ABSTRACT
In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.
Subject(s)
Exons , Proteome/analysis , RNA Processing, Post-Transcriptional , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Humans , Multiprotein Complexes/analysis , RNA Precursors/metabolism , RNA SplicingABSTRACT
Aberrant translational repression is a feature of multiple neurodegenerative diseases. The association between disease-linked proteins and stress granules further implicates impaired stress responses in neurodegeneration. However, our knowledge of the proteins that evade translational repression is incomplete. It is also unclear whether disease-linked proteins influence the proteome under conditions of translational repression. To address these questions, a quantitative proteomics approach was used to identify proteins that evade stress-induced translational repression in arsenite-treated cells expressing either wild-type or amyotrophic lateral sclerosis (ALS)-linked mutant FUS. This study revealed hundreds of proteins that are actively synthesized during stress-induced translational repression, irrespective of FUS genotype. In addition to proteins involved in RNA- and protein-processing, proteins associated with neurodegenerative diseases such as ALS were also actively synthesized during stress. Protein synthesis under stress was largely unperturbed by mutant FUS, although several proteins were found to be differentially expressed between mutant and control cells. One protein in particular, COPBI, was downregulated in mutant FUS-expressing cells under stress. COPBI is the beta subunit of the coat protein I (COPI), which is involved in Golgi to endoplasmic reticulum (ER) retrograde transport. Further investigation revealed reduced levels of other COPI subunit proteins and defects in COPBI-relatedprocesses in cells expressing mutant FUS. Even in the absence of stress, COPBI localization was altered in primary and human stem cell-derived neurons expressing ALS-linked FUS variants. Our results suggest that Golgi to ER retrograde transport may be important under conditions of stress and is perturbed upon the expression of disease-linked proteins such as FUS.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Motor Neurons/metabolism , Protein Biosynthesis , RNA-Binding Protein FUS/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Arsenites/pharmacology , Cell Line, Tumor , Coat Protein Complex I/metabolism , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Endoplasmic Reticulum/drug effects , Golgi Apparatus/drug effects , Humans , Mice , Motor Neurons/drug effects , Mutation , Protein Biosynthesis/drug effects , Proteomics , RNA-Binding Protein FUS/metabolismABSTRACT
Presentation of antigenic peptides on MHC-II molecules is essential for tolerance to self and for initiation of immune responses against foreign antigens. DO (HLA-DO in humans, H2-O in mice) is a nonclassical MHC-II protein that has been implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role of DO in selecting MHC-II epitopes, but previous studies examining the effect of DO on presentation of selected CD4 T cell epitopes have been contradictory. To understand how DO modulates MHC-II antigen presentation, we characterized the full spectrum of peptides presented by MHC-II molecules expressed by DO-sufficient and DO-deficient antigen-presenting cells in vivo and in vitro using quantitative mass spectrometry approaches. We found that DO controlled the diversity of the presented peptide repertoire, with a subset of peptides presented only when DO was expressed. Antigen-presenting cells express another nonclassical MHC-II protein, DM, which acts as a peptide editor by preferentially catalyzing the exchange of less stable MHC-II peptide complexes, and which is inhibited when bound to DO. Peptides presented uniquely in the presence of DO were sensitive to DM-mediated exchange, suggesting that decreased DM editing was responsible for the increased diversity. DO-deficient mice mounted CD4 T cell responses against wild-type antigen-presenting cells, but not vice versa, indicating that DO-dependent alterations in the MHC-II peptidome could be recognized by circulating T cells. These data suggest that cell-specific and regulated expression of HLA-DO serves to fine-tune MHC-II peptidomes, in order to enhance self-tolerance to a wide spectrum of epitopes while allowing focused presentation of immunodominant epitopes during an immune response.
Subject(s)
HLA-D Antigens/genetics , Histocompatibility Antigens Class II/chemistry , Peptides/metabolism , Animals , Antigen Presentation , Cell Line , Epitopes, T-Lymphocyte/metabolism , HLA-D Antigens/chemistry , Histocompatibility Antigens Class II/genetics , Humans , Immunodominant Epitopes/metabolism , Mice , Mice, Inbred C57BLABSTRACT
Human herpes virus 6B (HHV-6B) is a widespread virus that infects most people early in infancy and establishes a chronic life-long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV-6B, but antigenic targets and functional characteristics of the CD4 T-cell response are poorly understood. We identified 25 naturally processed MHC-II peptides, derived from six different HHV-6B proteins, and showed that they were recognized by CD4 T-cell responses in HLA-matched donors. The peptides were identified by mass spectrometry after elution from HLA-DR molecules isolated from HHV-6B-infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T-cell responses in vitro. T-cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3+ CD4+ , produced IFN-γ, TNF-α, and low levels of IL-2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide-pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long-term control of HHV-6B infection.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Herpesvirus 6, Human/physiology , Roseolovirus Infections/immunology , Antigen Presentation , Antigens, Viral/metabolism , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Cytotoxicity, Immunologic , Epitope Mapping , HLA-DR3 Antigen/metabolism , Humans , Immunodominant Epitopes , Interferon-gamma/metabolism , Lymphocyte Activation , Mass Spectrometry , Peptides/metabolismABSTRACT
Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.
Subject(s)
Brain/metabolism , Huntingtin Protein/biosynthesis , Huntington Disease/genetics , Trinucleotide Repeat Expansion/genetics , Adult , Age of Onset , Alleles , Autopsy , Brain/pathology , Female , Gene Expression Regulation , Humans , Huntingtin Protein/genetics , Huntington Disease/pathology , Male , RNA, Messenger/biosynthesisABSTRACT
Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Biomimetic Materials/pharmacology , Codon, Nonsense/drug effects , Furans/pharmacology , Nucleosides/pharmacology , Ovarian Neoplasms/drug therapy , Pyrimidine Nucleosides/pharmacology , Tumor Suppressor Protein p53/agonists , Animals , Antimetabolites, Antineoplastic/chemical synthesis , Antimetabolites, Antineoplastic/metabolism , Apoptosis/drug effects , Biomimetic Materials/chemical synthesis , Biomimetic Materials/metabolism , Cell Line, Tumor , Female , Furans/chemical synthesis , Furans/metabolism , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Mice , Mice, Nude , Nucleosides/chemical synthesis , Nucleosides/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Protein Biosynthesis , Pyrimidine Nucleosides/chemical synthesis , Pyrimidine Nucleosides/metabolism , Signal Transduction , Transcriptional Activation , Tumor Burden/drug effects , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.
Subject(s)
Codon, Nonsense/genetics , Oxadiazoles/pharmacology , RNA, Transfer/genetics , Ribosomes/drug effects , HEK293 Cells , Humans , Protein Biosynthesis/drug effects , RNA Stability/drug effects , RNA, Transfer/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Transcription, Genetic/drug effectsABSTRACT
Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis.
Subject(s)
Bacterial Proteins/metabolism , Lipid Metabolism/physiology , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Membrane Proteins/metabolism , Mycobacterium/metabolism , Mycobacterium/ultrastructureABSTRACT
Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization of the mRNA. Readthrough of a PTC allows ribosomal A-site insertion of a near-cognate tRNA, leading to synthesis of a full-length protein from otherwise defective mRNA. To understand the mechanism of such nonsense suppression, we developed a yeast system that allows purification and sequence analysis of full-length readthrough products arising as a consequence of endogenous readthrough or the compromised termination fidelity attributable to the loss of Upf (up-frameshift) factors, defective release factors, or the presence of the aminoglycoside gentamicin. Unlike classical "wobble" models, our analyses showed that three of four possible near-cognate tRNAs could mispair at position 1 or 3 of nonsense codons and that, irrespective of whether readthrough is endogenous or induced, the same sets of amino acids are inserted. We identified the insertion of Gln, Tyr, and Lys at UAA and UAG, whereas Trp, Arg, and Cys were inserted at UGA, and the frequency of insertion of individual amino acids was distinct for specific nonsense codons and readthrough-inducing agents. Our analysis suggests that the use of genetic or chemical means to increase readthrough does not promote novel or alternative mispairing events; rather, readthrough effectors cause quantitative enhancement of endogenous mistranslation events. Knowledge of the amino acids incorporated during readthrough not only elucidates the decoding process but also may allow predictions of the functionality of readthrough protein products.
Subject(s)
Base Pairing , Codon , RNA, Transfer/geneticsABSTRACT
Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification (Francisella), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.
Subject(s)
Acyltransferases/metabolism , Bacterial Proteins/metabolism , Francisella/metabolism , Lipopolysaccharides/metabolism , Acyltransferases/classification , Acyltransferases/genetics , Animals , Bacterial Proteins/genetics , Biological Evolution , Body Temperature , Cell Membrane Permeability/genetics , Francisella/genetics , Francisella/pathogenicity , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/physiopathology , Host-Pathogen Interactions , Kinetics , Lipid A/chemistry , Lipid A/metabolism , Lipopolysaccharides/chemistry , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Microbial Viability , Mutation , Phylogeny , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature , Virulence/geneticsABSTRACT
Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
Subject(s)
Blood Proteins/chemistry , Tandem Mass Spectrometry/methods , Blood Proteins/isolation & purification , Blood Proteins/metabolism , Humans , Immunoprecipitation , Peptide Mapping , Proteomics , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/standardsABSTRACT
Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.
Subject(s)
Glycoproteins/metabolism , Hexosyltransferases/metabolism , Membrane Proteins/metabolism , Proteomics , Glycosylation , HEK293 Cells , HSP70 Heat-Shock Proteins/metabolism , HeLa Cells , HumansABSTRACT
Upon inhibition of respiration, which occurs in hypoxic or nitric oxide-containing host microenvironments, Mycobacterium tuberculosis (Mtb) adopts a non-replicating "quiescent" state and becomes relatively unresponsive to antibiotic treatment. We used comprehensive mutant fitness analysis to identify regulatory and metabolic pathways that are essential for the survival of quiescent Mtb. This genetic study identified a protein acetyltransferase (Mt-Pat/Rv0998) that promoted survival and altered the flux of carbon from oxidative to reductive tricarboxylic acid (TCA) reactions. Reductive TCA requires malate dehydrogenase (MDH) and maintains the redox state of the NAD+/NADH pool. Genetic or chemical inhibition of MDH resulted in rapid cell death in both hypoxic cultures and in murine lung. These phenotypic data, in conjunction with significant structural differences between human and mycobacterial MDH enzymes that could be exploited for drug development, suggest a new strategy for eradicating quiescent bacteria.
Subject(s)
Hypoxia/metabolism , Lysine Acetyltransferases/metabolism , Mycobacterium tuberculosis/enzymology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Hypoxia/drug therapy , Hypoxia/genetics , Lysine Acetyltransferases/antagonists & inhibitors , Lysine Acetyltransferases/genetics , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolismABSTRACT
Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle.
Subject(s)
Cross-Linking Reagents/metabolism , DNA Topoisomerases, Type I/metabolism , DNA, Bacterial/metabolism , Amino Acid Sequence , Base Sequence , DNA Topoisomerases, Type I/chemistry , DNA, Superhelical/metabolism , Escherichia coli/enzymology , Lysine/metabolism , Micrococcal Nuclease/metabolism , Oligonucleotides/metabolism , Oxidation-Reduction , Peptides/chemistry , Peptides/metabolism , Substrate SpecificityABSTRACT
Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins.
Subject(s)
RNA-Binding Proteins/metabolism , Tauopathies/metabolism , Tauopathies/physiopathology , tau Proteins/metabolism , tau Proteins/toxicity , Animals , Brain/metabolism , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Dendrites/drug effects , Dendrites/metabolism , Mice, Inbred C57BL , Protein Binding/drug effects , Protein Folding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Stability/drug effects , Protein Synthesis Inhibitors/pharmacology , Protein Transport/drug effects , Proteome/metabolism , Solubility , T-Cell Intracellular Antigen-1ABSTRACT
Calcium signalling is important for differentiation-dependent gene expression, but is also involved in other cellular functions. Therefore, mechanisms must exist to distinguish calcium signalling relevant to differentiation. Calcineurin is a calcium-regulated phosphatase that is required for myogenic gene expression and skeletal muscle differentiation. Here, we demonstrate that inhibition of calcineurin blocks chromatin remodelling and that the Brg1 ATPase of the SWI/SNF chromatin remodelling enzyme, which is required for the activation of myogenic gene expression, is a calcineurin substrate. Furthermore, we identify the calcium-regulated classical protein kinase C ß (PKCß) as a repressor of myogenesis and as the enzyme that opposes calcineurin function. Replacement of endogenous Brg1 with a phosphomimetic mutant in primary myoblasts inhibits myogenesis, whereas replacement with a non-phosphorylatable mutant allows myogenesis despite inhibition of calcineurin signalling, demonstrating the functionality of calcineurin/PKC-modified residues. Thus, the Brg1 chromatin remodelling enzyme integrates two antagonistic calcium-dependent signalling pathways that control myogenic differentiation.
Subject(s)
Calcineurin/metabolism , Calcium Signaling , Chromatin Assembly and Disassembly , DNA Helicases/metabolism , Muscle Development , Nuclear Proteins/metabolism , Protein Kinase C beta/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Cell Line , Female , Male , Mice , Molecular Sequence Data , Muscle, Skeletal/cytologyABSTRACT
MALDI-TOF continues to be an important tool for many proteomic studies. Recently, a new rationally designed matrix 4-chloro-alpha-cyanocinnamic acid was introduced, which is reported to have superior performance as compared with the "gold standard" alpha-cyano-4-hydroxycinnamic acid (CHCA). In this study, the performance of this new matrix, using the Shimadzu Biotech Axima TOF(2) (Shimadzu Biotech, Manchester, UK), was investigated. The overall sequence coverage as well as sensitivity of this matrix were compared with CHCA using standard protein tryptic digests. The performance of this matrix with labile peptides, such as phosphopeptides and 4-sulfophenyl isothiocynate-derivatized peptides, to facilitate de novo sequencing was also explored. This matrix was found to be better performing than CHCA in overall sensitivity and showed better sequence coverage at low-digest levels, partly as a result of less of a bias for arginine-containing peptides. It also showed as much as a tenfold improvement in sensitivity with labile peptides on standard stainless-steel targets. In addition, as a result of the much cooler nature of this matrix, labile peptides are readily seen intact with much less fragmentation in mass spectrometry (MS) mode. This matrix was also evaluated in the MS/MS fragmentation modes of post-source decay (PSD) and collisional-induced dissociation (CID). It was found that fragmentation occurs readily in CID, however as a result of the very cool nature of this new matrix, the PSD fragments were quite weak. This matrix promises to be an important addition to the already extensive array of MALDI matrices.
Subject(s)
Cinnamates/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Animals , Cattle , Chickens , Fibrinogen/analysis , Fibrinogen/chemistry , Molecular Sequence Data , Ovalbumin/chemistry , Ovalbumin/metabolism , Phosphopeptides/analysis , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Trypsin/metabolismABSTRACT
Plasma membranes are organized into functional domains both by liquid-ordered packing into "lipid rafts," structures that resist Triton extraction, and by attachments to underlying cytoskeletal proteins in assemblies called "membrane skeletons." Although the actin cytoskeleton is implicated in many lipid raft-mediated signaling processes, little is known about the biochemical basis for actin involvement. We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H; d approximately 1.16 g/ml). By using matrix-assisted laser desorption/ionization time of flight and tandem mass spectrometry, we identified 19 major DRM-H proteins. Membrane skeleton proteins include fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, alpha-actinin 1, alpha-actinin 4, vimentin, and the F-actin-binding protein, supervillin. Other DRM-H components include lipid raft-associated integral membrane proteins (stomatin, flotillin 1, and flotillin 2), extracellular surface-bound and glycophosphatidylinositol-anchored proteins (IgM, membrane-type 6 matrix metalloproteinase), and intracellular dually acylated signaling proteins (Lyn kinase, Galpha(i-2)). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Galpha(i-2) also resist extraction with 0.1 m octyl glucoside. Supervillin, myosin-IG, and myosin-IIA resist extraction with 0.1 m sodium carbonate, a treatment that removes all detectable actin, suggesting that these cytoskeletal proteins are proximal to the DRM-H bilayer. Binding of supervillin to the DRM-H fragments is confirmed by co-immunoaffinity purification. In spreading neutrophils, supervillin localizes with F-actin in cell extensions and in discrete basal puncta that partially overlap with Galpha(i) staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains.