|
1
|
Kumar A, Boradia VM, Mahajan A, Kumaran S, Raje M, Raje CI. Mycobacterium tuberculosis H37Rv enolase (Rv1023)- expression, characterization and effect of host dependent modifications on protein functionality. Biochimie 2023; 214:102-113. [PMID: 37385399 DOI: 10.1016/j.biochi.2023.06.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2022] [Revised: 06/21/2023] [Accepted: 06/27/2023] [Indexed: 07/01/2023]
Abstract
Mycobacterium tuberculosis enolase is an essential glycolytic enzyme that catalyzes the conversion of 2, phosphoglycerate (PGA) to phosphoenol pyruvate (PEP). It is also a crucial link between glycolysis and the tricarboxylic acid (TCA) pathway. The depletion of PEP has recently been associated with the emergence of non-replicating drug resistant bacteria. Enolase is also known to exhibit multiple alternate functions, such as promoting tissue invasion via its role as a plasminogen (Plg) receptor. In addition, proteomic studies have identified the presence of enolase in the Mtb degradosome and in biofilms. However, the precise role in these processes has not been elaborated. The enzyme was recently identified as a target for 2-amino thiazoles - a novel class of anti-mycobacterials. In vitro assays and characterization of this enzyme were unsuccessful due to the inability to obtain functional recombinant protein. In the present study, we report the expression and characterization of enolase using Mtb H37Ra as a host strain. Our study demonstrates that the enzyme activity and alternate functions of this protein are significantly impacted by the choice of expression host (Mtb H37Ra or E. coli). Detailed analysis of the protein from each source revealed subtle differences in the post-translational modifications. Lastly, our study confirms the role of enolase in Mtb biofilm formation and describes the potential for inhibiting this process.
Collapse
Affiliation(s)
- Ajay Kumar
- Department of Biotechnology National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India
| | - Vishant Mahendra Boradia
- Department of Biotechnology National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India
| | - Apurwa Mahajan
- Council of Scientific and Industrial Research -Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India
| | - S Kumaran
- Council of Scientific and Industrial Research -Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India
| | - Manoj Raje
- Council of Scientific and Industrial Research -Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India
| | - Chaaya Iyengar Raje
- Department of Biotechnology National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
| |
Collapse
|
|
2
|
Wu X, Xu M, Geng M, Chen S, Little PJ, Xu S, Weng J. Targeting protein modifications in metabolic diseases: molecular mechanisms and targeted therapies. Signal Transduct Target Ther 2023; 8:220. [PMID: 37244925 PMCID: PMC10224996 DOI: 10.1038/s41392-023-01439-y] [Citation(s) in RCA: 91] [Impact Index Per Article: 45.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Revised: 03/01/2023] [Accepted: 04/06/2023] [Indexed: 05/29/2023] Open
Abstract
The ever-increasing prevalence of noncommunicable diseases (NCDs) represents a major public health burden worldwide. The most common form of NCD is metabolic diseases, which affect people of all ages and usually manifest their pathobiology through life-threatening cardiovascular complications. A comprehensive understanding of the pathobiology of metabolic diseases will generate novel targets for improved therapies across the common metabolic spectrum. Protein posttranslational modification (PTM) is an important term that refers to biochemical modification of specific amino acid residues in target proteins, which immensely increases the functional diversity of the proteome. The range of PTMs includes phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, glycosylation, palmitoylation, myristoylation, prenylation, cholesterylation, glutathionylation, S-nitrosylation, sulfhydration, citrullination, ADP ribosylation, and several novel PTMs. Here, we offer a comprehensive review of PTMs and their roles in common metabolic diseases and pathological consequences, including diabetes, obesity, fatty liver diseases, hyperlipidemia, and atherosclerosis. Building upon this framework, we afford a through description of proteins and pathways involved in metabolic diseases by focusing on PTM-based protein modifications, showcase the pharmaceutical intervention of PTMs in preclinical studies and clinical trials, and offer future perspectives. Fundamental research defining the mechanisms whereby PTMs of proteins regulate metabolic diseases will open new avenues for therapeutic intervention.
Collapse
Affiliation(s)
- Xiumei Wu
- Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, Anhui, 230001, China
- Department of Endocrinology and Metabolism, Guangdong Provincial Key Laboratory of Diabetology, The Third Affiliated Hospital of Sun Yat-sen University, 510000, Guangzhou, China
| | - Mengyun Xu
- Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, Anhui, 230001, China
| | - Mengya Geng
- Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, Anhui, 230001, China
| | - Shuo Chen
- Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, Anhui, 230001, China
| | - Peter J Little
- School of Pharmacy, University of Queensland, Pharmacy Australia Centre of Excellence, Woolloongabba, QLD, 4102, Australia
- Sunshine Coast Health Institute and School of Health and Behavioural Sciences, University of the Sunshine Coast, Birtinya, QLD, 4575, Australia
| | - Suowen Xu
- Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, Anhui, 230001, China
| | - Jianping Weng
- Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, Anhui, 230001, China.
- Department of Endocrinology and Metabolism, Guangdong Provincial Key Laboratory of Diabetology, The Third Affiliated Hospital of Sun Yat-sen University, 510000, Guangzhou, China.
- Bengbu Medical College, Bengbu, 233000, China.
| |
Collapse
|
|
3
|
Su P, McGee JP, Durbin KR, Hollas MAR, Yang M, Neumann EK, Allen JL, Drown BS, Butun FA, Greer JB, Early BP, Fellers RT, Spraggins JM, Laskin J, Camarillo JM, Kafader JO, Kelleher NL. Highly multiplexed, label-free proteoform imaging of tissues by individual ion mass spectrometry. SCIENCE ADVANCES 2022; 8:eabp9929. [PMID: 35947651 PMCID: PMC9365283 DOI: 10.1126/sciadv.abp9929] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 06/24/2022] [Indexed: 05/25/2023]
Abstract
Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by fourfold compared to reported methods and reveals tissue localization of proteoforms at <80-μm spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization with ion detection using individual ion mass spectrometry. We demonstrate highly multiplexed proteoform imaging of human kidney, annotating 169 of 400 proteoforms of <70 kDa using top-down MS and a database lookup of ~1000 kidney candidate proteoforms, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.
Collapse
Affiliation(s)
- Pei Su
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - John P. McGee
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - Kenneth R. Durbin
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - Michael A. R. Hollas
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - Manxi Yang
- Department of Chemistry, Purdue University, West Lafayette, IN, USA
| | - Elizabeth K. Neumann
- Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN, USA
| | - Jamie L. Allen
- Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN, USA
| | - Bryon S. Drown
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | | | - Joseph B. Greer
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - Bryan P. Early
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - Ryan T. Fellers
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
| | - Jeffrey M. Spraggins
- Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN, USA
- Departments of Chemistry and Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA
| | - Julia Laskin
- Department of Chemistry, Purdue University, West Lafayette, IN, USA
| | - Jeannie M. Camarillo
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
- Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA
| | - Jared O. Kafader
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
- Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA
| | - Neil L. Kelleher
- Departments of Molecular Biosciences, Chemistry, and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
- Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA
- Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
| |
Collapse
|
|
4
|
Chen J, Zhang L, Sun Z, Li H, Li J, Xue X, Zhu Q, Dong B, Wang Y, Yang Y, Dong Y, Guo G, Jiang H, Zhang A, Zhang G, Hou Z, Li X, Yang JH. Open Search-Based Proteomics Reveals Widespread Tryptophan Modifications Associated with Hypoxia in Lung Cancer. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2022; 2022:2590198. [PMID: 35535361 PMCID: PMC9078843 DOI: 10.1155/2022/2590198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Accepted: 03/08/2022] [Indexed: 11/17/2022]
Abstract
The tryptophan residue has a large hydrophobic surface that plays a unique role in the folded protein conformation and functions. Tryptophan modifications are presumably to be readily detected in proteins due to the vulnerability of the indole structure to electrophilic attacks. In this study, we report a systematic identification of sequence variations at tryptophan, termed tryptophan variants, from the proteome of patients with nonsmall cell lung cancer (NSCLC). Using shotgun proteomics and a modified open search algorithm, 25 tryptophan variants on 2481 sites in over 858 proteins were identified. Among these, 6 tryptophan variants are previously identified, 15 are newly annotated, and 4 are still unknown, most of which are involved in the cascade of oxidation in the blood microparticle. Remarkably, Trp313 of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-oxidized whereas Trp16 and Trp38 of hemoglobin (HBB) were down-oxidized in NCSLC tissues. The results were further supported by an independent cohort of 103 lung adenocarcinoma samples, reflecting a negative feedback and potential detoxification mechanism against tumor glycolysis and hypoxia. Overall, the study reports a quick approach to explore tryptophan variants at the proteomic scale. Our findings highlight the predominant role of tryptophan oxidation in regulating the redox balance of cancer cells and its potential role as prognostic biomarker for patients with NSCLC.
Collapse
Affiliation(s)
- Jinfeng Chen
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Lei Zhang
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Zhao Sun
- Departments of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Hongyi Li
- Departments of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Jingyi Li
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Xinli Xue
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Qingqing Zhu
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Bowen Dong
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Yuanyuan Wang
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Yang Yang
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Yongqiang Dong
- Departments of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Guangyu Guo
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Hongqiang Jiang
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - An Zhang
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| | - Guoqing Zhang
- Departments of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Zhichao Hou
- Departments of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Xiangnan Li
- Departments of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Jing-Hua Yang
- Clinical Systems Biology Key Laboratories of Henan, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan, China
| |
Collapse
|
|
5
|
Johnson KR, Greguš M, Kostas JC, Ivanov AR. Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples. Anal Chem 2022; 94:704-713. [PMID: 34983182 PMCID: PMC8770592 DOI: 10.1021/acs.analchem.1c02929] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra-sensitive CE-MS/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ∼10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein- and especially peptide-level identifications of the evaluated CE-MS- and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ∼60 min, CE-MS/MS identified ∼2000 ± 53 proteins on average from a single injection of ∼8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2- and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.
Collapse
Affiliation(s)
- Kendall R Johnson
- Department of Chemistry and Chemical Biology, Northeastern University, Barnett Institute of Chemical and Biological Analysis, 360 Huntington Avenue, Boston, Massachusetts 02115, United States
| | - Michal Greguš
- Department of Chemistry and Chemical Biology, Northeastern University, Barnett Institute of Chemical and Biological Analysis, 360 Huntington Avenue, Boston, Massachusetts 02115, United States
| | - James C Kostas
- Department of Chemistry and Chemical Biology, Northeastern University, Barnett Institute of Chemical and Biological Analysis, 360 Huntington Avenue, Boston, Massachusetts 02115, United States
| | - Alexander R Ivanov
- Department of Chemistry and Chemical Biology, Northeastern University, Barnett Institute of Chemical and Biological Analysis, 360 Huntington Avenue, Boston, Massachusetts 02115, United States
| |
Collapse
|
|
6
|
Stepwise oxidations play key roles in the structural and functional regulations of DJ-1. Biochem J 2021; 478:3505-3525. [PMID: 34515295 DOI: 10.1042/bcj20210245] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 09/06/2021] [Accepted: 09/10/2021] [Indexed: 01/03/2023]
Abstract
DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46 is the most reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe (Ctag) that selectively reacts with redox-sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with adjacent resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. Mutants (C46A and C53A), not forming Cys46-Cys53 disulfide cross-linking, increased oxidation of Cys106 to sulfinic and sulfonic acids. Furthermore, we found that DJ-1 C46A mutant has distorted unstable structure identified by biochemical assay and employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike WT DJ-1. These findings suggest that all three Cys residues including Cys46-Cys53 disulfide cross-linking are required for maintaining the structural integrity, the regulation process and cellular function as an antioxidant protein. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.
Collapse
|
|
7
|
Do QT, Huang TE, Liu YC, Tai JH, Chen SH. Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-Based Workflow. J Proteome Res 2020; 20:624-633. [PMID: 32951420 DOI: 10.1021/acs.jproteome.0c00578] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Catechol estrogens (CEs) are known to be toxic metabolites and the initiators of the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins, but their cellular protein targets remain unexplored. Here, we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an improved click chemistry-based workflow. Multiple comparative proteomics composed of an experimental pair (probe versus solvent) and two control pairs (solvent versus solvent and probe versus solvent without enrichment) were studied using stable isotope dimethyl labeling. The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free linker coupled with on-bead digestion and redigestion of the proteins cleaved from the beads was shown to greatly improve the recovery and identification of CE-adducted peptides. A total of 310 protein targets and 40 adduction sites were repeatedly (n ≥ 2) identified with D/H (probe/solvent) ratio >4 versus only one identified with D/H >4 from the two control pairs, suggesting that our workflow imposes only a very low background. Meanwhile, multiple comparative D/H ratios revealed that CEs may downregulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CE-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data will provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.
Collapse
Affiliation(s)
- Quynh-Trang Do
- Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan
| | - Ting-En Huang
- Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan
| | - Yi-Chen Liu
- Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan
| | - Jung-Hsiang Tai
- Division of Infectious Diseases and Immunology, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
| | - Shu-Hui Chen
- Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan
| |
Collapse
|
|
8
|
How Do the Different Proteomic Strategies Cope with the Complexity of Biological Regulations in a Multi-Omic World? Critical Appraisal and Suggestions for Improvements. Proteomes 2020; 8:proteomes8030023. [PMID: 32899323 PMCID: PMC7564458 DOI: 10.3390/proteomes8030023] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2020] [Revised: 08/30/2020] [Accepted: 09/01/2020] [Indexed: 12/12/2022] Open
Abstract
In this second decade of the 21st century, we are lucky enough to have different types of proteomic analyses at our disposal. Furthermore, other functional omics such as transcriptomics have also undergone major developments, resulting in mature tools. However, choice equals questions, and the major question is how each proteomic strategy is fit for which purpose. The aim of this opinion paper is to reposition the various proteomic strategies in the frame of what is known in terms of biological regulations in order to shed light on the power, limitations, and paths for improvement for the different proteomic setups. This should help biologists to select the best-suited proteomic strategy for their purposes in order not to be driven by raw availability or fashion arguments but rather by the best fitness for purpose. In particular, knowing the limitations of the different proteomic strategies helps in interpreting the results correctly and in devising the validation experiments that should be made downstream of the proteomic analyses.
Collapse
|
|
9
|
Marcus K, Lelong C, Rabilloud T. What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World? Proteomes 2020; 8:proteomes8030017. [PMID: 32781532 PMCID: PMC7563651 DOI: 10.3390/proteomes8030017] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 08/02/2020] [Accepted: 08/04/2020] [Indexed: 02/07/2023] Open
Abstract
Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics—a thing of the past. Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy interface with many powerful biochemistry techniques (including western blotting). This paper reviews where and why two-dimensional gel electrophoresis-based proteomics can still be profitably used. It emerges that, rather than being a thing of the past, two-dimensional gel electrophoresis-based proteomics is still highly valuable for many studies. Thus, its use cannot be dismissed on simple fashion arguments and, as usual, in science, the tree is to be judged by the fruit.
Collapse
Affiliation(s)
- Katrin Marcus
- Medizinisches Proteom-Center, Medical Faculty & Medical Proteome Analysis, Center for Proteindiagnostics (PRODI) Ruhr-University Bochum Gesundheitscampus, 4 44801 Bochum, Germany;
| | - Cécile Lelong
- CBM UMR CNRS5249, Université Grenoble Alpes, CEA, CNRS, 17 rue des Martyrs, CEDEX 9, 38054 Grenoble, France;
| | - Thierry Rabilloud
- Laboratory of Chemistry and Biology of Metals, UMR 5249, Université Grenoble Alpes, CNRS, 38054 Grenoble, France
- Correspondence: ; Tel.: +33-438-783-212
| |
Collapse
|
|
10
|
Li YQ, Ngo A, Hoffmann P, Ferrante A, Hii CS. Regulation of endothelial cell survival and death by the MAP kinase/ERK kinase kinase 3 - glyceraldehyde-3-phosphate dehydrogenase signaling axis. Cell Signal 2019; 58:20-33. [DOI: 10.1016/j.cellsig.2019.03.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 03/01/2019] [Accepted: 03/01/2019] [Indexed: 02/06/2023]
|
|
11
|
Tramutola A, Sharma N, Barone E, Lanzillotta C, Castellani A, Iavarone F, Vincenzoni F, Castagnola M, Butterfield DA, Gaetani S, Cassano T, Perluigi M, Di Domenico F. Proteomic identification of altered protein O-GlcNAcylation in a triple transgenic mouse model of Alzheimer's disease. Biochim Biophys Acta Mol Basis Dis 2018; 1864:3309-3321. [PMID: 30031227 DOI: 10.1016/j.bbadis.2018.07.017] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2018] [Revised: 06/22/2018] [Accepted: 07/16/2018] [Indexed: 12/23/2022]
Abstract
PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.
Collapse
Affiliation(s)
- Antonella Tramutola
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy
| | - Nidhi Sharma
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy
| | - Eugenio Barone
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy; Universidad Autònoma de Chile, Instituto de Ciencias Biomédicas, Facultad de alud, Providencia, Santiago, Chile
| | - Chiara Lanzillotta
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy
| | - Andrea Castellani
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy
| | - Federica Iavarone
- Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy
| | - Federica Vincenzoni
- Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy
| | - Massimo Castagnola
- Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy
| | - D Allan Butterfield
- Department of Chemistry and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA
| | - Silvana Gaetani
- Department of Physiology and Pharmacology "V. Erspamer", Sapienza University of Rome, Rome, Italy
| | - Tommaso Cassano
- Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy
| | - Marzia Perluigi
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy
| | - Fabio Di Domenico
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy.
| |
Collapse
|
|
12
|
Choi JE, Lee JJ, Kang W, Kim HJ, Cho JH, Han PL, Lee KJ. Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications. Mol Cell Proteomics 2018; 17:1803-1823. [PMID: 29959188 PMCID: PMC6126396 DOI: 10.1074/mcp.ra118.000835] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2018] [Revised: 06/20/2018] [Indexed: 01/08/2023] Open
Abstract
Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.
Collapse
Affiliation(s)
- Jung-Eun Choi
- From the ‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, and
| | - Jae-Jin Lee
- From the ‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, and
| | - Wonmo Kang
- From the ‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, and
| | - Hyun Jung Kim
- From the ‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, and
| | - Jin-Hwan Cho
- From the ‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, and
| | - Pyung-Lim Han
- §Department of Brain and Cognitive Sciences, Ewha Womans University, Seoul, Korea 03760
| | - Kong-Joo Lee
- From the ‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, and
| |
Collapse
|
|
13
|
Kim HJ, Lee JJ, Cho JH, Jeong J, Park AY, Kang W, Lee KJ. Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1. J Biol Chem 2017; 292:12801-12812. [PMID: 28592492 DOI: 10.1074/jbc.m117.774992] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2017] [Revised: 06/06/2017] [Indexed: 12/22/2022] Open
Abstract
When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys132 was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys132 in hnRNP K is critical for this inhibition.
Collapse
Affiliation(s)
- Hee-Jung Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
| | - Jae-Jin Lee
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
| | - Jin-Hwan Cho
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
| | - Jaeho Jeong
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
| | - A Young Park
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
| | - Wonmo Kang
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
| | - Kong-Joo Lee
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea.
| |
Collapse
|
|
14
|
Post-translational modifications of FDA-approved plasma biomarkers in glioblastoma samples. PLoS One 2017; 12:e0177427. [PMID: 28493947 PMCID: PMC5426747 DOI: 10.1371/journal.pone.0177427] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Accepted: 04/27/2017] [Indexed: 01/08/2023] Open
Abstract
Liquid chromatography-tandem mass spectrometry was used to analyze plasma proteins of volunteers (control) and patients with glioblastoma multiform (GBM). A database search was pre-set with a variable post-translational modification (PTM): phosphorylation, acetylation or ubiquitination. There were no significant differences between the control and the GBM groups regarding the number of protein identifications, sequence coverage or number of PTMs. However, in GBM plasma, we unambiguously observed a decreased fraction in post-translationally modified peptides identified with high quality. The disease-specific PTM patterns were extracted and mapped to the set of FDA-approved plasma protein markers. Decreases of 46% and 24% in the number of acetylated and ubiquitinated peptides, respectively, were observed in the GBM samples. Significance of capturing disease-associated patterns of protein modifications was envisaged.
Collapse
|
|
15
|
Koelmel JP, Kroeger NM, Gill EL, Ulmer CZ, Bowden JA, Patterson RE, Yost RA, Garrett TJ. Expanding Lipidome Coverage Using LC-MS/MS Data-Dependent Acquisition with Automated Exclusion List Generation. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2017; 28:908-917. [PMID: 28265968 PMCID: PMC5408749 DOI: 10.1007/s13361-017-1608-0] [Citation(s) in RCA: 149] [Impact Index Per Article: 18.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/23/2016] [Revised: 01/13/2017] [Accepted: 01/15/2017] [Indexed: 05/19/2023]
Abstract
Untargeted omics analyses aim to comprehensively characterize biomolecules within a biological system. Changes in the presence or quantity of these biomolecules can indicate important biological perturbations, such as those caused by disease. With current technological advancements, the entire genome can now be sequenced; however, in the burgeoning fields of lipidomics, only a subset of lipids can be identified. The recent emergence of high resolution tandem mass spectrometry (HR-MS/MS), in combination with ultra-high performance liquid chromatography, has resulted in an increased coverage of the lipidome. Nevertheless, identifications from MS/MS are generally limited by the number of precursors that can be selected for fragmentation during chromatographic elution. Therefore, we developed the software IE-Omics to automate iterative exclusion (IE), where selected precursors using data-dependent topN analyses are excluded in sequential injections. In each sequential injection, unique precursors are fragmented until HR-MS/MS spectra of all ions above a user-defined intensity threshold are acquired. IE-Omics was applied to lipidomic analyses in Red Cross plasma and substantia nigra tissue. Coverage of the lipidome was drastically improved using IE. When applying IE-Omics to Red Cross plasma and substantia nigra lipid extracts in positive ion mode, 69% and 40% more molecular identifications were obtained, respectively. In addition, applying IE-Omics to a lipidomics workflow increased the coverage of trace species, including odd-chained and short-chained diacylglycerides and oxidized lipid species. By increasing the coverage of the lipidome, applying IE to a lipidomics workflow increases the probability of finding biomarkers and provides additional information for determining etiology of disease. Graphical Abstract ᅟ.
Collapse
Affiliation(s)
- Jeremy P Koelmel
- Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA
| | - Nicholas M Kroeger
- College of Engineering, University of Florida, 412 Newell Drive, Gainesville, FL, 32611, USA
| | - Emily L Gill
- Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA
| | - Candice Z Ulmer
- Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA
- Hollings Marine Laboratory, National Institute of Standards and Technology, 331 Ft. Johnson Rd., Charleston, SC, 29412, USA
| | - John A Bowden
- Hollings Marine Laboratory, National Institute of Standards and Technology, 331 Ft. Johnson Rd., Charleston, SC, 29412, USA
| | - Rainey E Patterson
- Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA
| | - Richard A Yost
- Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA
- Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, PO BOX 13303, 1395 Center Dr, Gainesville, FL, 32610, USA
| | - Timothy J Garrett
- Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA.
- Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, PO BOX 13303, 1395 Center Dr, Gainesville, FL, 32610, USA.
| |
Collapse
|
|
16
|
Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination. Sci Rep 2016; 6:34432. [PMID: 27703196 PMCID: PMC5050490 DOI: 10.1038/srep34432] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Accepted: 09/14/2016] [Indexed: 02/07/2023] Open
Abstract
Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.
Collapse
|
|
17
|
Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells. Blood 2016; 128:e32-42. [PMID: 27405778 DOI: 10.1182/blood-2016-05-714816] [Citation(s) in RCA: 154] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2016] [Accepted: 07/08/2016] [Indexed: 02/07/2023] Open
Abstract
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.
Collapse
|
|
18
|
Kwiatkowski M, Wurlitzer M, Krutilin A, Kiani P, Nimer R, Omidi M, Mannaa A, Bussmann T, Bartkowiak K, Kruber S, Uschold S, Steffen P, Lübberstedt J, Küpker N, Petersen H, Knecht R, Hansen NO, Zarrine-Afsar A, Robertson WD, Miller RJD, Schlüter H. Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization. J Proteomics 2016; 134:193-202. [PMID: 26778141 PMCID: PMC4767054 DOI: 10.1016/j.jprot.2015.12.029] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Revised: 12/22/2015] [Accepted: 12/31/2015] [Indexed: 12/30/2022]
Abstract
Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. Biological significance Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen.
Collapse
Affiliation(s)
- M Kwiatkowski
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - M Wurlitzer
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - A Krutilin
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - P Kiani
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - R Nimer
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - M Omidi
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - A Mannaa
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - T Bussmann
- Beiersdorf AG, Research & Development, Unnastrasse 48, 20245, Hamburg, Germany
| | - K Bartkowiak
- University Medical Centre Hamburg-Eppendorf, Department of Tumor Biology, Martinistraße 52, 20246 Hamburg, Germany
| | - S Kruber
- Max Planck Institute for the Structure and Dynamics of Matter, Atomically Resolved Dynamics Division, Luruper Chaussee 149, 22761 Hamburg, Germany
| | - S Uschold
- Max Planck Institute for the Structure and Dynamics of Matter, Atomically Resolved Dynamics Division, Luruper Chaussee 149, 22761 Hamburg, Germany
| | - P Steffen
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - J Lübberstedt
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - N Küpker
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany
| | - H Petersen
- University Medical Centre Hamburg-Eppendorf, Department of Otorhinolaryngology, Head and Neck Surgery and Oncology, Martinistraße 52, 20246 Hamburg, Germany
| | - R Knecht
- University Medical Centre Hamburg-Eppendorf, Department of Otorhinolaryngology, Head and Neck Surgery and Oncology, Martinistraße 52, 20246 Hamburg, Germany
| | - N O Hansen
- Max Planck Institute for the Structure and Dynamics of Matter, Atomically Resolved Dynamics Division, Luruper Chaussee 149, 22761 Hamburg, Germany
| | - A Zarrine-Afsar
- Techna Institute for the Advancement of Technology for Health, University Health Network, Toronto, ON M5G-1P5, Canada & Department of Medical Biophysics, University of Toronto, 101 College Street Suite 15-701, Toronto, ON M5G 1L7, Canada
| | - W D Robertson
- Max Planck Institute for the Structure and Dynamics of Matter, Atomically Resolved Dynamics Division, Luruper Chaussee 149, 22761 Hamburg, Germany
| | - R J D Miller
- Max Planck Institute for the Structure and Dynamics of Matter, Atomically Resolved Dynamics Division, Luruper Chaussee 149, 22761 Hamburg, Germany
| | - H Schlüter
- University Medical Centre Hamburg-Eppendorf, Institute for Clinical Chemistry, Department for Mass Spectrometric Proteomics, Martinistraße 52, 20246 Hamburg, Germany.
| |
Collapse
|
|
19
|
Phadke M, Krynetskaia N, Mishra A, Barrero C, Merali S, Gothe SA, Krynetskiy E. Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions. World J Biol Chem 2015; 6:366-78. [PMID: 26629320 PMCID: PMC4657119 DOI: 10.4331/wjbc.v6.i4.366] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2015] [Revised: 09/01/2015] [Accepted: 09/29/2015] [Indexed: 02/05/2023] Open
Abstract
AIM To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding. RESULTS Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.
Collapse
|
|
20
|
White MR, Garcin ED. The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein. WILEY INTERDISCIPLINARY REVIEWS-RNA 2015; 7:53-70. [PMID: 26564736 DOI: 10.1002/wrna.1315] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Revised: 09/30/2015] [Accepted: 10/01/2015] [Indexed: 01/26/2023]
Abstract
The glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single-stranded RNA viruses. A growing body of evidence supports GAPDH-RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA-binding sequences. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function. WIREs RNA 2016, 7:53-70. doi: 10.1002/wrna.1315 For further resources related to this article, please visit the WIREs website.
Collapse
Affiliation(s)
- Michael R White
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, USA
| | - Elsa D Garcin
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, USA
| |
Collapse
|
|
21
|
Shortreed MR, Wenger CD, Frey BL, Sheynkman GM, Scalf M, Keller MP, Attie AD, Smith LM. Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search. J Proteome Res 2015; 14:4714-20. [PMID: 26418581 PMCID: PMC4642219 DOI: 10.1021/acs.jproteome.5b00599] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
![]()
Bottom-up
proteomics database search algorithms used for peptide
identification cannot comprehensively identify post-translational
modifications (PTMs) in a single-pass because of high false discovery
rates (FDRs). A new approach to database searching enables global
PTM (G-PTM) identification by exclusively looking for curated PTMs,
thereby avoiding the FDR penalty experienced during conventional variable
modification searches. We identified over 2200 unique, high-confidence
modified peptides comprising 26 different PTM types in a single-pass
database search.
Collapse
Affiliation(s)
- Michael R Shortreed
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Craig D Wenger
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Brian L Frey
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Gloria M Sheynkman
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Mark Scalf
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Mark P Keller
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Alan D Attie
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| | - Lloyd M Smith
- Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States
| |
Collapse
|
|
22
|
Evidence for the dimerization-mediated catalysis of methionine sulfoxide reductase A from Clostridium oremlandii. PLoS One 2015; 10:e0131523. [PMID: 26107511 PMCID: PMC4479559 DOI: 10.1371/journal.pone.0131523] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2015] [Accepted: 06/02/2015] [Indexed: 11/30/2022] Open
Abstract
Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.
Collapse
|
|
23
|
Kim MK, Cho JH, Lee JJ, Son MH, Lee KJ. Proteomic analysis of INS-1 rat insulinoma cells: ER stress effects and the protective role of exenatide, a GLP-1 receptor agonist. PLoS One 2015; 10:e0120536. [PMID: 25793496 PMCID: PMC4368701 DOI: 10.1371/journal.pone.0120536] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2014] [Accepted: 01/23/2015] [Indexed: 01/01/2023] Open
Abstract
Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.
Collapse
Affiliation(s)
- Mi-Kyung Kim
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul 120–750, Republic of Korea
- Dong-A ST Research Institute, Yongin-si, Gyeonggi-do 446–905, Republic of Korea
| | - Jin-Hwan Cho
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul 120–750, Republic of Korea
| | - Jae-Jin Lee
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul 120–750, Republic of Korea
| | - Moon-Ho Son
- Dong-A ST Research Institute, Yongin-si, Gyeonggi-do 446–905, Republic of Korea
| | - Kong-Joo Lee
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul 120–750, Republic of Korea
- * E-mail:
| |
Collapse
|
|
24
|
Lee JJ, Park YS, Lee KJ. Hydrogen-deuterium exchange mass spectrometry for determining protein structural changes in drug discovery. Arch Pharm Res 2015; 38:1737-45. [PMID: 25743629 DOI: 10.1007/s12272-015-0584-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2015] [Accepted: 02/25/2015] [Indexed: 12/11/2022]
Abstract
Protein structures are dynamically changed in response to post-translational modifications, ligand or chemical binding, or protein-protein interactions. Understanding the structural changes that occur in proteins in response to potential candidate drugs is important for predicting the modes of action of drugs and their functions and regulations. Recent advances in hydrogen/deuterium exchange mass spectrometry (HDX-MS) have the potential to offer a tool for obtaining such understanding similarly to other biophysical techniques, such as X-ray crystallography and high resolution NMR. We present here, a review of basic concept and methodology of HDX-MS, how it is being applied for identifying the sites and structural changes in proteins following their interactions with other proteins and small molecules, and the potential of this tool to help in drug discovery.
Collapse
Affiliation(s)
- Jae-Jin Lee
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul, 120-750, Republic of Korea
| | - Yeon Seung Park
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul, 120-750, Republic of Korea
| | - Kong-Joo Lee
- Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul, 120-750, Republic of Korea.
| |
Collapse
|
|
25
|
Kim HJ, Ha S, Lee HY, Lee KJ. ROSics: chemistry and proteomics of cysteine modifications in redox biology. MASS SPECTROMETRY REVIEWS 2015; 34:184-208. [PMID: 24916017 PMCID: PMC4340047 DOI: 10.1002/mas.21430] [Citation(s) in RCA: 84] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/04/2012] [Revised: 04/30/2013] [Accepted: 11/20/2013] [Indexed: 05/29/2023]
Abstract
Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1-2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, "ROSics," for the science which describes the principles of mode of action of ROS at molecular levels.
Collapse
Affiliation(s)
- Hee-Jung Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans UniversitySeoul, 120-750, Korea
| | - Sura Ha
- Department of Chemistry, Korea Advanced Institute of Science & Technology (KAIST)Daejeon, 305-701, Korea
| | - Hee Yoon Lee
- Department of Chemistry, Korea Advanced Institute of Science & Technology (KAIST)Daejeon, 305-701, Korea
| | - Kong-Joo Lee
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans UniversitySeoul, 120-750, Korea
| |
Collapse
|
|
26
|
Na S, Paek E. Software eyes for protein post-translational modifications. MASS SPECTROMETRY REVIEWS 2015; 34:133-147. [PMID: 24889695 DOI: 10.1002/mas.21425] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/26/2012] [Revised: 07/18/2013] [Accepted: 11/20/2013] [Indexed: 06/03/2023]
Abstract
Post-translational modifications (PTMs) are critical to almost all aspects of complex processes of the cell. Identification of PTMs is one of the biggest challenges for proteomics, and there have been many computational studies for the analysis of PTMs from tandem mass spectrometry (MS/MS). Most early PTM identification studies have been performed by matching MS/MS data to protein databases, using database search tools, but they are prohibitively slow when a large number of PTMs is given as a search parameter. In this article, we present recent developments to search for more types of PTMs and to speed up the search, and discuss many computational issues and solutions in terms of identifying multiply modified peptides or searching for all possible modifications at once in unrestrictive mode. Apart from the most common type of PTMs involving covalent addition of functional groups to proteins, PTMs such as disulfide linkage require dedicated software for the analysis because they may involve cross-linking between two different parts of proteins. Finally, methods for identification of protein disulfide bonds are presented.
Collapse
Affiliation(s)
- Seungjin Na
- Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA, 92093; Center for Computational Mass Spectrometry, University of California, San Diego, La Jolla, CA, 92093
| | | |
Collapse
|
|
27
|
Zhang JY, Zhang F, Hong CQ, Giuliano AE, Cui XJ, Zhou GJ, Zhang GJ, Cui YK. Critical protein GAPDH and its regulatory mechanisms in cancer cells. Cancer Biol Med 2015; 12:10-22. [PMID: 25859407 PMCID: PMC4383849 DOI: 10.7497/j.issn.2095-3941.2014.0019] [Citation(s) in RCA: 101] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Accepted: 12/26/2014] [Indexed: 02/04/2023] Open
Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and posttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 (HIF-1), p53, nitric oxide (NO), and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications (PTMs) occurring in GAPDH in cancer cells result in new activities unrelated to the original glycolytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described.
Collapse
Affiliation(s)
- Jin-Ying Zhang
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Fan Zhang
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Chao-Qun Hong
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Armando E Giuliano
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Xiao-Jiang Cui
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Guang-Ji Zhou
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Guo-Jun Zhang
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Yu-Kun Cui
- 1 Department of Physiology, Guangdong Medical College, Dongguan 523808, China ; 2 Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis and Treatment, Cancer Hospital of Shantou University Medical College, Shantou 515041, China ; 3 Department of Surgery, Women's Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| |
Collapse
|
|
28
|
Kim MJ, Jeong J, Jeong J, Hwang KY, Lee KJ, Kim HY. Mechanism of 1-Cys type methionine sulfoxide reductase A regeneration by glutaredoxin. Biochem Biophys Res Commun 2015; 457:567-71. [PMID: 25600814 DOI: 10.1016/j.bbrc.2015.01.025] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Accepted: 01/08/2015] [Indexed: 10/24/2022]
Abstract
Glutaredoxin (Grx), a major redox regulator, can act as a reductant of methionine sulfoxide reductase A (MsrA). However, the biochemical mechanisms involved in MsrA activity regeneration by Grx remain largely unknown. In this study, we investigated the regeneration mechanism of 1-Cys type Clostridium oremlandii MsrA (cMsrA) lacking a resolving Cys residue in a Grx-dependent assay. Kinetic analysis showed that cMsrA could be reduced by both monothiol and dithiol Grxs as efficiently as by in vitro reductant dithiothreitol. Our data revealed that the catalytic Cys sulfenic acid intermediate is not glutathionylated in the presence of the substrate, and that Grx instead directly formed a complex with cMsrA. Mass spectrometry analysis identified a disulfide bond between the N-terminal catalytic Cys of the active site of Grx and the catalytic Cys of cMsrA. This mixed disulfide bond could be resolved by glutathione. Based on these findings, we propose a model for regeneration of 1-Cys type cMsrA by Grx that involves no glutathionylation on the catalytic Cys of cMsrA. This mechanism contrasts with that of the previously known 1-Cys type MsrB.
Collapse
Affiliation(s)
- Moon-Jung Kim
- Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea
| | - Jaeho Jeong
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
| | - Jihye Jeong
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
| | - Kwang Yeon Hwang
- Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701, Republic of Korea
| | - Kong-Joo Lee
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea.
| | - Hwa-Young Kim
- Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea.
| |
Collapse
|
|
29
|
Lee JJ, Ha S, Kim HJ, Ha HJ, Lee HY, Lee KJ. Sulfhydryl-specific probe for monitoring protein redox sensitivity. ACS Chem Biol 2014; 9:2883-94. [PMID: 25354229 DOI: 10.1021/cb500839j] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Reactive oxygen species (ROS) regulate various biological processes by modifying reactive cysteine residues in the proteins participating in the relevant signaling pathways. Identification of ROS target proteins requires specific reagents that identify ROS-sensitive cysteine sulfhydryls that differ from the known alkylating agents, iodoacetamide and N-ethylmaleimide, which react nonspecifically with oxidized cysteines including sulfenic and sulfinic acid. We designed and synthesized a novel reagent, methyl-3-nitro-4-(piperidin-1-ylsulfonyl)benzoate (NPSB-1), that selectively and specifically reacts with the sulfhydryl of cysteines in model compounds. We validated the specificity of this reagent by allowing it to react with recombinant proteins followed by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS), and mutant studies employed it to identify cellular proteins containing redox-sensitive cysteine residues. We also obtained proteins from cells treated with various concentrations of hydrogen peroxide, labeled them with biotinylated NPSB-1 (NPSB-B), pulled them down with streptavidin beads, and identified them with MS/MS. We grouped these proteins into four families: (1) those having reactive cysteine residues easily oxidized by hydrogen peroxide, (2) those with cysteines reactive only under mild oxidative stress, (3) those with cysteines reactive only after exposure to oxidative stress, and (4) those with cysteines that are reactive regardless of oxidative stress. These results confirm that NPSBs can serve as novel chemical probes for specifically capturing reactive cysteine residues and as powerful tools for measuring their oxidative sensitivity and can help to understand the function of cysteine modifications in ROS-mediated signaling pathways.
Collapse
Affiliation(s)
- Jae-Jin Lee
- Graduate
School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea 120-750
| | - Sura Ha
- Department of Chemistry, Korea Advanced Institute of Science & Technology (KAIST), Daejeon, Korea 305-701
| | - Hee-Jung Kim
- Graduate
School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea 120-750
| | - Hyun Joo Ha
- Graduate
School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea 120-750
| | - Hee-Yoon Lee
- Department of Chemistry, Korea Advanced Institute of Science & Technology (KAIST), Daejeon, Korea 305-701
| | - Kong-Joo Lee
- Graduate
School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea 120-750
| |
Collapse
|
|
30
|
White MR, Khan MM, Deredge D, Ross CR, Quintyn R, Zucconi BE, Wysocki VH, Wintrode PL, Wilson GM, Garcin ED. A dimer interface mutation in glyceraldehyde-3-phosphate dehydrogenase regulates its binding to AU-rich RNA. J Biol Chem 2014; 290:1770-85. [PMID: 25451934 DOI: 10.1074/jbc.m114.618165] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3'-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD(+) binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.
Collapse
Affiliation(s)
- Michael R White
- From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250
| | - Mohd M Khan
- From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250
| | - Daniel Deredge
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
| | - Christina R Ross
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and
| | - Royston Quintyn
- Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210
| | - Beth E Zucconi
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and
| | - Vicki H Wysocki
- Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210
| | - Patrick L Wintrode
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
| | - Gerald M Wilson
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and
| | - Elsa D Garcin
- From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250,
| |
Collapse
|
|
31
|
Warmoes MO, Locasale JW. Heterogeneity of glycolysis in cancers and therapeutic opportunities. Biochem Pharmacol 2014; 92:12-21. [PMID: 25093285 PMCID: PMC4254151 DOI: 10.1016/j.bcp.2014.07.019] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2014] [Revised: 07/21/2014] [Accepted: 07/21/2014] [Indexed: 12/19/2022]
Abstract
Upregulated glycolysis, both in normoxic and hypoxic environments, is a nearly universal trait of cancer cells. The enormous difference in glucose metabolism offers a target for therapeutic intervention with a potentially low toxicity profile. The past decade has seen a steep rise in the development and clinical assessment of small molecules that target glycolysis. The enzymes in glycolysis have a highly heterogeneous nature that allows for the different bioenergetic, biosynthetic, and signaling demands needed for various tissue functions. In cancers, these properties enable them to respond to the variable requirements of cell survival, proliferation and adaptation to nutrient availability. Heterogeneity in glycolysis occurs through the expression of different isoforms, posttranslational modifications that affect the kinetic and regulatory properties of the enzyme. In this review, we will explore this vast heterogeneity of glycolysis and discuss how this information might be exploited to better target glucose metabolism and offer possibilities for biomarker development.
Collapse
Affiliation(s)
- Marc O Warmoes
- Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States
| | - Jason W Locasale
- Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States.
| |
Collapse
|
|
32
|
Bell RAV, Smith JC, Storey KB. Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus. PeerJ 2014; 2:e634. [PMID: 25374779 PMCID: PMC4217184 DOI: 10.7717/peerj.634] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2014] [Accepted: 10/01/2014] [Indexed: 01/19/2023] Open
Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40-60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.
Collapse
Affiliation(s)
- Ryan A V Bell
- Department of Chemistry, Carleton University , Ottawa, Ontario , Canada
| | - Jeffrey C Smith
- Department of Chemistry, Carleton University , Ottawa, Ontario , Canada
| | - Kenneth B Storey
- Department of Chemistry, Carleton University , Ottawa, Ontario , Canada
| |
Collapse
|
|
33
|
Yuan ZF, Lin S, Molden RC, Garcia BA. Evaluation of proteomic search engines for the analysis of histone modifications. J Proteome Res 2014; 13:4470-8. [PMID: 25167464 PMCID: PMC4184451 DOI: 10.1021/pr5008015] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.
Collapse
Affiliation(s)
- Zuo-Fei Yuan
- Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania , 3400 Civic Center, Building 421, Philadelphia, Pennsylvania 19104, United States
| | | | | | | |
Collapse
|
|
34
|
Identification of Posttranslational Modifications in Peroxisome Proliferator-Activated Receptor γ Using Mass Spectrometry. PPAR Res 2014; 2014:468925. [PMID: 25061449 PMCID: PMC4099357 DOI: 10.1155/2014/468925] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Accepted: 05/19/2014] [Indexed: 01/15/2023] Open
Abstract
Posttranslational modification (PTM) of proteins is critical for various cellular processes. However, there are few studies examining PTMs in specific proteins using unbiased approaches. Here we report the attempt to identify the PTMs in peroxisome proliferator-activated receptor γ (PPARγ) proteins using our previously established PTM analysis system. In this study, we identified several PTMs in exogenously expressed PPARγ2 proteins from 293T cells as well as endogenous PPARγ1 proteins from a Caco-2 colon cancer cell line. The identified PTMs include phosphorylation of serine 112 and serine 81 in PPARγ2 and PPARγ1, respectively, both of which are well-known mitogen-activated protein kinase- (MAP kinase-) mediated PTMs in PPARγ proteins, thus confirming our experimental approach. Furthermore, previously unknown PTMs were also identified, demonstrating that this method can be applied to find previously unidentified PTMs in PPARγ proteins and other proteins including nuclear receptors.
Collapse
|
|
35
|
Kim HJ, Kim HJ, Jeong JE, Baek JY, Jeong J, Kim S, Kim YM, Kim Y, Nam JH, Huh SH, Seo J, Jin BK, Lee KJ. N-terminal truncated UCH-L1 prevents Parkinson's disease associated damage. PLoS One 2014; 9:e99654. [PMID: 24959670 PMCID: PMC4069018 DOI: 10.1371/journal.pone.0099654] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2014] [Accepted: 05/16/2014] [Indexed: 11/20/2022] Open
Abstract
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. In vivo experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H2O2, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD.
Collapse
Affiliation(s)
- Hee-Jung Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Hyun Jung Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Jae-Eun Jeong
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Jeong Yeob Baek
- Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, Kyung Hee University, Seoul, Korea
| | - Jaeho Jeong
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Sun Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Young-Mee Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Youhwa Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Jin Han Nam
- Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, Kyung Hee University, Seoul, Korea
| | - Sue Hee Huh
- Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, Kyung Hee University, Seoul, Korea
| | - Jawon Seo
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
| | - Byung Kwan Jin
- Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, Kyung Hee University, Seoul, Korea
- * E-mail: (KJL); (BKJ)
| | - Kong-Joo Lee
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea
- * E-mail: (KJL); (BKJ)
| |
Collapse
|
|
36
|
Bennike T, Birkelund S, Stensballe A, Andersen V. Biomarkers in inflammatory bowel diseases: Current status and proteomics identification strategies. World J Gastroenterol 2014; 20:3231-3244. [PMID: 24696607 PMCID: PMC3964395 DOI: 10.3748/wjg.v20.i12.3231] [Citation(s) in RCA: 84] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Revised: 01/13/2014] [Accepted: 02/20/2014] [Indexed: 02/06/2023] Open
Abstract
Unambiguous diagnosis of the two main forms of inflammatory bowel diseases (IBD): Ulcerative colitis (UC) and Crohn’s disease (CD), represents a challenge in the early stages of the diseases. The diagnosis may be established several years after the debut of symptoms. Hence, protein biomarkers for early and accurate diagnostic could help clinicians improve treatment of the individual patients. Moreover, the biomarkers could aid physicians to predict disease courses and in this way, identify patients in need of intensive treatment. Patients with low risk of disease flares may avoid treatment with medications with the concomitant risk of adverse events. In addition, identification of disease and course specific biomarker profiles can be used to identify biological pathways involved in the disease development and treatment. Knowledge of disease mechanisms in general can lead to improved future development of preventive and treatment strategies. Thus, the clinical use of a panel of biomarkers represents a diagnostic and prognostic tool of potentially great value. The technological development in recent years within proteomic research (determination and quantification of the complete protein content) has made the discovery of novel biomarkers feasible. Several IBD-associated protein biomarkers are known, but none have been successfully implemented in daily use to distinguish CD and UC patients. The intestinal tissue remains an obvious place to search for novel biomarkers, which blood, urine or stool later can be screened for. When considering the protein complexity encountered in intestinal biopsy-samples and the recent development within the field of mass spectrometry driven quantitative proteomics, a more thorough and accurate biomarker discovery endeavor could today be performed than ever before. In this review, we report the current status of the proteomics IBD biomarkers and discuss various emerging proteomic strategies for identifying and characterizing novel biomarkers, as well as suggesting future targets for analysis.
Collapse
|
|
37
|
Paleoproteomics explained to youngsters: how did the wedding of two-dimensional electrophoresis and protein sequencing spark proteomics on: let there be light. J Proteomics 2014; 107:5-12. [PMID: 24657497 DOI: 10.1016/j.jprot.2014.03.011] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2014] [Revised: 02/26/2014] [Accepted: 03/04/2014] [Indexed: 11/22/2022]
Abstract
UNLABELLED Taking the opportunity of the 20th anniversary of the word "proteomics", this young adult age is a good time to remember how proteomics came from enormous progress in protein separation and protein microanalysis techniques, and from the conjugation of these advances into a high performance and streamlined working setup. However, in the history of the almost three decades that encompass the first attempts to perform large scale analysis of proteins to the current high throughput proteomics that we can enjoy now, it is also interesting to underline and to recall how difficult the first decade was. Indeed when the word was cast, the battle was already won. This recollection is mostly devoted to the almost forgotten period where proteomics was being conceived and put to birth, as this collective scientific work will never appear when searched through the keyword "proteomics". BIOLOGICAL SIGNIFICANCE The significance of this manuscript is to recall and review the two decades that separated the first attempts of performing large scale analysis of proteins from the solid technical corpus that existed when the word "proteomics" was coined twenty years ago. This recollection is made within the scientific historical context of this decade, which also saw the blossoming of DNA cloning and sequencing. This article is part of a Special Issue entitled: 20 years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini , Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.
Collapse
|
|
38
|
Fields PA, Eurich C, Gao WL, Cela B. Changes in protein expression in the salt marsh mussel Geukensia demissa: evidence for a shift from anaerobic to aerobic metabolism during prolonged aerial exposure. ACTA ACUST UNITED AC 2014; 217:1601-12. [PMID: 24501137 DOI: 10.1242/jeb.101758] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
During aerial exposure (emersion), most sessile intertidal invertebrates experience cellular stress caused by hypoxia, and the amount and types of hypoxia-induced stress will differ as exposure time increases, likely leading to altered metabolic responses. We examined proteomic responses to increasing emersion times and decreasing recovery (immersion) times in the mussel Geukensia demissa, which occurs in salt marshes along the east coast of North America. Individuals are found above mean tide level, and can be emersed for over 18 h during spring tides. We acclimated mussels to full immersion at 15°C for 4 weeks, and compared changes in gill protein expression between groups of mussels that were continually immersed (control), were emersed for 6 h and immersed during recovery for 18 h (6E/18R), were emersed for 12 h and recovered for 12 h (12E/12R), or were emersed for 18 h with a 6 h recovery (18E/6R). We found clear differences in protein expression patterns among the treatments. Proteins associated with anaerobic fermentation increased in abundance in 6E/18R but not in 12E/12R or 18E/6R. Increases in oxidative stress proteins were most apparent in 12E/12R, and in 18E/6R changes in cytoskeletal protein expression predominated. We conclude that G. demissa alters its strategy for coping with emersion stress over time, relying on anaerobic metabolism for short- to medium-duration exposure, but switching to an air-gaping strategy for long-term exposure, which reduces hypoxia stress but may cause structural damage to gill tissue.
Collapse
Affiliation(s)
- Peter A Fields
- Biology Department, Franklin and Marshall College, Lancaster, PA 17603, USA
| | | | | | | |
Collapse
|
|
39
|
Sheokand N, Malhotra H, Kumar S, Tillu VA, Chauhan AS, Raje CI, Raje M. Moonlighting cell surface GAPDH recruits Apo Transferrin to effect iron egress from mammalian cells. J Cell Sci 2014; 127:4279-91. [DOI: 10.1242/jcs.154005] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Iron homeostasis is a tightly regulated process with precise control of its influx and egress from cells. Though mechanisms of its import into cells via iron carrier molecules are well characterized, iron export remains poorly understood. The current paradigm envisages unique functions associated with specialized macromolecules for its cellular import (transferrin receptors) or export (ferroportin) Earlier studies have revealed that, iron depleted cells recruit Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a moonlighting protein to their surface for internalization of the iron carrier holo transferrin (holo Tf). Here we report that under the contrary condition of intracellular iron excess, cells switch the isoform of GAPDH on their surface which now recruits iron free apo transferrin in close association with ferroportin to facilitate efflux of iron. Increased surface GAPDH expression synchronized with increased apo Tf binding and enhanced iron export from cells, a capability lost in GAPDH knockdown cells. These findings were confirmed in vivo utilizing a rodent model of iron overload. Besides identifying for the first time an apo transferrin receptor, our work uncovers two-way switching of multifunctional molecules for managing cellular micronutrient requirements.
Collapse
|
|
40
|
Abstract
Two-dimensional electrophoresis has nurtured the birth of proteomics. It is however no longer the exclusive setup used in proteomics, with the development of shotgun proteomics techniques that appear more fancy and fashionable nowadays.Nevertheless, 2D gel-based proteomics still has valuable features, and sometimes unique ones, which make it often an attractive choice when a proteomics strategy must be selected. These features are detailed in this chapter, as is the rationale for selecting or not 2D gel-based proteomics as a proteomic strategy.
Collapse
|
|
41
|
Xu G, Deglincerti A, Paige JS, Jaffrey SR. Profiling lysine ubiquitination by selective enrichment of ubiquitin remnant-containing peptides. Methods Mol Biol 2014; 1174:57-71. [PMID: 24947374 DOI: 10.1007/978-1-4939-0944-5_4] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Protein ubiquitination plays critical roles in many biological processes. However, functional studies of protein ubiquitination in eukaryotic cells are limited by the ability to identify protein ubiquitination sites. Unbiased high-throughput screening methods are necessary to discover novel ubiquitination sites that play important roles in cellular regulation. Here, we describe an immunopurification approach that enriches ubiquitin remnant-containing peptides to facilitate downstream mass spectrometry (MS) identification of lysine ubiquitination sites. This approach can be utilized to identify ubiquitination sites from proteins in a complex mixture.
Collapse
Affiliation(s)
- Guoqiang Xu
- Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China
| | | | | | | |
Collapse
|
|
42
|
|
|
43
|
Kim MK, Cho JH, Lee JJ, Cheong YH, Son MH, Lee KJ. Differential protective effects of exenatide, an agonist of GLP-1 receptor and Piragliatin, a glucokinase activator in beta cell response to streptozotocin-induced and endoplasmic reticulum stresses. PLoS One 2013; 8:e73340. [PMID: 24069189 PMCID: PMC3777936 DOI: 10.1371/journal.pone.0073340] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2013] [Accepted: 07/29/2013] [Indexed: 12/27/2022] Open
Abstract
Background Agonists of glucagon-like peptide-1 receptor (GLP-1R) and glucokinase activators (GKA) act as antidiabetic agents by their ability protect beta cells, and stimulate insulin secretion. Oxidative and endoplasmic reticulum (ER) stresses aggravate type 2 diabetes by causing beta cell loss. It was shown that GLP-1R agonists protect beta cells from oxidative and ER stresses. On the other hand, little is known regarding how GKAs protect beta cells. We hypothesized that GKAs protect beta cells by mechanisms distinct from those underlying GLP-1R agonist and tested our hypothesis by comparing the molecular effects of exenatide, a GLP-1R agonist, and piragliatin, a GKA, on INS-1 cells under oxidative and ER-induced stresses. Methods Beta cells were treated with streptozotocin (STZ) to induce oxidative stress and with palmitate or thapsigargin (Tg) to induce ER stress respectively, and the effects of exenatide and piragliatin on these cells were investigated by: a) characterizing the kinases involved employing specific kinase inhibitors, and b) by identifying the differentially regulated proteins in response to stresses with proteomic analysis. Results Exenatide protected INS-1 cells from both ER and STZ-induced death. In contrast, piragliatin rescued the cells only from STZ-induced stress. Akt activation by exenatide appeared to contribute to its protective effects of beta cells while enhanced glucose utilization was the contributing factor in the case of piragliatin. Also, exenatide, not piragliatin, blocked changes in proteins 14-3-3β, ε and θ, and preserved the 14-3-3θ levels under the ER stress. Isoform-specific modifications of 14-3-3, and the reduction of 14-3-3θ, commonly associated with beta cell death were assessed. Conclusions Exenatide and piragliatin exert distinct effects on beta cell survival and thus on type 2 diabetes. This study which confirmed our hypothesis is also the first to observe specific modulation of 14-3-3 isoform in stress-induced beta cell death associated with progressive deterioration of type 2 diabetes.
Collapse
Affiliation(s)
- Mi-Kyung Kim
- College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea
- Dong-A ST Research Institute, Yongin-si, Gyeonggi-do, Republic of Korea
| | - Jin-Hwan Cho
- College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea
| | - Jae-Jin Lee
- College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea
| | - Ye-Hwang Cheong
- Dong-A ST Research Institute, Yongin-si, Gyeonggi-do, Republic of Korea
| | - Moon-Ho Son
- Dong-A ST Research Institute, Yongin-si, Gyeonggi-do, Republic of Korea
| | - Kong-Joo Lee
- College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea
- * E-mail:
| |
Collapse
|
|
44
|
Nagai K, Morimoto K, Ikegami H, Kimura H, Yotsukura N. Investigation of proteomic profiles of lamina of Ecklonia kurome (Laminariales): homology-based cross-species protein identification and analysis of the post-translational processing of vanadium-dependent bromoperoxidases using MALDI-TOF/TOF. MARINE BIOTECHNOLOGY (NEW YORK, N.Y.) 2013; 15:487-98. [PMID: 23547002 DOI: 10.1007/s10126-013-9498-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/12/2012] [Accepted: 03/13/2013] [Indexed: 06/02/2023]
Abstract
Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4-7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.
Collapse
Affiliation(s)
- Kouhei Nagai
- Department of Genetic Engineering, Faculity of Biology-Oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649-6493, Japan
| | | | | | | | | |
Collapse
|
|
45
|
Yau Y, Leong RW, Zeng M, Wasinger VC. Proteomics and metabolomics in inflammatory bowel disease. J Gastroenterol Hepatol 2013; 28:1076-86. [PMID: 23489082 DOI: 10.1111/jgh.12193] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/01/2013] [Indexed: 12/16/2022]
Abstract
Genome-wide studies in inflammatory bowel disease (IBD) have allowed us to understand Crohn's disease and ulcerative colitis as forms of related autoinflammatory disorders that arise from a multitude of pathogenic origins. Proteomics and metabolomics are the offspring of genomics that possess unprecedented possibilities to characterize unknown pathogenic pathways. It has been about a decade since proteomics was first applied to IBD, and 5 years for metabolomics. These techniques have yielded novel and potentially important findings, but turning these results into beneficial patient outcomes remains challenging. This review recounts the history and context of clinical IBD developments before and after proteomics and metabolomics IBD in this field, discusses the challenges in consolidating high complexity data with physiological understanding, and provides an outlook on the emerging principles that will help interface the bioanalytical laboratory with IBD prognosis.
Collapse
Affiliation(s)
- Yunki Yau
- Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, The University of New South Wales, Sydney, Australia
| | | | | | | |
Collapse
|
|
46
|
Edgell TA, Rombauts LJF, Salamonsen LA. Assessing receptivity in the endometrium: the need for a rapid, non-invasive test. Reprod Biomed Online 2013; 27:486-96. [PMID: 23933033 DOI: 10.1016/j.rbmo.2013.05.014] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2012] [Revised: 04/30/2013] [Accepted: 05/30/2013] [Indexed: 01/11/2023]
Abstract
Successful implantation of an embryo into the uterus requires synchrony between the blastocyst and the endometrium. Endometrial preparedness, or receptivity, occurs only for a very short time during the mid-secretory phase of the menstrual cycle in fertile women. Failure to achieve receptivity results in infertility and is a rate-limiting step for IVF success. Frozen embryo transfer in non-stimulation cycles is already improving live birth rates. However, an important tool that is missing in the armoury of reproductive specialists is a means to rapidly assess endometrial receptivity, either during initial assessment or immediately prior to embryo transfer. The development of a wealth of omics technologies now opens the way for identifying potential receptivity markers, although validation of these is still a major issue. This review assesses the current state of the field and the requirements to proceed to a valid clinical test.
Collapse
Affiliation(s)
- Tracey A Edgell
- Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia.
| | | | | |
Collapse
|
|
47
|
Mikkat S, Kischstein T, Kreutzer M, Glocker MO. Mass spectrometric peptide analysis of 2DE-separated mouse spinal cord and rat hippocampus proteins suggests an NGxG motif of importance for in vivo deamidation. Electrophoresis 2013; 34:1610-8. [DOI: 10.1002/elps.201200682] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2012] [Revised: 02/11/2013] [Accepted: 02/20/2013] [Indexed: 12/16/2022]
Affiliation(s)
| | - Timo Kischstein
- Oscar Langendorff Institute of Physiology; University Medicine Rostock; Rostock; Germany
| | - Michael Kreutzer
- Proteome Center Rostock; University Medicine Rostock; Rostock; Germany
| | | |
Collapse
|
|
48
|
Kim MS, Jeong J, Jeong J, Shin DH, Lee KJ. Structure of Nm23-H1 under oxidative conditions. ACTA CRYSTALLOGRAPHICA SECTION D: BIOLOGICAL CRYSTALLOGRAPHY 2013; 69:669-80. [PMID: 23519676 DOI: 10.1107/s0907444913001194] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/26/2012] [Accepted: 01/11/2013] [Indexed: 01/06/2023]
Abstract
Nm23-H1/NDPK-A, a tumour metastasis suppressor, is a multifunctional housekeeping enzyme with nucleoside diphosphate kinase activity. Hexameric Nm23-H1 is required for suppression of tumour metastasis and it is dissociated into dimers under oxidative conditions. Here, the crystal structure of oxidized Nm23-H1 is presented. It reveals the formation of an intramolecular disulfide bond between Cys4 and Cys145 that triggers a large conformational change that destabilizes the hexameric state. The dependence of the dissociation dynamics on the H2O2 concentration was determined using hydrogen/deuterium-exchange experiments. The quaternary conformational change provides a suitable environment for the oxidation of Cys109 to sulfonic acid, as demonstrated by peptide sequencing using nanoUPLC-ESI-q-TOF tandem MS. From these and other data, it is proposed that the molecular and cellular functions of Nm23-H1 are regulated by a series of oxidative modifications coupled to its oligomeric states and that the modified cysteines are resolvable by NADPH-dependent reduction systems. These findings broaden the understanding of the complicated enzyme-regulatory mechanisms that operate under oxidative conditions.
Collapse
Affiliation(s)
- Mi-Sun Kim
- The Center for Cell Signaling and Drug Discovery Research, College of Pharmacy, Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea
| | | | | | | | | |
Collapse
|
|
49
|
Liddy KA, White MY, Cordwell SJ. Functional decorations: post-translational modifications and heart disease delineated by targeted proteomics. Genome Med 2013; 5:20. [PMID: 23445784 PMCID: PMC3706772 DOI: 10.1186/gm424] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The more than 300 currently identified post-translational modifications (PTMs) provides great scope for subtle or dramatic alteration of protein structure and function. Furthermore, the rapid and transient nature of many PTMs allows efficient signal transmission in response to internal and environmental stimuli. PTMs are predominantly added by enzymes, and the enzymes responsible (such as kinases) are thus attractive targets for therapeutic interventions. Modifications can be grouped according to their stability or transience (reversible versus irreversible): irreversible types (such as irreversible redox modifications or protein deamidation) are often associated with aging or tissue injury, whereas transient modifications are associated with signal propagation and regulation. This is particularly important in the setting of heart disease, which comprises a diverse range of acute (such as ischemia/reperfusion), chronic (such as heart failure, dilated cardiomyopathy) and genetic (such as hypertrophic cardiomyopathy) disease states, all of which have been associated with protein PTM. Recently the interplay between diverse PTMs has been suggested to also influence cellular function, with cooperation or competition for sites of modification possible. Here we discuss the utility of proteomics for examining PTMs in the context of the molecular mechanisms of heart disease.
Collapse
Affiliation(s)
- Kiersten A Liddy
- School of Molecular Bioscience, The University of Sydney, 2006 Sydney, Australia
| | - Melanie Y White
- School of Molecular Bioscience, The University of Sydney, 2006 Sydney, Australia ; Discipline of Pathology, School of Medical Sciences, The University of Sydney, 2006 Sydney, Australia
| | - Stuart J Cordwell
- School of Molecular Bioscience, The University of Sydney, 2006 Sydney, Australia ; Discipline of Pathology, School of Medical Sciences, The University of Sydney, 2006 Sydney, Australia
| |
Collapse
|
|
50
|
Seidler NW. Dynamic oligomeric properties. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 985:207-47. [PMID: 22851451 DOI: 10.1007/978-94-007-4716-6_7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
This chapter provides a foundation for further research into the relationship between dynamic oligomeric properties and functional diversity. The structural basis that underlies the conformational sub-states of the GAPDH oligomer is discussed. The issue of protein stability is given a thorough analysis, since it is well-established that the primary strategy for protein oligomerization is to stabilize conformation. Several factors that affect oligomerization are described, including chemical modification by synthetic reagents. The effects of native substrates and coenzymes are also discussed. The curious feature of chloride ions having a de-stabilizing effect on native GAPDH structure is described. Additionally, the role of adenine dinucleotides in tetramer-dimer equilibrium dynamics is suggested to be a major part of the physiological regulation of GAPDH structure and function. This chapter also contends that a vast amount of useful information can come from comparative analyses of diverse species, particularly regarding protein stability and subunit-subunit interaction. Lastly, the concept of domain exchange is introduced as a means of understanding the stabilization of dynamic oligomers, suggesting that inter-subunit contacts may also be a way of masking docking sites to other proteins.
Collapse
Affiliation(s)
- Norbert W Seidler
- Department of Biochemistry, Kansas City University of Medicine and Biosciences, Kansas City, MO, USA
| |
Collapse
|