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
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15
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Hamidi R, Ataei F, Hosseinkhani S. Inhibition of noncaspase proteases, calpain and proteasome, via ALLN and Bortezomib contributes to cell death through low degradation of pro-/anti-apoptotic proteins and apoptosis induction. Med Oncol 2022; 39:125. [PMID: 35716322 DOI: 10.1007/s12032-022-01716-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 03/25/2022] [Indexed: 10/18/2022]
Abstract
Dysfunction at any regulatory point along the apoptotic signaling pathway is closely related to many diseases including cancers. The apoptotic protein expression level is an important cause of cancer-related death, and the correct degradation of apoptotic proteins is involved in tumor development. Therefore, understanding of a regulatory point that underlying cancer-related death may help the development of new strategies to overcome the clinical challenges. Here, proteasome inhibitor Bortezomib and calpain inhibitor ALLN were examined on protein levels of caspase-3, caspase-9, XIAP, and E3-ligase PARC in HEK293T cells overexpressing XIAP and caspase-9. ATP depletion and caspase-3 activation were as a consequence of Bortezomib and ALLN function. Higher numbers of PI-stained cells provided evidence of cell death by both inhibitors. Western blotting analysis showed that both ALLN and Bortezomib equally inhibited degradation of XIAP, but only ALLN was effective at inhibiting caspase proteolytic degradation. Moreover, treatment of cells with both types of inhibitors significantly increased the level of E3-ligase PARC. Our findings showed that inhibition of proteasome and calpains enhanced the level of anti-apoptotic, XIAP and PARC, and pro-apoptotic, caspase-9 and 3 proteins, which totally promote cell death significantly.
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Affiliation(s)
- Roghaye Hamidi
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
| | - Farangis Ataei
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
| | - Saman Hosseinkhani
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
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16
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Staikou A, Feidantsis K, Gkanatsiou O, Bibos MN, Hatziioannou M, Storey KB, Michaelidis B. Seasonal cellular stress phenomena and phenotypic plasticity in land snail Helix lucorum populations from different altitudes. J Exp Biol 2021; 224:273728. [PMID: 34796901 DOI: 10.1242/jeb.243298] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Accepted: 11/15/2021] [Indexed: 11/20/2022]
Abstract
Temperature, a major abiotic environmental factor, regulates various physiological functions in land snails and therefore determines their biogeographical distribution. Thus, species with different distributions may present different thermal tolerance limits. Additionally, the intense reactivation of snail metabolic rate upon arousal from hibernation or estivation may provoke stress. Land snails, Helix lucorum, display a wide altitudinal distribution resulting in populations being exposed to different seasonal temperature variations. The aim of the present study was to investigate the expression of heat shock proteins (Hsps), mitogen activated protein kinases (MAPKs) and proteins that are related to apoptosis (Bcl-2, ubiquitin), that have 'cytoprotective' roles and are also considered to be reliable indicators of stress because of their crucial role in maintaining cellular homeostasis. These proteins were assessed in H. lucorum individuals from two different populations, one at Axios (sea level, 0 m) and the other at Kokkinopilos (Olympus, 1250 m), as well as after mutual population exchanges, in order to find out whether the different responses of these stress-related proteins depend solely on the environmental temperature. The results showed seasonally altered levels in all studied proteins in the hepatopancreas and foot of snails, both among different populations and between the same populations exposed to varying altitudes. However, individuals of the same population in their native habitat or acclimatized to a different habitat showed a relatively similar pattern of expression, supporting the induction of the specific proteins according to the life history of each species.
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Affiliation(s)
- Alexandra Staikou
- Laboratory of Zoology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Konstantinos Feidantsis
- Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Ourania Gkanatsiou
- Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Modestos Nakos Bibos
- Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Marianthi Hatziioannou
- Department of Ichthyology and Aquatic Environment, Faculty of Agricultural Sciences, University of Thessaly, Fytoko street, GR-38445 Volos, Greece
| | - Kenneth B Storey
- Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada, K1S 5B6
| | - Basile Michaelidis
- Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
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17
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Xu DC, Yang L, Zhang PQ, Yan D, Xue Q, Huang QT, Li XF, Hao YL, Tang DL, Ping Dou Q, Chen X, Liu JB. Pharmacological characterization of a novel metal-based proteasome inhibitor Na-AuPT for cancer treatment. Acta Pharmacol Sin 2021; 43:2128-2138. [PMID: 34893683 PMCID: PMC9343436 DOI: 10.1038/s41401-021-00816-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Accepted: 11/04/2021] [Indexed: 11/09/2022]
Abstract
The ubiquitin-proteasome system (UPS) is essential for maintaining cell homeostasis by orchestrating the protein degradation, but is impaired in various diseases, including cancers. Several proteasome inhibitors, such as bortezomib, are currently used in cancer treatment, but associated toxicity limits their widespread application. Recently metal complex-based drugs have attracted great attention in tumor therapy; however, their application is hindered by low water-solubility and poor absorbency. Herein, we synthesized a new type of gold (I) complex named Na-AuPT, and further characterized its anticancer activity. Na-AuPT is highly water-soluble (6 mg/mL), and it was able to potently inhibit growth of a panel of 11 cancer cell lines (A549, SMMC7721, H460, HepG2, BEL7402, LNCap, PC3, MGC-803, SGC-7901, U266, and K562). In A549 and SMMC7721 cells, Na-AuPT (in a range of 2.5-20 μM) inhibited the UPS function in a dose-dependent fashion by targeting and inhibiting both 20 S proteasomal proteolytic peptidases and 19 S proteasomal deubiquitinases. Furthermore, Na-AuPT induced caspase-dependent apoptosis in A549 and SMMC7721 cells, which was prevented by the metal chelator EDTA. Administration of Na-AuPT (40 mg · kg-1 · d-1, ip) in nude mice bearing A549 or SMMC7721 xenografts significantly inhibited the tumor growth in vivo, accompanied by increased levels of total ubiquitinated proteins, cleaved caspase 3 and Bax protein in tumor tissue. Moreover, Na-AuPT induced cell death of primary mononuclear cells from 5 patients with acute myeloid leukemia ex vivo with an average IC50 value of 2.46 μM. We conclude that Na-AuPT is a novel metal-based proteasome inhibitor that may hold great potential for cancer therapy.
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18
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Zhang Y, Liu Q, Wei W, Zhang G, Yan S, Dai R, Sun Y, Su D, Lv S, Xia Y, Li J, Li C. Bortezomib potentiates antitumor activity of mitoxantrone through dampening Wnt/β-catenin signal pathway in prostate cancer cells. BMC Cancer 2021; 21:1101. [PMID: 34645397 PMCID: PMC8515742 DOI: 10.1186/s12885-021-08841-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2021] [Accepted: 10/05/2021] [Indexed: 12/25/2022] Open
Abstract
BACKGROUND Bortezomib (BZM), alone or in combination with other chemotherapies, has displayed strong anticancer effects in several cancers. The efficacy of the combination of BZM and mitoxantrone (MTX) in treating prostate cancer remains unknown. METHODS Anticancer effects of combination of BZM and MTX were determined by apoptosis and proliferation assay in vivo and in vitro. Expression of β-Catenin and its target genes were characterized by western blot and Real-time PCR. RESULTS BZM significantly enhanced MTX-induced antiproliferation in vivo and in vitro. Mice administered a combination of BZM and MTX displayed attenuated tumor growth and prolonged survival. BZM significantly attenuated MTX-induced apoptosis. Moreover, the combination of BZM and MTX contributed to inhibition of the Wnt/β-Catenin signaling pathway compared to monotherapy. CONCLUSIONS This study demonstrates that BZM enhances MTX-induced anti-tumor effects by inhibiting the Wnt/β-Catenin signaling pathway in prostate cancer cells.
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Affiliation(s)
- Ying Zhang
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Qiuzi Liu
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Wei Wei
- Center for Experimental Medicine, School of Public Health, Jining Medical University, Jining, 272067, China
| | - Guoan Zhang
- Institute of Cancer Pathology Research, Jining Medical University, Jining, 272067, China
| | - Siyuan Yan
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Rongrong Dai
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Ying Sun
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Dubo Su
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Shun Lv
- Laboratory animal center, Jining Medical University, Jining, 272067, China
| | - Yong Xia
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Jing Li
- Department of Histology and Embryology, School of Medicine, Nankai University, Tianjin, 300071, China
| | - Changlin Li
- Institute of Precision Medicine, Jining Medical University, Jining, 272067, China.
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Loh D, Reiter RJ. Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders. Antioxidants (Basel) 2021; 10:1483. [PMID: 34573116 PMCID: PMC8465482 DOI: 10.3390/antiox10091483] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Revised: 09/10/2021] [Accepted: 09/13/2021] [Indexed: 12/12/2022] Open
Abstract
Biomolecular condensates are membraneless organelles (MLOs) that form dynamic, chemically distinct subcellular compartments organizing macromolecules such as proteins, RNA, and DNA in unicellular prokaryotic bacteria and complex eukaryotic cells. Separated from surrounding environments, MLOs in the nucleoplasm, cytoplasm, and mitochondria assemble by liquid-liquid phase separation (LLPS) into transient, non-static, liquid-like droplets that regulate essential molecular functions. LLPS is primarily controlled by post-translational modifications (PTMs) that fine-tune the balance between attractive and repulsive charge states and/or binding motifs of proteins. Aberrant phase separation due to dysregulated membrane lipid rafts and/or PTMs, as well as the absence of adequate hydrotropic small molecules such as ATP, or the presence of specific RNA proteins can cause pathological protein aggregation in neurodegenerative disorders. Melatonin may exert a dominant influence over phase separation in biomolecular condensates by optimizing membrane and MLO interdependent reactions through stabilizing lipid raft domains, reducing line tension, and maintaining negative membrane curvature and fluidity. As a potent antioxidant, melatonin protects cardiolipin and other membrane lipids from peroxidation cascades, supporting protein trafficking, signaling, ion channel activities, and ATPase functionality during condensate coacervation or dissolution. Melatonin may even control condensate LLPS through PTM and balance mRNA- and RNA-binding protein composition by regulating N6-methyladenosine (m6A) modifications. There is currently a lack of pharmaceuticals targeting neurodegenerative disorders via the regulation of phase separation. The potential of melatonin in the modulation of biomolecular condensate in the attenuation of aberrant condensate aggregation in neurodegenerative disorders is discussed in this review.
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Affiliation(s)
- Doris Loh
- Independent Researcher, Marble Falls, TX 78654, USA
| | - Russel J. Reiter
- Department of Cellular and Structural Biology, UT Health Science Center, San Antonio, TX 78229, USA
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Wang J, Wang Y, He S, Wang Z, Deng Q, Liang H. Proteasome inhibition induces macrophage apoptosis via mitochondrial dysfunction. J Biochem Mol Toxicol 2021; 35:e22894. [PMID: 34418242 DOI: 10.1002/jbt.22894] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2020] [Revised: 07/15/2021] [Accepted: 08/11/2021] [Indexed: 11/10/2022]
Abstract
Dysfunction of the ubiquitin-proteasome system has been linked to the pathogenesis of a variety of diseases. Proteasome inhibition not only exerts antitumor effects but also affects inflammatory signaling pathways. MG132, a proteasome inhibitor, has been shown to induce tumor cell apoptosis. However, its role in the induction of macrophage apoptosis remains unknown. In our study, we investigated the mechanism of the proapoptotic effects of MG132 in macrophages. Our data showed that MG132 treatment induced mitochondrial reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential in macrophages. We found that proteasome inhibition induced a significant increase in the apoptosis rate, as evidenced by cleavage of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenyl-phosphonium chloride (Mito-TEMPO) attenuated MG132-induced apoptosis. In conclusion, proteasome inhibition by MG132 can induce macrophage apoptosis by promoting the production of ROS and mitochondrial dysfunction.
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Affiliation(s)
- Jieyan Wang
- Department of Urology, The People's Hospital of Longhua, The Affiliated Hospital of Southern Medical University, Shenzhen, Guangdong, China
| | - Yingling Wang
- Department of Pediatrics, The Second Hospital of Zhuzhou, Zhuzhou, Hunan, China
| | - Shihan He
- The First School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong, China
| | - Zhu Wang
- Department of Urology, The People's Hospital of Longhua, The Affiliated Hospital of Southern Medical University, Shenzhen, Guangdong, China
| | - Qiong Deng
- Department of Urology, The People's Hospital of Longhua, The Affiliated Hospital of Southern Medical University, Shenzhen, Guangdong, China
| | - Hui Liang
- Department of Urology, The People's Hospital of Longhua, The Affiliated Hospital of Southern Medical University, Shenzhen, Guangdong, China
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21
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Signaling Pathways Regulated by UBR Box-Containing E3 Ligases. Int J Mol Sci 2021; 22:ijms22158323. [PMID: 34361089 PMCID: PMC8346999 DOI: 10.3390/ijms22158323] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 07/28/2021] [Accepted: 07/29/2021] [Indexed: 12/31/2022] Open
Abstract
UBR box E3 ligases, also called N-recognins, are integral components of the N-degron pathway. Representative N-recognins include UBR1, UBR2, UBR4, and UBR5, and they bind destabilizing N-terminal residues, termed N-degrons. Understanding the molecular bases of their substrate recognition and the biological impact of the clearance of their substrates on cellular signaling pathways can provide valuable insights into the regulation of these pathways. This review provides an overview of the current knowledge of the binding mechanism of UBR box N-recognin/N-degron interactions and their roles in signaling pathways linked to G-protein-coupled receptors, apoptosis, mitochondrial quality control, inflammation, and DNA damage. The targeting of these UBR box N-recognins can provide potential therapies to treat diseases such as cancer and neurodegenerative diseases.
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22
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Cai B, Hou M, Zhang S, Xin Z, Huang J, Yang J, Wang Y, Cai X, Xie S, Zhang C, Huang Y. Dual Targeting of Endoplasmic Reticulum by Redox-Deubiquitination Regulation for Cancer Therapy. Int J Nanomedicine 2021; 16:5193-5209. [PMID: 34354353 PMCID: PMC8331122 DOI: 10.2147/ijn.s321612] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Accepted: 07/22/2021] [Indexed: 12/15/2022] Open
Abstract
Background Recently, nanocatalyst-induced endoplasmic reticulum (ER) stress for cancer therapy has been attracting considerable attention. However, cancer cells are often able to overcome ER stress-induced death by activating the unfolded protein response (UPR), making nanocatalytic monotherapy a poor defense against cancer progression. Purpose In this study, to improve the nanocatalytic treatment efficacy, a phase change material (PCM) was used to encapsulate the upstream ER stress initiator, iron oxide nanoparticles (Fe3O4 NPs), and the downstream UPR modulator, PR-619. Subsequently, the tumor-homing peptide tLyP-1 was coupled to it to form tLyP-1/PR-619/Fe3O4@PCM (tPF@PCM) theranostic platform. Materials and Methods tPF@PCM was synthesized using nanoprecipitation and resolidification methods followed by the EDC/NHS cross-linking method. The targeting capacity of tPF@PCM was evaluated in vitro and in vivo using flow cytometry and magnetic resonance imaging, respectively. The therapeutic efficacy of tPF@PCM was investigated in a renal cell carcinoma mouse model. Moreover, we explored the synergistic anti-tumor mechanism by examining the intracellular reactive oxygen species (ROS), aggregated proteins, ER stress response levels, and type of cell death. Results tPF@PCM had excellent tumor-targeting properties and exhibited satisfactory photothermal-enhanced tumor inhibition efficacy both in vitro and in vivo. Specifically, the phase transition temperature (45 °C) maintained using 808 nm laser irradiation significantly increased the release and catalytic activity of the peroxidase mimic Fe3O4 NPs. This strongly catalyzed the generation of hydroxyl radicals (•OH) via the Fenton reaction in the acidic tumor microenvironment. The redox imbalance subsequently resulted in an increase in the level of damaged proteins in the ER and initiated ER stress. Moreover, the pan-deubiquitinase inhibitor PR-619 blocked the “adaptive” UPR-mediated degradation of these damaged proteins, exacerbating the ER burden. Consequently, irremediable ER stress activated the “terminal” UPR, leading to apoptosis in cancer cells. Conclusion This ER stress-exacerbating strategy effectively suppresses tumorigenesis, offering novel directions for advances in the treatment of conventional therapy-resistant cancers.
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Affiliation(s)
- Biao Cai
- Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Mengfei Hou
- School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, People's Republic of China
| | - Shijun Zhang
- Department of Ultrasound, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Zhixiang Xin
- Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Jiwei Huang
- Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Jingxing Yang
- School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, People's Republic of China
| | - Yueming Wang
- Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Xingyun Cai
- Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Shaowei Xie
- Department of Ultrasound, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
| | - Chunfu Zhang
- School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, People's Republic of China
| | - Yiran Huang
- Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People's Republic of China
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Marizomib sensitizes primary glioma cells to apoptosis induced by a latest-generation TRAIL receptor agonist. Cell Death Dis 2021; 12:647. [PMID: 34168123 PMCID: PMC8225658 DOI: 10.1038/s41419-021-03927-x] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Revised: 05/28/2021] [Accepted: 06/07/2021] [Indexed: 12/26/2022]
Abstract
Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood–brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.
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24
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Kaur G, Iyer LM, Burroughs AM, Aravind L. Bacterial death and TRADD-N domains help define novel apoptosis and immunity mechanisms shared by prokaryotes and metazoans. eLife 2021; 10:70394. [PMID: 34061031 PMCID: PMC8195603 DOI: 10.7554/elife.70394] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Accepted: 05/23/2021] [Indexed: 12/12/2022] Open
Abstract
Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter.
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Affiliation(s)
- Gurmeet Kaur
- Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, United States
| | - Lakshminarayan M Iyer
- Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, United States
| | - A Maxwell Burroughs
- Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, United States
| | - L Aravind
- Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, United States
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25
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Xiong C, Zhou L, Tan J, Song S, Bao X, Zhang N, Ding H, Zhao J, He JX, Miao ZH, Zhang A. Development of Potent NEDD8-Activating Enzyme Inhibitors Bearing a Pyrimidotriazole Scaffold. J Med Chem 2021; 64:6161-6178. [PMID: 33857374 DOI: 10.1021/acs.jmedchem.1c00242] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The ubiquitin-like protein NEDD8 is a critical signaling molecule implicated in the functional maintenance and homeostasis of cells. Dysregulation of this process is involved in a variety of human diseases, including cancer. Therefore, NEDD8-activating enzyme E1 (NAE), the only activation enzyme of the neddylation pathway, has been an emergent anticancer target. In view of the single-agent modest response of the clinical NAE inhibitor, pevonedistat (compound 1, MLN4924), efforts on development of new inhibitors with both high potency and better safety profiles are urgently needed. Here, we report a structural hopping strategy by optimizing the central deazapurine framework and the solvent interaction region of compound 1, leading to compound 26 bearing a pyrimidotriazole scaffold. Compound 26 not only has compatible potency in the biochemical and cell assays but also possesses improved pharmacokinetic (PK) properties than compound 1. In vivo, compound 26 showed significant antitumor efficacy and good safety in xenograft models.
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Affiliation(s)
- Chaodong Xiong
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lina Zhou
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jing Tan
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shanshan Song
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xubin Bao
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Ning Zhang
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Huaqian Ding
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jiannan Zhao
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.,School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Jin-Xue He
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Ze-Hong Miao
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Ao Zhang
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China.,Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.,University of Chinese Academy of Sciences, Beijing 100049, China.,Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.,State Key Laboratory of Esophageal Cancer Prevention and Treatment, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, China
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26
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Gutierrez-Castillo E, Ming H, Foster B, Gatenby L, Mak CK, Pinto C, Bondioli K, Jiang Z. Effect of vitrification on global gene expression dynamics of bovine elongating embryos. Reprod Fertil Dev 2021; 33:338-348. [PMID: 33602389 PMCID: PMC8049512 DOI: 10.1071/rd20285] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 01/13/2021] [Indexed: 12/27/2022] Open
Abstract
Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.
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Affiliation(s)
| | - Hao Ming
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA, USA
| | - Brittany Foster
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA, USA
| | - Lauren Gatenby
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA, USA
| | - Chun Kuen Mak
- Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA
| | - Carlos Pinto
- Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA
| | - Kenneth Bondioli
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA, USA; and Corresponding authors. ;
| | - Zongliang Jiang
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA, USA; and Corresponding authors. ;
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27
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Kablau A, Eckert JH, Pistorius J, Sharbati S, Einspanier R. Effects of selected insecticidal substances on mRNA transcriptome in larvae of Apis mellifera. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 2020; 170:104703. [PMID: 32980071 DOI: 10.1016/j.pestbp.2020.104703] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Revised: 09/03/2020] [Accepted: 09/06/2020] [Indexed: 06/11/2023]
Abstract
For the last decade, scientists have reported a loss of honeybee colonies. Multiple factors like parasites, pathogens and pesticides are dealt as possible drivers of honeybee losses. In particular, insecticides are considered as a major factor of pollinator poisoning. We applied sublethal concentrations of four insecticidal substances to honeybee larval food and analyzed the effects on transcriptome. The aim was to identify candidate genes indicating early negative impacts after application of insecticidal substances. Honeybee larvae were kept in-vitro under hive conditions (34-35 °C) and fed with dimethoate, fenoxycarb, chlorantraniliprole and flupyradifurone in sublethal concentrations between day 3-6 after grafting. Larvae at day 4, 6 and 8 were sampled and their transcriptome analyzed. By use of a RT-qPCR array differences in gene expression of selected gene families (immune system, development detoxification) were measured. Targets mainly involved in development, energy metabolism and the immune system were significantly affected by the insecticidal substances tested, selectively inducing genes of the detoxification system, immune response and nutritional stress.
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Affiliation(s)
- Arne Kablau
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany
| | - Jakob H Eckert
- Julius Kühn-Institute, Institute for Bee Protection, Braunschweig, Germany
| | - Jens Pistorius
- Julius Kühn-Institute, Institute for Bee Protection, Braunschweig, Germany
| | - Soroush Sharbati
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany
| | - Ralf Einspanier
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany.
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28
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Huang Y, Nawatha M, Livneh I, Rogers JM, Sun H, Singh SK, Ciechanover A, Brik A, Suga H. Affinity Maturation of Macrocyclic Peptide Modulators of Lys48‐Linked Diubiquitin by a Twofold Strategy. Chemistry 2020; 26:8022-8027. [DOI: 10.1002/chem.202000273] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Indexed: 12/15/2022]
Affiliation(s)
- Yichao Huang
- Department of ChemistrySchool of ScienceThe University of Tokyo 7-3-1 Hongo Bunkyo Tokyo 113-0033 Japan
| | - Mickal Nawatha
- Schulich Faculty of ChemistryTechnion-Israel Institute of Technology Haifa 3200008 Israel
| | - Ido Livneh
- The Rappaport Faculty of Medicine and Research InstituteTechnion-Israel Institute of Technology Haifa 3200008 Israel
| | - Joseph M. Rogers
- Department of ChemistrySchool of ScienceThe University of Tokyo 7-3-1 Hongo Bunkyo Tokyo 113-0033 Japan
| | - Hao Sun
- Schulich Faculty of ChemistryTechnion-Israel Institute of Technology Haifa 3200008 Israel
| | - Sumeet K. Singh
- Schulich Faculty of ChemistryTechnion-Israel Institute of Technology Haifa 3200008 Israel
| | - Aaron Ciechanover
- The Rappaport Faculty of Medicine and Research InstituteTechnion-Israel Institute of Technology Haifa 3200008 Israel
| | - Ashraf Brik
- Schulich Faculty of ChemistryTechnion-Israel Institute of Technology Haifa 3200008 Israel
| | - Hiroaki Suga
- Department of ChemistrySchool of ScienceThe University of Tokyo 7-3-1 Hongo Bunkyo Tokyo 113-0033 Japan
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29
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Tian CB, Li YY, Huang J, Chu WQ, Wang ZY, Liu H. Comparative Transcriptome and Proteome Analysis of Heat Acclimation in Predatory Mite Neoseiulus barkeri. Front Physiol 2020; 11:426. [PMID: 32411020 PMCID: PMC7201100 DOI: 10.3389/fphys.2020.00426] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2019] [Accepted: 04/07/2020] [Indexed: 11/24/2022] Open
Abstract
In our previous study, we reported a high temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri was artificially selected via a long-term heat acclimation (35°C) and frequent heat hardenings. To understand the molecular basis of heat acclimation, 'omics' analyses were performed to compare the differences between HTAS female adults to conventional strain (CS) at transcriptional and translational levels. We obtained a total of 5,374 differentially expressed genes and 500 differentially expressed proteins. Among them, 119 transcripts had concurrent transcription and translation profiles. It's conserved that some processes, such as high expression of heat shock protein (HSP) genes, involved in heat tolerance of transcriptome analyses, while many protective enzymes including glutathione S-transferase, superoxide dismutase, peroxidase, and cytochrome P450 displayed down-regulated expression. KEGG analysis mapped 4,979 and 348 differentially expressed genes and proteins, to 299 and 253 pathways, respectively. The mitogen-activated protein kinases (MAPK) signaling pathway may provide new insights for the investigation of the molecular mechanisms of heat tolerance. Correlation enriched pathways indicated that there were four pathways associated with heat acclimation involving in energy metabolism and immunity. In addition, the expression patterns of ten randomly selected genes including HSP were consistent with the transcriptome results obtained through quantitative real-time PCR. Comparisons between transcriptome and proteome results indicated the upregulation of HSPs and genes participated in ATP production, immunity and energy metabolism process. A majority of antioxidant-related genes and detoxication-related genes were down-regulated suggesting a fitness cost of heat acclimation. Our results demonstrated that heat tolerance during a long-time acclimation of N. barkeri is a fairly complicated process of physiological regulations. These findings also contribute to a better understanding of the mechanisms of thermal responses of phytoseiid mites which could provide useful information for biological control through natural enemies.
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Affiliation(s)
| | | | | | | | | | - Huai Liu
- Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing, China
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30
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Zhang X, Linder S, Bazzaro M. Drug Development Targeting the Ubiquitin-Proteasome System (UPS) for the Treatment of Human Cancers. Cancers (Basel) 2020; 12:cancers12040902. [PMID: 32272746 PMCID: PMC7226376 DOI: 10.3390/cancers12040902] [Citation(s) in RCA: 66] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Revised: 04/01/2020] [Accepted: 04/02/2020] [Indexed: 12/12/2022] Open
Abstract
Cancer cells are characterized by a higher rate of protein turnover and greater demand for protein homeostasis compared to normal cells. In this scenario, the ubiquitin-proteasome system (UPS), which is responsible for the degradation of over 80% of cellular proteins within mammalian cells, becomes vital to cancer cells, making the UPS a critical target for the discovery of novel cancer therapeutics. This review systematically categorizes all current reported small molecule inhibitors of the various essential components of the UPS, including ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), the 20S proteasome catalytic core particle (20S CP) and the 19S proteasome regulatory particles (19S RP), as well as their mechanism/s of action and limitations. We also discuss the immunoproteasome which is considered as a prospective therapeutic target of the next generation of proteasome inhibitors in cancer therapies.
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Affiliation(s)
- Xiaonan Zhang
- Masonic Cancer Center and Department of Obstetrics, Gynecology and Women’s Health, University of Minnesota, Minneapolis, MN 55455, USA;
- Department of Oncology-Pathology, Karolinska Institutet, 171 77 Stockholm, Sweden;
- Department of Immunology, Genetics, and Pathology, Uppsala University, 751 05 Uppsala, Sweden
| | - Stig Linder
- Department of Oncology-Pathology, Karolinska Institutet, 171 77 Stockholm, Sweden;
- Department of Medical and Health Sciences, Linköping University, SE-58183 Linköping, Sweden
| | - Martina Bazzaro
- Masonic Cancer Center and Department of Obstetrics, Gynecology and Women’s Health, University of Minnesota, Minneapolis, MN 55455, USA;
- Correspondence:
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31
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The Ubiquitin Proteasome System in Ischemic and Dilated Cardiomyopathy. Int J Mol Sci 2019; 20:ijms20246354. [PMID: 31861129 PMCID: PMC6940920 DOI: 10.3390/ijms20246354] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2019] [Revised: 12/06/2019] [Accepted: 12/10/2019] [Indexed: 12/21/2022] Open
Abstract
Dilated (DCM) and ischemic cardiomyopathies (ICM) are associated with cardiac remodeling, where the ubiquitin–proteasome system (UPS) holds a central role. Little is known about the UPS and its alterations in patients suffering from DCM or ICM. The aim of this study is to characterize the UPS activity in human heart tissue from cardiomyopathy patients. Myocardial tissue from ICM (n = 23), DCM (n = 28), and control (n = 14) patients were used to quantify ubiquitinylated proteins, E3-ubiquitin-ligases muscle-atrophy-F-box (MAFbx)/atrogin-1, muscle-RING-finger-1 (MuRF1), and eukaryotic-translation-initiation-factor-4E (eIF4E), by Western blot. Furthermore, the proteasomal chymotrypsin-like and trypsin-like peptidase activities were determined fluorometrically. Enzyme activity of NAD(P)H oxidase was assessed as an index of reactive oxygen species production. The chymotrypsin- (p = 0.71) and caspase-like proteasomal activity (p = 0.93) was similar between the groups. Trypsin-like proteasomal activity was lower in ICM (0.78 ± 0.11 µU/mg) compared to DCM (1.06 ± 0.08 µU/mg) and control (1.00 ± 0.06 µU/mg; p = 0.06) samples. Decreased ubiquitin expression in both cardiomyopathy groups (ICM vs. control: p < 0.001; DCM vs. control: p < 0.001), as well as less ubiquitin-positive deposits in ICM-damaged tissue (ICM: 4.19% ± 0.60%, control: 6.28% ± 0.40%, p = 0.022), were detected. E3-ligase MuRF1 protein expression (p = 0.62), NADPH-oxidase activity (p = 0.63), and AIF-positive cells (p = 0.50). Statistical trends were detected for reduced MAFbx protein expression in the DCM-group (p = 0.07). Different levels of UPS components, E3 ligases, and UPS activation markers were observed in myocardial tissue from patients affected by DCM and ICM, suggesting differential involvement of the UPS in the underlying pathologies.
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32
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Guo J, Yi GZ, Liu Z, Sun X, Yang R, Guo M, Li Y, Li K, Li K, Wang X, Song H, Qi S, Huang G, Liu Y. Quantitative Proteomics Analysis Reveals Nuclear Perturbation in Human Glioma U87 Cells treated with Temozolomide. Cell Biochem Funct 2019; 38:185-194. [PMID: 31833081 DOI: 10.1002/cbf.3459] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2019] [Revised: 09/23/2019] [Accepted: 10/24/2019] [Indexed: 01/12/2023]
Abstract
Glioblastoma (GBM) is the most malignant and aggressive glioma, which has a very poor prognosis. Temozolomide (TMZ) is still a first-line treatment, but resistance is inevitable even in MGMT-deficient glioblastoma cells. The aims of this study were to comprehend the effect of TMZ on nucleus and the underlying mechanism of acquired TMZ resistance in MGMT-deficient GBM. We show the changes of nuclear proteome in the MGMT-deficient GBM U87 cells treated with TMZ for 1 week. Label-free-based quantitative proteomics were used to investigate nuclear protein abundance change. Subsequently, gene ontology function annotation, KEGG pathway analysis, protein-protein interaction (PPI) network construction analysis of DAPs, and immunofluorescence were applied to validate the quality of proteomics. In total, 457 (455 gene products) significant DAPs were identified, of which 327 were up-regulated and 128 were down-regulated. Bioinformatics analysis uncovered RAD50, MRE11, UBR5, MSH2, MSH6, DDB1, DDB2, RPA1, RBX1, CUL4A, and CUL4B mainly enriched in DNA damage repair related pathway and constituted a protein-protein interaction network. Ribosomal proteins were down-regulated. Cells were in a stress-responsive state, while the entire metabolic level was lowered. SIGNIFICANCE OF THE STUDY: In U87 cell treated with TMZ for 1 week, which resulted in DNA damage, we found various proteins dysregulated in the nucleus. Some proteins related to the DNA damage repair pathway were up-regulated, and there was a strong interaction. We believe this is the potential clues of chemotherapy resistance in tumour cells. These proteins can be used as indicators of tumour resistance screening in the future.
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Affiliation(s)
- Jinglin Guo
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Guo-Zhong Yi
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China.,Nanfang Glioma Center, Guangzhou, China
| | - Zhifeng Liu
- Guangdong Provincial Key Laboratory of Molecular Oncologic Pathology, Guangzhou, China
| | - Xuegang Sun
- The Key Laboratory of Molecular Biology, State Administration of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangdong, China
| | - Runwei Yang
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Manlan Guo
- The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yaomin Li
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Ke Li
- The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Kaishu Li
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xiran Wang
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Haimin Song
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Songtao Qi
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China.,Nanfang Glioma Center, Guangzhou, China
| | - Guanglong Huang
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Nanfang Glioma Center, Guangzhou, China
| | - Yawei Liu
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.,The Laboratory for Precision Neurosurgery Nanfang Hospital, Southern Medical University, Guangzhou, China
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33
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Khetani S, Kollath VO, Eastick E, Debert C, Sen A, Karan K, Sanati-Nezhad A. Single-step functionalization of poly-catecholamine nanofilms for ultra-sensitive immunosensing of ubiquitin carboxyl terminal hydrolase-L1 (UCHL-1) in spinal cord injury. Biosens Bioelectron 2019; 145:111715. [DOI: 10.1016/j.bios.2019.111715] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2019] [Revised: 09/17/2019] [Accepted: 09/17/2019] [Indexed: 02/06/2023]
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34
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Marino A, Camponovo A, Degl'Innocenti A, Bartolucci M, Tapeinos C, Martinelli C, De Pasquale D, Santoro F, Mollo V, Arai S, Suzuki M, Harada Y, Petretto A, Ciofani G. Multifunctional temozolomide-loaded lipid superparamagnetic nanovectors: dual targeting and disintegration of glioblastoma spheroids by synergic chemotherapy and hyperthermia treatment. NANOSCALE 2019; 11:21227-21248. [PMID: 31663592 PMCID: PMC6867905 DOI: 10.1039/c9nr07976a] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/18/2023]
Abstract
Aiming at finding new solutions for fighting glioblastoma multiforme, one of the most aggressive and lethal human cancer, here an in vitro validation of multifunctional nanovectors for drug delivery and hyperthermia therapy is proposed. Hybrid magnetic lipid nanoparticles have been fully characterized and tested on a multi-cellular complex model resembling the tumor microenvironment. Investigations of cancer therapy based on a physical approach (namely hyperthermia) and on a pharmaceutical approach (by exploiting the chemotherapeutic drug temozolomide) have been extensively carried out, by evaluating its antiproliferative and pro-apoptotic effects on 3D models of glioblastoma multiforme. A systematic study of transcytosis and endocytosis mechanisms has been moreover performed with multiple complimentary investigations, besides a detailed description of local temperature increments following hyperthermia application. Finally, an in-depth proteomic analysis corroborated the obtained findings, which can be summarized in the preparation of a versatile, multifunctional, and effective nanoplatform able to overcome the blood-brain barrier and to induce powerful anti-cancer effects on in vitro complex models.
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Affiliation(s)
- Attilio Marino
- Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy.
| | - Alice Camponovo
- Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
| | - Andrea Degl'Innocenti
- Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy.
| | - Martina Bartolucci
- IRCCS Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genova, Italy
| | - Christos Tapeinos
- Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy.
| | - Chiara Martinelli
- Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy.
| | - Daniele De Pasquale
- Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy. and Scuola Superiore Sant'Anna, The Biorobotics Institute, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy
| | - Francesca Santoro
- Istituto Italiano di Tecnologia, Center for Advanced Biomaterials for Health Care, Largo Barsanti e Matteucci 53, 80125 Naples, Italy
| | - Valentina Mollo
- Istituto Italiano di Tecnologia, Center for Advanced Biomaterials for Health Care, Largo Barsanti e Matteucci 53, 80125 Naples, Italy
| | - Satoshi Arai
- Kanazawa University, Nano Life Science Institute (WPI-NanoLSI), Kakuma-Machi, 920-1192 Kanazawa, Japan and Waseda University, Research Institute for Science and Engineering, 3-4-1 Ohkubo, Shinjuku-ku, 169-8555 Tokyo, Japan
| | - Madoka Suzuki
- Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Suita-Shi, 565-0871 Osaka, Japan and PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, 332-0012 Saitama, Japan
| | - Yoshie Harada
- Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Suita-Shi, 565-0871 Osaka, Japan
| | - Andrea Petretto
- IRCCS Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genova, Italy
| | - Gianni Ciofani
- Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy. and Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
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35
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Kam A, Loo S, Fan JS, Sze SK, Yang D, Tam JP. Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation. J Biol Chem 2019; 294:19604-19615. [PMID: 31727740 DOI: 10.1074/jbc.ra119.010796] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2019] [Revised: 11/02/2019] [Indexed: 12/21/2022] Open
Abstract
Disulfide-rich plant peptides with molecular masses of 2-6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family) that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, six-cysteine hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is >100-fold more stable against protease degradation than its S-alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBα degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies.
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Affiliation(s)
- Antony Kam
- School of Biological Sciences, Nanyang Technological University, Singapore 637551
| | - Shining Loo
- School of Biological Sciences, Nanyang Technological University, Singapore 637551
| | - Jing-Song Fan
- Department of Biological Sciences, National University of Singapore, Singapore 117543
| | - Siu Kwan Sze
- School of Biological Sciences, Nanyang Technological University, Singapore 637551
| | - Daiwen Yang
- Department of Biological Sciences, National University of Singapore, Singapore 117543
| | - James P Tam
- School of Biological Sciences, Nanyang Technological University, Singapore 637551
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36
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Ethiraj P, Sambandam Y, Hathaway-Schrader JD, Haque A, Novince CM, Reddy SV. RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma. J Cell Physiol 2019; 235:1663-1673. [PMID: 31309556 DOI: 10.1002/jcp.29086] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Accepted: 06/21/2019] [Indexed: 12/29/2022]
Abstract
Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.
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Affiliation(s)
- Purushoth Ethiraj
- Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina
| | - Yuvaraj Sambandam
- Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina
| | - Jessica D Hathaway-Schrader
- Department of Oral Health Sciences, College of Dental Medicine, Medical University of South Carolina, Charleston, South Carolina
| | - Azizul Haque
- Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina.,Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina
| | - Chad M Novince
- Department of Oral Health Sciences, College of Dental Medicine, Medical University of South Carolina, Charleston, South Carolina
| | - Sakamuri V Reddy
- Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina
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37
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De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains. Nat Chem 2019; 11:644-652. [PMID: 31182821 DOI: 10.1038/s41557-019-0278-x] [Citation(s) in RCA: 57] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2018] [Accepted: 04/30/2019] [Indexed: 12/12/2022]
Abstract
A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.
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38
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Wang L, Lin Z, Triviño M, Nowack MK, Franklin-Tong VE, Bosch M. Self-incompatibility in Papaver pollen: programmed cell death in an acidic environment. JOURNAL OF EXPERIMENTAL BOTANY 2019; 70:2113-2123. [PMID: 30481323 PMCID: PMC7116307 DOI: 10.1093/jxb/ery406] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/28/2018] [Accepted: 11/07/2018] [Indexed: 05/18/2023]
Abstract
Self-incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilization and thus encourages outbreeding and genetic diversity. During pollination, most SI systems utilize cell-cell recognition to reject incompatible pollen. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy), which involves the interaction between the two S-determinants, a stigma-expressed secreted protein (PrsS) and a pollen-expressed plasma membrane-localized protein (PrpS). This interaction is the critical step in determining acceptance of compatible pollen or rejection of incompatible pollen. Cognate PrpS-PrsS interaction triggers a signalling network causing rapid growth arrest and eventually programmed cell death (PCD) in incompatible pollen. In this review, we provide an overview of recent advances in our understanding of the major components involved in the SI-induced PCD (SI-PCD). In particular, we focus on the importance of SI-induced intracellular acidification and consequences for protein function, and the regulation of soluble inorganic pyrophosphatase (Pr-p26.1) activity by post-translational modification. We also discuss attempts to identify protease(s) involved in the SI-PCD process. Finally, we outline future opportunities made possible by the functional transfer of the P. rhoeas SI system to Arabidopsis.
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Affiliation(s)
- Ludi Wang
- Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Plas Gogerddan, Aberystwyth, UK
| | - Zongcheng Lin
- Department of Plant Biotechnology and Genetics, Ghent University, Ghent, Belgium
- VIB Center for Plant Systems Biology, Ghent, Belgium
| | - Marina Triviño
- Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Plas Gogerddan, Aberystwyth, UK
- Department of Plant Biotechnology and Genetics, Ghent University, Ghent, Belgium
- VIB Center for Plant Systems Biology, Ghent, Belgium
| | - Moritz K Nowack
- Department of Plant Biotechnology and Genetics, Ghent University, Ghent, Belgium
- VIB Center for Plant Systems Biology, Ghent, Belgium
| | - Vernonica E Franklin-Tong
- School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, UK
| | - Maurice Bosch
- Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Plas Gogerddan, Aberystwyth, UK
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39
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Ōmura S, Crump A. Lactacystin: first-in-class proteasome inhibitor still excelling and an exemplar for future antibiotic research. J Antibiot (Tokyo) 2019; 72:189-201. [PMID: 30755736 PMCID: PMC6760633 DOI: 10.1038/s41429-019-0141-8] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2018] [Revised: 12/12/2018] [Accepted: 01/07/2019] [Indexed: 01/28/2023]
Abstract
Lactacystin exemplifies the role that serendipity plays in drug discovery and why “finding things without actually looking for them” retains such a pivotal role in the search for the useful properties of chemicals. The first proteasome inhibitor discovered, lactacystin stimulated new possibilities in cancer control. New and innovative uses are regularly being found for lactacystin, including as a model to study dementia, while new formulations and delivery systems may facilitate its use clinically as an anticancer agent. All this provides yet more evidence that we need a comprehensive, collaborative and coordinated programme to fully investigate all new and existing chemical compounds, especially those of microbial origin. We need to do so in order to avoid failing to detect and successfully exploit unsought yet potentially life-saving or extremely advantageous properties of microbial metabolites.
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Affiliation(s)
- Satoshi Ōmura
- Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
| | - Andy Crump
- Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan
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40
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Woo SM, Kwon TK. E3 ubiquitin ligases and deubiquitinases as modulators of TRAIL-mediated extrinsic apoptotic signaling pathway. BMB Rep 2019. [PMID: 30638181 PMCID: PMC6443324 DOI: 10.5483/bmbrep.2019.52.2.011] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) initiates the extrinsic apoptotic pathway through formation of the death-inducing signaling complex (DISC), followed by activation of effector caspases. TRAIL receptors are composed of death receptors (DR4 and DR5), decoy receptors (DcR1 and DcR2), and osteoprotegerin. Among them, only DRs activate apoptotic signaling by TRAIL. Since the levels of DR expressions are higher in cancer cells than in normal cells, TRAIL selectively activates apoptotic signaling pathway in cancer cells. However, multiple mechanisms, including down-regulation of DR expression and pro-apoptotic proteins, and up-regulation of anti-apoptotic proteins, make cancer cells TRAIL-resistant. Therefore, many researchers have investigated strategies to overcome TRAIL resistance. In this review, we focus on protein regulation in relation to extrinsic apoptotic signaling pathways via ubiquitination. The ubiquitin proteasome system (UPS) is an important process in control of protein degradation and stabilization, and regulates proliferation and apoptosis in cancer cells. The level of ubiquitination of proteins is determined by the balance of E3 ubiquitin ligases and deubiquitinases (DUBs), which determine protein stability. Regulation of the UPS may be an attractive target for enhancement of TRAIL-induced apoptosis. Our review provides insight to increasing sensitivity to TRAIL-mediated apoptosis through control of post-translational protein expression. [BMB Reports 2019; 52(2): 119-126].
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Affiliation(s)
- Seon Min Woo
- Department of Immunology, School of Medicine, Keimyung University, Daegu 42601, Korea
| | - Taeg Kyu Kwon
- Department of Immunology, School of Medicine, Keimyung University, Daegu 42601, Korea
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41
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Fuchs O. Treatment of Lymphoid and Myeloid Malignancies by Immunomodulatory Drugs. Cardiovasc Hematol Disord Drug Targets 2019; 19:51-78. [PMID: 29788898 DOI: 10.2174/1871529x18666180522073855] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 05/05/2018] [Accepted: 05/14/2018] [Indexed: 06/08/2023]
Abstract
Thalidomide and its derivatives (lenalidomide, pomalidomide, avadomide, iberdomide hydrochoride, CC-885 and CC-90009) form the family of immunomodulatory drugs (IMiDs). Lenalidomide (CC5013, Revlimid®) was approved by the US FDA and the EMA for the treatment of multiple myeloma (MM) patients, low or intermediate-1 risk transfusion-dependent myelodysplastic syndrome (MDS) with chromosome 5q deletion [del(5q)] and relapsed and/or refractory mantle cell lymphoma following bortezomib. Lenalidomide has also been studied in clinical trials and has shown promising activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). Lenalidomide has anti-inflammatory effects and inhibits angiogenesis. Pomalidomide (CC4047, Imnovid® [EU], Pomalyst® [USA]) was approved for advanced MM insensitive to bortezomib and lenalidomide. Other IMiDs are in phases 1 and 2 of clinical trials. Cereblon (CRBN) seems to have an important role in IMiDs action in both lymphoid and myeloid hematological malignancies. Cereblon acts as the substrate receptor of a cullin-4 really interesting new gene (RING) E3 ubiquitin ligase CRL4CRBN. This E3 ubiquitin ligase in the absence of lenalidomide ubiquitinates CRBN itself and the other components of CRL4CRBN complex. Presence of lenalidomide changes specificity of CRL4CRBN which ubiquitinates two transcription factors, IKZF1 (Ikaros) and IKZF3 (Aiolos), and casein kinase 1α (CK1α) and marks them for degradation in proteasomes. Both these transcription factors (IKZF1 and IKZF3) stimulate proliferation of MM cells and inhibit T cells. Low CRBN level was connected with insensitivity of MM cells to lenalidomide. Lenalidomide decreases expression of protein argonaute-2, which binds to cereblon. Argonaute-2 seems to be an important drug target against IMiDs resistance in MM cells. Lenalidomide decreases also basigin and monocarboxylate transporter 1 in MM cells. MM cells with low expression of Ikaros, Aiolos and basigin are more sensitive to lenalidomide treatment. The CK1α gene (CSNK1A1) is located on 5q32 in commonly deleted region (CDR) in del(5q) MDS. Inhibition of CK1α sensitizes del(5q) MDS cells to lenalidomide. CK1α mediates also survival of malignant plasma cells in MM. Though, inhibition of CK1α is a potential novel therapy not only in del(5q) MDS but also in MM. High level of full length CRBN mRNA in mononuclear cells of bone marrow and of peripheral blood seems to be necessary for successful therapy of del(5q) MDS with lenalidomide. While transfusion independence (TI) after lenalidomide treatment is more than 60% in MDS patients with del(5q), only 25% TI and substantially shorter duration of response with occurrence of neutropenia and thrombocytopenia were achieved in lower risk MDS patients with normal karyotype treated with lenalidomide. Shortage of the biomarkers for lenalidomide response in these MDS patients is the main problem up to now.
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Affiliation(s)
- Ota Fuchs
- Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic
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42
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Wang L, Li H, Yang S, Ma W, Liu M, Guo S, Zhan J, Zhang H, Tsang SY, Zhang Z, Wang Z, Li X, Guo YD, Li X. Cyanidin-3-o-glucoside directly binds to ERα36 and inhibits EGFR-positive triple-negative breast cancer. Oncotarget 2018; 7:68864-68882. [PMID: 27655695 PMCID: PMC5356596 DOI: 10.18632/oncotarget.12025] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2015] [Accepted: 09/02/2016] [Indexed: 12/31/2022] Open
Abstract
Anthocyanins have been shown to inhibit the growth and metastatic potential of breast cancer (BC) cells. However, the effects of individual anthocyanins on triple-negative breast cancer (TNBC) have not yet been studied. In this study, we found that cyanidin-3-o-glucoside (Cy-3-glu) preferentially promotes the apoptosis of TNBC cells, which co-express the estrogen receptor alpha 36 (ERα36) and the epidermal growth factor receptor (EGFR). We demonstrated that Cy-3-glu directly binds to the ligand-binding domain (LBD) of ERα36, inhibits EGFR/AKT signaling, and promotes EGFR degradation. We also confirmed the therapeutic efficacy of Cy-3-glu on TNBC in the xenograft mouse model. Our data indicates that Cy-3-glu could be a novel preventive/therapeutic agent against the TNBC co-expressed ERα36/EGFR.
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Affiliation(s)
- Li Wang
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Haifeng Li
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Shiping Yang
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Wenqiang Ma
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Mei Liu
- Department of General Surgery, The 301th Hospital of PLA, Beijing, China
| | - Shichao Guo
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Jun Zhan
- Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, China
| | - Hongquan Zhang
- Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, China
| | - Suk Ying Tsang
- School of Life Sciences and State Key Laboratory of Agro-Biotechnology, Chinese University of Hong Kong, Hong Kong, China
| | - Ziding Zhang
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Zhaoyi Wang
- Beijing Shenogen Pharma Group, Beijing, China
| | - Xiru Li
- Department of General Surgery, The 301th Hospital of PLA, Beijing, China
| | - Yang-Dong Guo
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
| | - Xiangdong Li
- State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China
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43
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Li J, Basler M, Alvarez G, Brunner T, Kirk CJ, Groettrup M. Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation. Kidney Int 2018; 93:670-680. [DOI: 10.1016/j.kint.2017.09.023] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2017] [Revised: 09/13/2017] [Accepted: 09/21/2017] [Indexed: 11/16/2022]
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44
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Wang S, Xia W, Qiu M, Wang X, Jiang F, Yin R, Xu L. Atlas on substrate recognition subunits of CRL2 E3 ligases. Oncotarget 2018; 7:46707-46716. [PMID: 27107416 PMCID: PMC5216831 DOI: 10.18632/oncotarget.8732] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Accepted: 04/02/2016] [Indexed: 12/16/2022] Open
Abstract
The Cullin2-type ubiquitin ligases belong to the Cullin-Ring Ligase (CRL) family, which is a crucial determinant of proteasome-based degradation processes in eukaryotes. Because of the finding of von Hippel-Lindau tumor suppressor (VHL), the Cullin2-type ubiquitin ligases gain focusing in the research of many diseases, especially in tumors. These multisubunit enzymes are composed of the Ring finger protein, the Cullin2 scaffold protein, the Elongin B&C linker protein and the variant substrate recognition subunits (SRSs), among which the Cullin2 scaffold protein is the determining factor of the enzyme mechanism. Substrate recognition of Cullin2-type ubiquitin ligases depends on SRSs and results in the degradation of diseases associated substrates by intracellular signaling events. This review focuses on the diversity and the multifunctionality of SRSs in the Cullin2-type ubiquitin ligases, including VHL, LRR-1, FEM1b, PRAME and ZYG11. Recently, as more SRSs are being discovered and more aspects of substrate recognition have been illuminated, insight into the relationship between Cul2-dependent SRSs and substrates provides a new area for cancer research.
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Affiliation(s)
- Siwei Wang
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China.,The Fourth Clinical College of Nanjing Medical University, Nanjing, China
| | - Wenjia Xia
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China.,The Fourth Clinical College of Nanjing Medical University, Nanjing, China
| | - Mantang Qiu
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China.,The Fourth Clinical College of Nanjing Medical University, Nanjing, China
| | - Xin Wang
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China.,The Fourth Clinical College of Nanjing Medical University, Nanjing, China
| | - Feng Jiang
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China
| | - Rong Yin
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China
| | - Lin Xu
- Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital, Jiangsu Key Laboratory of Molecular and Translational Cancer Research, Cancer Institute of Jiangsu Province, Nanjing, China
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45
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Guo Q, Chen Y, Wu Y. Enhancing Apoptosis and Overcoming Resistance of Gemcitabine in Pancreatic Cancer with Bortezomib: A Role of Death-Associated Protein Kinase-Related Apoptosis-Inducing Protein Kinase 1. TUMORI JOURNAL 2018; 95:796-803. [DOI: 10.1177/030089160909500624] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Aims and Background To investigate the role of the apoptosis gene, DAP (death-associated protein) kinase-related apoptosis-inducing protein kinase 1 (DRAK1), which is involved in enhancing cell sensitivity and overcoming cell resistance to gemcitabine in pancreatic cancer cells by the proteasome inhibitor bortezomib. Methods Cultured human pancreatic cancer gemcitabine-sensitive cell lines (bxpc-3) and gemcitabine-resistant (panc-1) cell lines were divided into four groups: control, treatment with bortezomib, treatment with gemcitabine, and the two-drug combination. Expression of DRAK1 genes in each group was detected by using reverse transcription-polymerase chain reaction and western blot. Apoptosis in the pancreatic cancer cell lines was measured by flow cytometry. Results We found that the effects of growth inhibition and apoptosis of gemcitabine on both pancreatic cancer cell lines were enhanced by bortezomib. Treatment of panc-1 and bxpc-3 cells with bortezomib (100 nM) and gemcitabine (50 μg/ml and 0.05 μg/ml, respectively) induced an increase in the levels of DRAK1 mRNA compared with the control and single-agent treatment. Furthermore, immunblotting analysis in panc-1 but not bxpc-3 cells showed similar changes in the expression of DRAK1 protein produced by combination therapy. Conclusions Our results demonstrated that bortezomib enhanced cell sensitivity and overcame cell resistance to gemcitabine in pancreatic cancer cells, which may be attributed to DRAK1 induced by bortezomib and the combination with gemcitabine.
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Affiliation(s)
- Qingqu Guo
- Department of Surgery, Second Affiliated Hospital, College of Medicine, Zhejiang University, Cancer Institute of Zhejiang University, P.R. China
| | - Ying Chen
- Department of Surgery, Second Affiliated Hospital, College of Medicine, Zhejiang University, Cancer Institute of Zhejiang University, P.R. China
| | - Yulian Wu
- Department of Surgery, Second Affiliated Hospital, College of Medicine, Zhejiang University, Cancer Institute of Zhejiang University, P.R. China
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Transcriptome Analysis of Flounder (Paralichthys olivaceus) Gill in Response to Lymphocystis Disease Virus (LCDV) Infection: Novel Insights into Fish Defense Mechanisms. Int J Mol Sci 2018; 19:ijms19010160. [PMID: 29304016 PMCID: PMC5796109 DOI: 10.3390/ijms19010160] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 01/01/2018] [Accepted: 01/02/2018] [Indexed: 12/21/2022] Open
Abstract
Lymphocystis disease virus (LCDV) infection may induce a variety of host gene expression changes associated with disease development; however, our understanding of the molecular mechanisms underlying host-virus interactions is limited. In this study, RNA sequencing (RNA-seq) was employed to investigate differentially expressed genes (DEGs) in the gill of the flounder (Paralichthys olivaceus) at one week post LCDV infection. Transcriptome sequencing of the gill with and without LCDV infection was performed using the Illumina HiSeq 2500 platform. In total, RNA-seq analysis generated 193,225,170 clean reads aligned with 106,293 unigenes. Among them, 1812 genes were up-regulated and 1626 genes were down-regulated after LCDV infection. The DEGs related to cellular process and metabolism occupied the dominant position involved in the LCDV infection. A further function analysis demonstrated that the genes related to inflammation, the ubiquitin-proteasome pathway, cell proliferation, apoptosis, tumor formation, and anti-viral defense showed a differential expression. Several DEGs including β actin, toll-like receptors, cytokine-related genes, antiviral related genes, and apoptosis related genes were involved in LCDV entry and immune response. In addition, RNA-seq data was validated by quantitative real-time PCR. For the first time, the comprehensive gene expression study provided valuable insights into the host-pathogen interaction between flounder and LCDV.
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Temporal-Spatial Profiling of Pedunculopontine Galanin-Cholinergic Neurons in the Lactacystin Rat Model of Parkinson's Disease. Neurotox Res 2017; 34:16-31. [PMID: 29218504 DOI: 10.1007/s12640-017-9846-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Revised: 11/22/2017] [Accepted: 11/22/2017] [Indexed: 12/17/2022]
Abstract
Parkinson's disease (PD) is conventionally seen as resulting from single-system neurodegeneration affecting nigrostriatal dopaminergic neurons. However, accumulating evidence indicates multi-system degeneration and neurotransmitter deficiencies, including cholinergic neurons which degenerate in a brainstem nucleus, the pedunculopontine nucleus (PPN), resulting in motor and cognitive impairments. The neuropeptide galanin can inhibit cholinergic transmission, while being upregulated in degenerating brain regions associated with cognitive decline. Here we determined the temporal-spatial profile of progressive expression of endogenous galanin within degenerating cholinergic neurons, across the rostro-caudal axis of the PPN, by utilizing the lactacystin-induced rat model of PD. First, we show progressive neuronal death affecting nigral dopaminergic and PPN cholinergic neurons, reflecting that seen in PD patients, to facilitate use of this model for assessing the therapeutic potential of bioactive peptides. Next, stereological analyses of the lesioned brain hemisphere found that the number of PPN cholinergic neurons expressing galanin increased by 11%, compared to sham-lesioned controls, and increasing by a further 5% as the neurodegenerative process evolved. Galanin upregulation within cholinergic PPN neurons was most prevalent closest to the intra-nigral lesion site, suggesting that galanin upregulation in such neurons adapt intrinsically to neurodegeneration, to possibly neuroprotect. This is the first report on the extent and pattern of galanin expression in cholinergic neurons across distinct PPN subregions in both the intact rat CNS and lactacystin-lesioned rats. The findings pave the way for future work to target galanin signaling in the PPN, to determine the extent to which upregulated galanin expression could offer a viable treatment strategy for ameliorating PD symptoms associated with cholinergic degeneration.
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Soave CL, Guerin T, Liu J, Dou QP. Targeting the ubiquitin-proteasome system for cancer treatment: discovering novel inhibitors from nature and drug repurposing. Cancer Metastasis Rev 2017; 36:717-736. [PMID: 29047025 PMCID: PMC5722705 DOI: 10.1007/s10555-017-9705-x] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
In the past 15 years, the proteasome has been validated as an anti-cancer drug target and 20S proteasome inhibitors (such as bortezomib and carfilzomib) have been approved by the FDA for the treatment of multiple myeloma and some other liquid tumors. However, there are shortcomings of clinical proteasome inhibitors, including severe toxicity, drug resistance, and no effect in solid tumors. At the same time, extensive research has been conducted in the areas of natural compounds and old drug repositioning towards the goal of discovering effective, economical, low toxicity proteasome-inhibitory anti-cancer drugs. A variety of dietary polyphenols, medicinal molecules, metallic complexes, and metal-binding compounds have been found to be able to selectively inhibit tumor cellular proteasomes and induce apoptotic cell death in vitro and in vivo, supporting the clinical success of specific 20S proteasome inhibitors bortezomib and carfilzomib. Therefore, the discovery of natural proteasome inhibitors and researching old drugs with proteasome-inhibitory properties may provide an alternative strategy for improving the current status of cancer treatment and even prevention.
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Affiliation(s)
- Claire L Soave
- Barbara Ann Karmanos Cancer Institute, and Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, 540.1 HWCRC, 4100 John R Road, Detroit, MI, 48201-2013, USA
| | - Tracey Guerin
- Barbara Ann Karmanos Cancer Institute, and Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, 540.1 HWCRC, 4100 John R Road, Detroit, MI, 48201-2013, USA
| | - Jinbao Liu
- Key Laboratory of Protein Modification and Degradation, School of Basic Medical Sciences, and Affiliated Cancer Hospital & Institute, Guangzhou Medical University, Guangzhou, 511436, People's Republic of China
| | - Q Ping Dou
- Barbara Ann Karmanos Cancer Institute, and Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, 540.1 HWCRC, 4100 John R Road, Detroit, MI, 48201-2013, USA.
- Key Laboratory of Protein Modification and Degradation, School of Basic Medical Sciences, and Affiliated Cancer Hospital & Institute, Guangzhou Medical University, Guangzhou, 511436, People's Republic of China.
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Lv DW, Zhong J, Zhang K, Pandey A, Li R. Understanding Epstein-Barr Virus Life Cycle with Proteomics: A Temporal Analysis of Ubiquitination During Virus Reactivation. OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY 2017; 21:27-37. [PMID: 28271981 DOI: 10.1089/omi.2016.0158] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Epstein-Barr virus (EBV) is a human γ-herpesvirus associated with cancer, including Burkitt lymphoma, nasopharyngeal, and gastric carcinoma. EBV reactivation in latently infected B cells is essential for persistent infection whereby B cell receptor (BCR) activation is a physiologically relevant stimulus. Yet, a global view of BCR activation-regulated protein ubiquitination is lacking when EBV is actively replicating. We report here, for the first time, the long-term effects of IgG cross-linking-regulated protein ubiquitination and offer a basis for dissecting the cellular environment during the course of EBV lytic replication. Using the Akata-BX1 (EBV+) and Akata-4E3 (EBV-) Burkitt lymphoma cells, we monitored the dynamic changes in protein ubiquitination using quantitative proteomics. We observed temporal alterations in the level of ubiquitination at ∼150 sites in both EBV+ and EBV- B cells post-IgG cross-linking, compared with controls with no cross-linking. The majority of protein ubiquitination was downregulated. The upregulated ubiquitination events were associated with proteins involved in RNA processing. Among the downregulated ubiquitination events were proteins involved in apoptosis, ubiquitination, and DNA repair. These comparative and quantitative proteomic observations represent the first analysis on the effects of IgG cross-linking at later time points when the majority of EBV genes are expressed and the viral genome is actively being replicated. In all, these data enhance our understanding of mechanistic linkages connecting protein ubiquitination, RNA processing, apoptosis, and the EBV life cycle.
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Affiliation(s)
- Dong-Wen Lv
- 1 Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University , Richmond, Virginia
| | - Jun Zhong
- 2 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland
| | - Kun Zhang
- 1 Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University , Richmond, Virginia
| | - Akhilesh Pandey
- 2 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland.,3 Institute of Bioinformatics , International Technology Park, Bangalore, India .,4 Diana Helis Henry Medical Research Foundation , New Orleans, Louisiana
| | - Renfeng Li
- 1 Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University , Richmond, Virginia.,5 Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University , Richmond, Virginia.,6 Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia
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Disrupting CCT-β : β-tubulin selectively kills CCT-β overexpressed cancer cells through MAPKs activation. Cell Death Dis 2017; 8:e3052. [PMID: 28906489 PMCID: PMC5636972 DOI: 10.1038/cddis.2017.425] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2017] [Revised: 07/12/2017] [Accepted: 07/24/2017] [Indexed: 12/21/2022]
Abstract
We have previously demonstrated the ability of I-Trp to disrupt the protein–protein interaction of β-tubulin with chaperonin-containing TCP-1β (CCT-β). This caused more severe apoptosis in multidrug-resistant MES-SA/Dx5, compared to MES-SA, due to its higher CCT-β overexpression. In this study, we screened a panel of cancer cell lines, finding CCT-β overexpression in the triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell lines Colo205 and HCT116, and a gastric cancer cell line MKN-45. Thus, I-Trp killed these cancers with sub- to low-μM EC50, whereas it was non-toxic to MCF-10A. We then synthesized analogs of I-Trp and evaluated their cytotoxicity. Furthermore, apoptotic mechanism investigations revealed the activation of both protein ubiquitination/degradation and ER-associated protein degradation pathways. These pathways proceeded through activation of MAPKs at the onset of CCT-β : β-tubulin complex disruption. We thus establish an effective strategy to treat CCT-β overexpressed cancers by disrupting the CCT-β : β-tubulin complex.
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