RESUMO
Protein S-acylation is an important lipid modification characteristic for heterogeneity in the acyl chain and dynamicity in the acylation/deacylation cycle. Most S-acylproteomic research has been limited by indirect identification of modified proteins/peptides without attached fatty acids, resulting in the failure to precisely characterize S-acylated sites with attached fatty acids. The study of S-acylation turnover is still limited at the protein level. Herein, aiming to site-specifically profile both the heterogeneity and the turnover of S-acylation, we first developed a site-specific strategy for intact S-acylated peptide analysis by introducing an acid cleavable bioorthogonal tag into a metabolic labelling method (ssMLCC). The cleavable bioorthogonal tag allowed for the selective enrichment and efficient MS analysis of intact S-acylated peptides so that S-acylated sites and their attached fatty acids could be directly analysed, enabling the precise mapping of S-acylated sites, as well as circumventing false positives from previous studies. Moreover, 606 S-palmitoylated (C16:0) sites of 441 proteins in HeLa cells were identified. All types of S-acylated peptides were further characterized by an open search, providing site-specific profiling of acyl chain heterogeneity, including S-myristoylation, S-palmitoylation, S-palmitoleylation, and S-oleylation. Furthermore, site-specific monitoring of S-palmitoylation turnover was achieved by coupling with pulse-chase methods, facilitating the detailed observation of the dynamic event at each site in multi-palmitoylated proteins, and 85 rapidly cycling palmitoylated sites in 79 proteins were identified. This study provided a strategy for the precise and comprehensive analysis of protein S-acylation based on intact S-acylated peptide analysis, contributing to the further understanding of its complexity and biological functions.
Assuntos
Ácidos Graxos , Proteínas , Humanos , Células HeLa , Proteínas/metabolismo , Ácidos Graxos/metabolismo , Peptídeos/metabolismo , AcilaçãoRESUMO
S-acylation is a widespread lipidation form in eukaryotes in which various fatty acids can be covalently attached to specific cysteine residues. However, due to the low reactivity of the lipid moieties and lack of specific antibodies, purification of intact S-acylated peptides remains challenging. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of the enrichment procedure as well as the LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in a restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate, and octanoate chain, respectively, providing a global insight into the endogenously heterogeneous modification state. Notably, we found and validated that S-palmitoleoylation (C16:1) provided less affinity toward lipid rafts compared with S-palmitoylation (C16:0). This study developed the first straightforward way to characterize endogenously intact S-acylated peptides on a proteome-wide scale, providing the modified residues together with their attached lipid moieties simultaneously, which paves the way for further understanding of protein S-acylation.
Assuntos
Fluoretos , Espectrometria de Massas em Tandem , Animais , Humanos , Cromatografia Líquida , Células HeLa , Acilação , Diferenciação Celular , MamíferosRESUMO
Comprehensive analysis of protein glycation is important for better understanding of its formation mechanism and biological significance. The current preconcentration methods of glycated proteome mainly depend on the reversible combination of boronic acid and cis-dihydroxy group by pH adjustment, but it has inherent limitations (e.g., poor specificity and time-consuming). Herein, for the first time, a novel enrichment method for glycated peptides is proposed based on the reversible chemical reaction between aldehyde and 1,2-aminothiol groups, in which oxidized glycated peptides are captured onto the magnetic nanoparticles via thiazolidine chemistry and then released by palladium-mediated cleavage. The method is rapid, with excellent selectivity (even at a 1:1000 molar ratio of glycated peptides/nonglycated peptides) and high sensitivity (1 fmol/µL). As a good evidence, 1549 glycated peptides were identified from glycated human serum with 94.6% specificity, providing a powerful technique for high-throughput analysis of glycated peptides.
Assuntos
Paládio , Proteoma , Glicosilação , Humanos , Peptídeos , TiazolidinasRESUMO
Glycation plays a pathogenic role in many age-related degenerative pathological conditions, such as diabetes, end-stage renal diseases, and cardiovascular diseases. Mass spectrometry-based qualitative and quantitative analysis methods have been greatly developed and contribute to our understanding of protein glycation. However, it is still challenging to sensitively and accurately quantify endogenous glycated proteome in biological samples. Herein, we proposed an integrated and robust quantitative strategy for comprehensive profiling of early-stage glycated proteome. In this strategy, a filter-assisted sample preparation method was applied to reduce sample loss and improve reproducibility of sample preparation, contributing to high-throughput analysis and accurate quantification of endogenous glycated proteins with low abundance. Standard glycated peptides were spiked and performed the subsequent process together with complex samples both in label-free quantification and multiple reaction monitoring (MRM) analysis, contributing to the improvement of quantitative accuracy. In parallel, a novel approach was developed for the synthesis of heavy isotope-labeled glycated peptides used in MRM analysis. By this way, a total of 1128 endogenous glycated peptides corresponding to 203 serum proteins were identified from 60 runs of 10 pairs of hemodialysis patients with and without cardiovascular complications, and 234 glycated peptides corresponding to 63 proteins existed in >70% runs, among which 17 peptides were discovered to be differentially glycated (P < 0.05, fold-change > 1.5 or <0.67). Furthermore, we validated the glycation difference of four target peptides in 46 serum samples using MRM analysis, which were consistent with our results of label-free quantification.
Assuntos
Proteínas Sanguíneas , Diálise Renal , Biomarcadores , Humanos , Espectrometria de Massas , Reprodutibilidade dos TestesRESUMO
Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.
Assuntos
Aminas/metabolismo , Lisina/metabolismo , Resinas Sintéticas/metabolismo , Acetilação , Aminas/química , Células HeLa , Humanos , Lisina/química , Resinas Sintéticas/análiseRESUMO
S-Nitrosylation is an important post-translational modification that occurs on cysteine amino acid and regulates signal transduction in diverse cell processes. Dysregulation of protein nitrosylation has shown close association with cardiovascular and neurological diseases, thus demanding further precise and in-depth understanding. Mass spectrometry-based proteomics has been the method of choice for analyzing S-nitrosylated (SNO-) proteins. However, due to their extremely low expression level and rapid turnover rate, quantitative analysis of the S-nitrosylation at the proteomic level remains challenging. Herein, we developed a novel approach termed FluoroTRAQ, which combined the fluorous solid-phase extraction of SNO-peptides and iTRAQ labeling for the quantitative analysis of the SNO-proteome with high sensitivity and specificity. This new analytical strategy was subsequently applied to examine the dynamic SNO-proteome changes of human umbilical vein endothelial cells upon in vitro S-nitrosoglutathione induction. Our data identified a number of novel SNO-proteins and revealed their temporal modulation as validated by biotin switch assay. Our study offered a practical approach for quantitative analysis of protein S-nitrosylation.
Assuntos
Espectrometria de Massas , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/isolamento & purificação , Proteínas/metabolismo , Extração em Fase Sólida , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Cinética , ProteômicaRESUMO
It is well established that palmitoylation plays a key role in the regulation of immune checkpoints, but the technical challenges in detecting protein palmitoylation have significantly prohibited further researches in this field. Till now, different approaches have been proposed, such as mutagenesis, antibody-based methods, bioinformatic prediction, "palmitate-centric" approaches, and "cysteine-centric" approaches. Of specific importance, high-throughput methods that allow the unbiased discovery of palmitoylation in the whole proteome should be further improved and employed. This chapter will summarize the methodological progresses for detecting protein palmitoylation, aiming to facilitate future researches in the lipid modification of immune checkpoint proteins.
Assuntos
Lipoilação , Palmitatos/metabolismo , Processamento de Proteína Pós-Traducional , Pontos de Checagem do Ciclo Celular/imunologia , Biologia Computacional , Cisteína/metabolismo , Humanos , Proteoma/química , Proteoma/metabolismoRESUMO
The ligands and receptors in immune checkpoint signaling are typically transmembrane proteins, which may be regulated by palmitoylation as a reversible lipid modification. Our recent work demonstrated that palmitoylation reduces the lysosomal degradation of PD-L1 trafficking and may present a new therapeutic target. To facilitate future investigations on palmitoylation and immune checkpoints, here we summarize the molecular roles of palmitoylation on protein stability, trafficking, membrane association, and protein-protein interaction. The biological effects of palmitoylation are exemplified by well-studied substrates such as Ras, EGFR, and Wnt proteins. Finally, the strategies for targeting protein palmitoylation are discussed to facilitate future translational studies.
Assuntos
Lipoilação , Palmitatos/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais , Humanos , Lisossomos/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMO
Analysis of protein C termini is very important for functional annotations of proteomes, while proteome-wide C termini analysis still poses substantial challenges. Here we described a simple and robust strategy for specific isolation of protein C termini based on LysC digestion and site-selective dimethylation to deplete N-terminal and internal peptides by scavenger materials. The performance of LysC digestion and conditions of site-selective dimethylation and resin coupling were discussed in detail. Then the strategy was successfully applied to the characterization of protein C termini of HeLa cells. A total of 781 protein C termini were identified with a 300 µg digest in our study, among which 38.9% were actually not identifiable using current trypsin digestion-based methods due to their inappropriate peptide length for MS analysis, indicating that our method was highly complementary to the existing methods. The enrichment procedure was rapid and easy to operate and could afford a very good identification efficiency by obtaining the largest C termini data set of the human proteome with the least sample loading. This method was without bias toward physicochemical properties of peptides. Moreover, a peptide-centric database was first introduced to analyze protein C termini, which effectively improved the accuracy and speed of the database search. Therefore, our method can be used to effectively and selectively isolate protein C termini and contributes to the global annotation of C terminomes.
Assuntos
Proteína C/química , Serina Endopeptidases/metabolismo , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
4-Hydroxy-2-nonenal (HNE)-modified proteins are closely associated with cellular functions and diseases, so qualitative and quantitative analysis of HNE-modified proteins is very necessary in order to further understand their structures and molecular functions. In this study, we described a six-plex isobaric labeling affinity purification (SiLAP) method based on the interaction of aminoxyTMT six-plex and anti-TMT antibody resin to identify and quantify the HNE modifications simultaneously. The labeling efficiency, ionization efficiency of the aminoxyTMT-tagged peptides, and reliability of the quantification method were investigated in detail. The mass tags were labeled on the modification sites, which could also significantly increase the ionization efficiency, contributing to site-specific identification and quantification of HNE peptides. The SiLAP strategy possessed high sensitivity, accuracy, and good reproducibility to qualitatively and quantitatively analyze HNE-modified proteins/peptides, which could be used to analyze both endogenously and exogenously modified proteins. Using the SiLAP strategy, 2257 HNE-modified peptides mapping 1121 proteins were collectively quantified, which was the largest data set of HNE-modified proteins with detailed modification sites, and 101 proteins were found to be differentially modified by HNE in six liver cell lines. At the same time, 33 endogenously HNE-modified peptides mapping 33 proteins were identified with modification sites.
Assuntos
Aldeídos/química , Proteínas de Neoplasias/análise , Aldeídos/isolamento & purificação , Células Hep G2 , Humanos , Fígado/química , Modelos Moleculares , Estrutura Molecular , Proteínas de Neoplasias/metabolismo , Células Tumorais CultivadasRESUMO
Palmitoylation is one of the most important protein translational modifications and plays vital roles in many key biological processes. Aberrant palmitoylation has been associated with a variety of human diseases. So it is of great significance to profile the palmitoylated proteomes qualitatively and quantitatively. Here, we described a novel method based on the cysteine-stable isotope labeling in cell culture (cysteine-SILAC) to facilitate the quantitation of palmitoylated proteins by mass spectrometry (MS), in which "light" or "heavy" samples could be pooled and subjected to the subsequent analysis procedures simultaneously, minimizing systematic errors caused by parallel operations and improving quantitative accuracy and precision. The mass tags lay on the cysteine residues, which were the potential palmitoylated sites, indicating that all the putative modified sites/peptides could be quantified, including the C-terminal peptide of one protein. Due to the isotopically labeled cysteine, the nonspecifically adsorbed peptide without cysteine was singlet in MS spectra, whereas pair peaks should be the signals of putative palmitoylated peptides, which could reduce spectral complexity and achieve double verification for the putative palmitoylated peptides. Finally, the palmitoylome in hepatocellular carcinoma (HCC) cells with different metastasis potentials (MHCC-97L and HCC-LM3 cells) were analyzed for the first time. Totally, 151 proteins were found to be differentially palmitoylated with high confidence, including many important proteins involved in a variety of biological processes, such as protein palmitoylation, cell proliferation, signal transduction, regulation of cell migration, and so on.
Assuntos
Cisteína/metabolismo , Marcação por Isótopo , Lipoilação , Proteínas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de MassasRESUMO
Palmitoylation, a type of fatty acylation, has vital roles in many biological processes. For ultradeep identification of protein palmitoylation, an enrichment approach based on a novel magnetic microsphere modified with 2,2'-dithiodipyridine (Fe3O4/SiO2-SSPy microsphere) is presented in this study. The Fe3O4/SiO2-SSPy microspheres were synthesized by directly coating thiol-containing silane coupling agent onto the magnetic supraparticles in aqueous solution at room temperature. Due to the intrinsic magnetic properties, high surface-to-volume ratios, and abundant reactive functional groups on the surface, these microspheres enabled direct capture of palmitoylated targets and convenient isolation, contributing to remarkable enrichment selectivity (purifying palmitoylated peptides from mixtures with nonpalmitoylated peptides even at a 1:500 molar ratio) and sensitivity (the detection limit was at femtomole level), thus enabling a global annotation of protein palmitoylation for complex biological samples. We successfully identified 1304 putative palmitoylated proteins from mouse brain tissues by using this method, which is the largest mouse palmitoylome data set to date. Except for those known members, many new proteins and pathways were also found to be regulated by palmitoylation.
Assuntos
Nanopartículas de Magnetita/química , Proteínas/análise , Piridinas/química , Compostos de Sulfidrila/química , Acilação , Estrutura Molecular , Tamanho da Partícula , Proteínas/metabolismo , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Proteome-wide quantitative analysis of protein ubiquitination is important to gain insight into its various cellular functions. However, it is still challenging to monitor how ubiquitination at each individual lysine residue is independently regulated, especially the whereabouts of peptides containing more than one ubiquitination site. In recent years, isobaric peptide termini labeling has been considered a promising strategy in quantitative proteomics, benefiting from its high accuracy by quantifying with a series of b, y fragment ion pairs. Herein, we extended the concept of isobaric peptide termini labeling to large-scale quantitative analysis of protein ubiquitination. A novel MS2 fragment ion based quantitative approach was developed, allowing the quantification of ubiquitination at site level via isobaric K-ε-GG peptide labeling, which combined metabolic labeling, K-ε-GG immunoaffinity enrichment, and site-selective N-terminus dimethylation. The feasibility of this proposed strategy was demonstrated through the ubiquitin proteome analysis of differently labeled MCF-7 cell digests. As a result, 2970 unique K-ε-GG peptides of 1383 proteins containing 2874 ubiquitinated sites were confidently quantified with high accuracy and sensitivity. In addition, we demonstrated that quantification on MS2 fragment ion level makes it possible to precisely quantify each individual ubiquitinated lysine residue in 39 K-ε-GG peptides bearing two ubiquitination sites by the use of specific ubiquitinated b, y ion pairs. It is expected that this proposed approach will serve as a powerful tool to quantify ubiquitination at the site level, especially for those multiubiquitinated peptides.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Células MCF-7 , Coloração e RotulagemRESUMO
Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA+ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA+ATPase for its replicase assembly. IMPORTANCE: Almost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA+ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA+ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Hepacivirus/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/fisiologia , Cromatografia de Afinidade/métodos , Genoma Viral/genética , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , RNA Viral/genética , Replicon/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Protein palmitoylation plays a significant role in a wide range of biological processes such as cell signal transduction, metabolism, apoptosis, and carcinogenesis. For high-throughput analysis of protein palmitoylation, approaches based on the acyl-biotin exchange or metabolic labeling of azide/alkynyl-palmitate analogs are commonly used. No palmitoylation antibody has been reported. Here, the palmitoylated proteome of human colon cancer cell lines SW480 was analyzed via a TS-6B-based method. In total, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Except for 3 known palmitoylated transmembrane proteins, ATP1A1, ZDHHC5, and PLP2, some important proteins including kinases, ion channels, receptors, and cytoskeletal proteins were also identified, such as CLIC1, PGK1, PPIA, FKBP4, exportin-2, etc. More importantly, the pan antipalmitoylation antibody was developed and verified for the first time. Our homemade pan antipalmitoylation antiserum could differentiate well protein palmitoylation from mouse brain membrane fraction and SW480 cells, which affords a new technique for analyzing protein palmitoylation by detecting the palmitic acid moiety directly. Furthermore, the candidate protein transitional endoplasmic reticulum ATPase (VCP) identified in SW480 cells was validated to be palmitoylated by Western blotting with anti-VCP antibody and the homemade pan antipalmitoylation antibody.
Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/enzimologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Soros Imunes/química , Soros Imunes/isolamento & purificação , Immunoblotting , Lipoilação , Camundongos , Ácido Palmítico/metabolismo , Processamento de Proteína Pós-Traducional , CoelhosRESUMO
Selective capture of protein C-termini is still challenging in view of the lower reactivity of the carboxyl group relative to amino groups and difficulties in site-specifically labeling the carboxyl group on the C-terminus rather than that on the side chains of acidic amino acids. For highly efficient purification of C-terminus peptides, a novel positive enrichment approach based on the oxazolone chemistry has been developed in this study. A bifunctional group reagent containing biotin and arginine was incorporated into the C-terminus of protein. Together with a streptavidin affinity strategy, the C-terminal peptides could be readily purified and analyzed by mass spectrometry (MS). Unlike the negative enrichment approach, C-terminal peptides, other than non-C-terminal peptides, were captured directly from the peptide mixture in this new method. The labeling efficiency (higher than 90%), enrichment selectivity (purifying C-terminal peptides from mixtures of non-C-terminal peptides at a 1:50 molar ratio), and ionization efficiencies in MS were dramatically improved. Moreover, the highly efficient identification of C-terminal peptides was further achieved by defining biotin as the 21st amino acid and optimizing the database search strategy. We have successfully identified 183 C-terminal peptides from Thermoanaerobacter tengcongensis using this creative method, which affords a highly selective and efficient purification approach for C-terminomics study.
Assuntos
Oxazolona/química , Peptídeos/isolamento & purificação , Proteínas/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biotinilação , Cromatografia Líquida de Alta Pressão , Cavalos , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Thermoanaerobacter/químicaRESUMO
RATIONALE: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. OBJECTIVE: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. METHODS AND RESULTS: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. CONCLUSIONS: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.
Assuntos
Bases de Dados de Proteínas , Bases de Conhecimento , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteômica/métodos , Biologia de Sistemas , Integração de Sistemas , Acesso à Informação , Animais , Caenorhabditis elegans , Difusão de Inovações , Drosophila , Humanos , Camundongos , Design de Software , Fluxo de TrabalhoRESUMO
Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.
Assuntos
Absorção Fisico-Química , Imãs/química , Metais/química , Microesferas , Proteínas/química , Animais , Cromatografia de Afinidade , Fígado/química , RatosRESUMO
BACKGROUND: Isoflurane has been demonstrated to limit myocardial ischemic injury. This effect is hypothesized to be mediated in part via effects on mitochondria. We investigated the hypothesis that isoflurane maintains mitochondrial respiratory chain functionality, in turn limiting mitochondrial damage and mitochondrial membrane disintegration during myocardial ischemic injury. METHODS: Mice (9-12 weeks of age) received isoflurane (1.0 minimum alveolar concentration) 36 hours before a 30-minute coronary artery occlusion that was followed by 24 hours of reperfusion. Cardiac mitochondria were isolated at a time point corresponding to 4 hours of reperfusion. 2,3,5-Triphenyltetrazoliumchloride staining was used to determine myocardial infarct size. Mitochondrial respiratory chain functionality was investigated using blue native polyacrylamide gel electrophoresis, as well as specific biochemical assays. Mitochondrial lipid peroxidation was quantified via the formation of malondialdehyde; mitochondrial membrane integrity was assessed by Ca-induced swelling. Protein identification was achieved via liquid chromatography mass spectrometry/mass spectrometry. RESULTS: Thirty-one mice were studied. Mice receiving isoflurane displayed a reduced myocardial infarct size (P = 0.0011 versus ischemia/reperfusion [I/R]), accompanied by a preserved activity of respiratory complex III (P = 0.0008 versus I/R). Isoflurane stabilized mitochondrial supercomplexes consisting of oligomers from complex III/IV (P = 0.0086 versus I/R). Alleviation of mitochondrial damage after isoflurane treatment was further demonstrated as decreased malondialdehyde formation (P = 0.0019 versus I/R) as well as a diminished susceptibility to Ca-induced swelling (P = 0.0010 versus I/R). CONCLUSIONS: Our findings support the hypothesis that isoflurane protects the heart from ischemic injury by maintaining the in vivo functionality of the mitochondrial respiratory chain. These effects may result in part from the preservation of mitochondrial supramolecular organization and minimized oxidative damage, circumventing the loss of mitochondrial membrane integrity.
Assuntos
Anestésicos Inalatórios/farmacologia , Cardiotônicos/farmacologia , Isoflurano/farmacologia , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Consumo de Oxigênio/efeitos dos fármacos , Animais , Transporte de Elétrons/efeitos dos fármacos , Precondicionamento Isquêmico Miocárdico/métodos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , PermeabilidadeRESUMO
Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.