ABSTRACT
The aim of this review is to present an overview of protein sulfation in the context of 'modificomics', i.e. post-translational modification-specific proteome research. In addition to a short introduction to the biology of protein sulfation (part 1), we will provide detailed discussion regarding (i) methods and tools for prediction of protein tyrosine sulfation sites (part 2), (ii) biochemical techniques used for protein sulfation analysis (part 3.1), and (iii) mass spectrometric strategies and methods applied to protein sulfation analysis (part 3.2). We will highlight strengths and limitations of different strategies and approaches (including references), providing a primer for newcomers to protein sulfation analysis.
Subject(s)
Protein Processing, Post-Translational , Proteins/metabolism , Sulfotransferases/metabolism , Tyrosine/analogs & derivatives , Animals , Electron Transport , Factor VIII/metabolism , Gas Chromatography-Mass Spectrometry/methods , Humans , Mass Spectrometry/methods , Membrane Glycoproteins/metabolism , Receptors, CCR5/metabolism , Sequence Analysis, Protein/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tyrosine/biosynthesis , von Willebrand Factor/metabolismABSTRACT
A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.
Subject(s)
Antigens, Surface/chemistry , Bacterial Outer Membrane Proteins/chemistry , CD4-Positive T-Lymphocytes/immunology , Lipoproteins/chemistry , Lyme Disease/immunology , Amino Acid Sequence , Antigens, Surface/genetics , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines , Clone Cells , HLA-DR Antigens/metabolism , HLA-DRB1 Chains , Humans , Lipoproteins/genetics , Lipoproteins/immunology , Lymphocyte Activation , Molecular Sequence Data , Peptide Fragments/metabolism , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , Synovial Fluid/chemistry , Synovial Fluid/cytology , Synovial Fluid/immunologySubject(s)
Endopeptidases/chemistry , Protease Inhibitors/metabolism , Sulfones/chemistry , Ubiquitin/chemistry , Vinyl Compounds/chemistry , Amino Acid Sequence , Endopeptidases/genetics , Endopeptidases/metabolism , Molecular Structure , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Processing, Post-Translational , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Specific ProteasesABSTRACT
A major cause of aging is thought to result from the cumulative effects of cell loss over time. In yeast, caloric restriction (CR) delays aging by activating the Sir2 deacetylase. Here we show that expression of mammalian Sir2 (SIRT1) is induced in CR rats as well as in human cells that are treated with serum from these animals. Insulin and insulin-like growth factor 1 (IGF-1) attenuated this response. SIRT1 deacetylates the DNA repair factor Ku70, causing it to sequester the proapoptotic factor Bax away from mitochondria, thereby inhibiting stress-induced apoptotic cell death. Thus, CR could extend life-span by inducing SIRT1 expression and promoting the long-term survival of irreplaceable cells.
Subject(s)
Apoptosis , Caloric Restriction , Cell Survival , Histone Deacetylases/metabolism , Proto-Oncogene Proteins c-bcl-2 , Sirtuins/metabolism , Acetylation , Adipose Tissue/metabolism , Alleles , Animals , Antigens, Nuclear/metabolism , Cell Line , DNA-Binding Proteins/metabolism , Histone Deacetylases/genetics , Humans , Insulin/metabolism , Insulin/pharmacology , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/pharmacology , Kidney/metabolism , Ku Autoantigen , Liver/metabolism , Male , Mitochondria/metabolism , Mutation , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering , Rats , Rats, Inbred F344 , Sirtuin 1 , Sirtuins/genetics , bcl-2-Associated X ProteinABSTRACT
Apoptosis is a key tumor suppression mechanism that can be initiated by activation of the proapoptotic factor Bax. The Ku70 DNA end-joining protein has recently been shown to suppress apoptosis by sequestering Bax from mitochondria. The mechanism by which Bax is regulated remains unknown. Here, we identify eight lysines in Ku70 that are targets for acetylation in vivo. Five of these, K539, K542, K544, K533, and K556, lie in the C-terminal linker domain of Ku70 adjacent to the Bax interaction domain. We show that CBP and PCAF efficiently acetylate K542 in vitro and associate with Ku70 in vivo. Mimicking acetylation of K539 or K542 or treating cells with deacetylase inhibitors abolishes the ability of Ku70 to suppress Bax-mediated apoptosis. We demonstrate that increased acetylation of Ku70 disrupts the Ku70-Bax interaction and coincides with cytoplasmic accumulation of CBP. These results shed light on the role of acetyltransferases as tumor suppressors.