RESUMO
Organophosphorus compounds (OPs) such as chemical agents and pesticides are posing critical threats to civilians due to their irreversible phosphonylation of diverse amino acids residues forming different protein adducts. However, traditional analytical approaches are quite limited in capturing the myriad of post-translational events that affect protein functions, especially in identifying the low-abundance OP adducts. Herein a systematic proteomic strategy based on a typical click-enrich-release-identify bioorthogonal operation was firstly developed by employing an alkynyl-tagged V-type agent probe (AVP) and a biotin-based azido-enrichment linker (BTP-N3 ). AVP targeting peptides from human serum albumin (HSA) or plasma were captured by BTP-N3 via CuAAC click reaction, enriched by streptavidin beads, released by selective alkaline hydrolysis of phenacyl ester bond, and subsequently sequenced by LC-MS/MS. This strategy has helped identifying 1115 unique OP adduction sites on 163 proteins in human plasma, and covers lots of OP adducts that cannot be achieved by traditional detection methods. The comprehensive coverage of novel OP substrates provided a general and sensitive approach to retrospective verification and/or dose assessment of toxic OPs.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Estudos Retrospectivos , Espectrometria de Massas em Tandem/métodos , Proteínas/metabolismoRESUMO
Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, play key roles in signal transduction and protein homeostasis. The crosstalk of PTMs greatly expands the components of proteome and protein functions. Multi-level proteome analysis, which involves proteome investigations of total lysate and PTMs in this context, provides a comprehensive approach to explore the PTM crosstalk of a biological system under diverse disturbances. However, multi-level proteome practice remains technically challenging. Here we intended to build a strategy for multi-level proteome analysis, in which we focus on the serial profiling the total proteome, ubiquitinome and phosphoproteome from the microscale of starting material. We started by evaluating five common lysis buffers and found that the sodium deoxycholate buffer provided the best overall performance. We then developed an approach for serial enrichment and profiling of the multi-level proteome. To expand the depth of identification, we customized the variable windows to perform data-independent acquisition (DIA) sequencing for each proteome. In total, we identified 6465 proteins, â¼20,000 GlyGly sites (class 1), and â¼ 19,000 phosphosites (class 1) sequentially using 1 mg of HeLa digest by three DIA measurements. We applied this strategy to analyze MG132-treated HeLa cells and observed the crosstalk between ubiquitination and phosphorylation. Our method can be referenced for other multi-level proteome studies with microscale samples. SIGNIFICANCE: Lysis buffer containing sodium deoxycholate provided the best overall performance in multi-level proteome analysis. One step of ubiquitination enrichment before phosphorylation enrichment does not reduce the reproducibility of phosphoproteome. Customized isolation windows were established for DIA analysis on each level of proteome. Combined the serial enrichment approach and the customized single-shot DIA method enabled the multi-level proteome of microscale protein samples.
Assuntos
Processamento de Proteína Pós-Traducional , Proteoma , Humanos , Células HeLa , Reprodutibilidade dos Testes , FosforilaçãoRESUMO
Hydroxyprolines are highly abundant in nature as they are components of many structural proteins and osmolytes. Anaerobic degradation of trans-4-hydroxy-l-proline (t4L-HP) was previously found to involve the glycyl radical enzyme (GRE) t4L-HP dehydratase (HypD). Here, we report a pathway for anaerobic hydroxyproline degradation that involves a new GRE, trans-4-hydroxy-d-proline (t4D-HP) C-N-lyase (HplG). In this pathway, cis-4-hydroxy-l-proline (c4L-HP) is first isomerized to t4D-HP, followed by radical-mediated ring opening by HplG to give 2-amino-4-ketopentanoate (AKP), the first example of a ring opening reaction catalyzed by a GRE 1,2-eliminase. Subsequent cleavage by AKP thiolase (OrtAB) yields acetyl-CoA and d-alanine. We report a crystal structure of HplG in complex with t4D-HP at a resolution of 2.7 Å, providing insights into its catalytic mechanism. Different from HypD commonly identified in proline-reducing Clostridia, HplG is present in other types of fermenting bacteria, including propionate-producing bacteria, underscoring the diversity of enzymatic radical chemistry in the anaerobic microbiome.
Assuntos
Prolina , Proteínas , Anaerobiose , Hidroxiprolina/química , Prolina/metabolismo , Proteínas/metabolismoRESUMO
Aminoethylsulfonate (taurine) is widespread in the environment and highly abundant in the human body. Taurine and other aliphatic sulfonates serve as sulfur sources for diverse aerobic bacteria, which carry out cleavage of the inert sulfonate C-S bond through various O2-dependent mechanisms. Taurine also serves as a sulfur source for certain strict anaerobic fermenting bacteria. However, the mechanism of C-S cleavage by these bacteria has long been a mystery. Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C-S cleavage by the O2-sensitive isethionate sulfo-lyase IseG, recently identified in sulfate- and sulfite-reducing bacteria. Homologs of the enzymes described in this study have a sporadic distribution in diverse strict and facultative anaerobic bacteria, from both the environment and the taurine-rich human gut, and may enable sulfonate sulfur acquisition in certain nutrient limiting conditions.
Assuntos
Proteínas de Bactérias , Clostridium butyricum , Microbioma Gastrointestinal , Intestinos/microbiologia , Família Multigênica , Taurina , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium butyricum/genética , Clostridium butyricum/metabolismo , Humanos , Ácido Isetiônico/metabolismo , Sulfatos/metabolismo , Taurina/biossíntese , Taurina/genéticaRESUMO
Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H2S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H2S in the disease-associated gut bacterium Bilophila wadsworthia. The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Desulfovibrio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Taurina/metabolismo , Acetiltransferases/genética , Acetiltransferases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ácidos e Sais Biliares/metabolismo , Bilophila/metabolismo , Ensaios Enzimáticos , Microbioma Gastrointestinal/fisiologia , Sulfeto de Hidrogênio/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mercaptoetanol/análogos & derivados , Mercaptoetanol/metabolismo , Redes e Vias Metabólicas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Enxofre/metabolismoRESUMO
Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.