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《Molecular & cellular proteomics : MCP》2018,17(12):2496-2507
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- •Quantitative (phospho)proteome analysis of antibiotic treatment in E. coli.
- •Largest bacterial phosphorylation catalogue.
- •Specific phosphorylation motifs changes during resistance development.
- •Phosphorylation mediated signaling could be a potential target for drug design.
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Kenneth P. Smith Julia G. Fields Richard D. Voogt Bin Deng Ying‐Wai Lam Keith P. Mintz 《Proteomics》2015,15(11):1859-1867
Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results in pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with MS was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild‐type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild‐type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the MorC mutant strain is discussed. 相似文献
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为探索伤寒沙门菌Ⅵ型分泌系统(type 6 secretion system, T6SS)对SopE表达的影响,阐明其中的分子机制,本研究利用自杀质粒同源重组法构建携带sopE∷3×Flag的伤寒沙门菌野生株(WT-pBAD33)、T6SS功能分子缺陷株(ΔsciG-pBAD33)和回补株(C-ΔsciG),用构建成功的菌株感染巨噬细胞THP-1并在模拟巨噬细胞内环境(LPM培养基、微需氧)下培养,通过实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)与蛋白质印迹法(Western blot, WB)试验探索T6SS对SopE表达的影响,采用β-半乳糖苷酶活性检测来进一步验证sopE基因启动子的转录水平,Ni柱纯化T6SS功能蛋白SciG,电泳迁移率变动分析(electrophoretic mobility shift assay,EMSA)研究SciG蛋白对sopE基因启动子的调控作用。结果显示：ΔsciG-pBAD33中SopE表达水平较于WT-pBAD33明显降低;SciG蛋白不能直接与sopE基因启动子区域结合。本研究结果表明,伤寒沙门菌T6SS影响了SopE的表达,但是其功能蛋白SciG不能直接调控sopE基因的表达。 相似文献
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Misra SK Milohanic E Aké F Mijakovic I Deutscher J Monnet V Henry C 《Proteomics》2011,11(21):4155-4165
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Jun Ito Nicolas L. Taylor Ian Castleden Wolfram Weckwerth A. Harvey Millar Joshua L. Heazlewood 《Proteomics》2009,9(17):4229-4240
Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2‐DE separations of Arabidopsis mitochondrial proteins and affinity‐enriched phosphoproteins using the Pro‐Q Diamond phospho‐specific in‐gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis. 相似文献
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Recent phosphoproteomics studies of several bacterial species have firmly established protein phosphorylation on Ser/Thr/Tyr residues as a PTM in bacteria. In particular, our recent reports on the Ser/Thr/Tyr phosphoproteomes of bacterial model organisms Bacillus subtilis and Escherichia coli detected over 100 phosphorylation events in each of the bacterial species. Here we extend our analyses to Lactococcus lactis, a lactic acid bacterium widely employed by the food industry, in which protein phosphorylation at Ser/Thr/Tyr residues was barely studied at all. Despite the lack of almost any prior evidence of Ser/Thr/Tyr protein phosphorylation in L. lactis, we identified a phosphoproteome of a size comparable to that of E. coli and B. subtilis, with 73 phosphorylation sites distributed over 63 different proteins. The presence of several multiply phosphorylated proteins, as well as over-representation of phosphothreonines seems to be the distinguishing features of the L. lactis phosphoproteome. Evolutionary comparison and the conservation of phosphorylation sites in different bacterial organisms indicate that a majority of the detected phosphorylation sites are species-specific, and therefore have probably co-evolved with the adaptation of the bacterial species to their present-day ecological niches. 相似文献
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许多革兰氏阴性菌借助Ⅲ型分泌系统黏附在宿主细胞表面,然后跨越胞膜将特异性蛋白注入宿主细胞内,破坏宿主细胞内的多种信号通路,从而有利于细菌的感染及定殖。在肠致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)中,除了肠细胞脱落位点(Locus of entericyte effacement,LEE)毒力岛编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)外,在分析肠出血性大肠杆菌O157:H7的基因组序列时发现一个新的Ⅲ型分泌系统,大肠杆菌Ⅲ型分泌系统2(Escherichia coli type Ⅲ secretion system 2,ETT2)毒力岛。研究显示,ETT2可能在大多数菌株中不具有完整的分泌系统功能,但是其对于细菌毒力的发挥具有重要作用。因此,本文简要综述了大肠杆菌ETT2的基因特征、ETT2的分布与流行、ETT2的功能与机制等方面的主要研究进展。 相似文献
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Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO(2) -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence. 相似文献
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《Cell reports》2023,42(7):112781
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《Molecular & cellular proteomics : MCP》2019,18(3):408-422
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- •Quantitative phosphoproteomics of cells treated with sphingolipid analogs or PP2A inhibitor identify novel protein targets of PP2A.
- •PP2A substrates include several nutrient transporter proteins, GTPase regulators and proteins associated with actin cytoskeletal remodeling.
- •Differential regulation of Akt and Gsk3b account for the difference in vacuolating phenotype observed between SH-BC-893 and C2-ceramide.
- •Dynamic phosphoproteomics enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.
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Sherry AE Inglis NF Stevenson A Fraser-Pitt D Everest P Smith DG Roberts M 《Proteomics》2011,11(3):361-370
Salmonella enterica has two pathogenicity islands encoding separate type three secretion systems (T3SS). Proteins secreted through these systems facilitate invasion and survival. After entry, Salmonella reside within a membrane bound vacuole, the Salmonella containing vacuole (SCV), where translocation of a second set of effectors by the Salmonella pathogenicity island 2 (SPI-2) T3SS is initiated. SPI-2 secretion in vitro can be induced by conditions that mimic the Salmonella containing vacuole. Utilising high-throughput mass spectrometry, we mapped the surface-attached proteome of S. Typhimurium SL1344 grown in vitro under SPI-2-inducing conditions and identified 108 proteins; using secretion signal prediction software, 43% of proteins identified contained a signal sequence. Of these proteins, 13 were known secreted effector proteins including SPI-2 effector proteins SseB, SseC, SseD, SseL, PipB2 and SteC, although surprisingly five were SPI-1 proteins, SipA, SipB, SipC, SipD and SopD, while 2 proteins SteA and SlrP are secreted by both T3SSs. This is the first in vitro study to demonstrate dual secretion of SPI-1 and SPI-2 proteins by S. Typhimurium and demonstrates the potential of high-throughput LC-ESI/MS/MS sequencing for the identification of novel proteins, providing a platform for subsequent comparative proteomic analysis, which should greatly assist understanding of the pathogenesis and inherent variation between serovars of Salmonella and ultimately help towards development of novel control strategies. 相似文献
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Juraj Lenco Vojtech Tambor Marek Link Jana Klimentova Jiri Dresler Miroslav Peterek Alain Charbit Jiri Stulik 《Proteomics》2014,14(21-22):2400-2409
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The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter- and intra-species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well-characterised core proteins necessary for effector delivery (TssA-M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS-dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ-independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins. 相似文献
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Phosphorylation is the predominant mechanism of post-translational modification for regulation of protein function. With central roles in virtually every cellular process, and strong linkages with many diseases, there is a considerable interest in defining, and ultimately controlling, kinase activities. Investigations of human cellular phosphorylation events, which includes over 500 different kinases and tens of thousands of phosphorylation targets, represent a daunting challenge for proteomic researchers and cell biologists alike. As such, there is a priority to develop tools that enable the evaluation of cellular phosphorylation events in a high-throughput, and biologically relevant, fashion. Towards this objective, two distinct, but functionally related, experimental approaches have emerged; phosphoproteome investigations, which focus on the sub-population of proteins which undergo phosphorylation and kinome analysis, which considers the activities of the kinase enzymes mediating these phosphorylation events. Within kinome analysis, peptide arrays have demonstrated considerable potential as a cost-effective, high-throughput approach for defining phosphorylation-mediated signal transduction activity. In particular, a number of recent advances in the application of peptide arrays for kinome analysis have enabled researchers to tackle increasingly complex biological problems in a wider range of species. In this review, recent advances in kinomic analysis utilizing peptides arrays including several of the biological questions studied by our group, as well as outstanding challenges still facing this technology, are discussed. 相似文献