Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y

© 2019, The Author(s). Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen

A comprehensive guide for performing sample preparation and top-down protein analysis

© 2017 by the authors. Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform0s abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism0s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer

N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen

© 2017 The Author(s). Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function

OPTIMALISASI KINERJA PERSEDIAAN SUKU CADANG UNTUK PELAKSANAAN MAINTENANCE REPAIR OVERHAUL (MRO) DENGAN PENERAPAN COMPUTERIZED MAINTENANCE MANAGEMENT SYSTEMS (CMMS) (Studi Kasus di PT. Indonesia Asahan Aluminium)

PT.Inalum is a manufacturing company engaged in the aluminum smelting industry. PT.Inalum pengendaliaan system requires an inventory of spare parts to conduct maintenance, repair and overhaul (MRO). CMMS (Computerized Maintenance Management System) is a computerized maintenance system running on a LAN (Local Area Network). CMMS is influenced by three variables, namely an integrated maintenance, inventory and procurement so as to create a good maintenance should be supported by the inventory control of spare parts used in maintenance. CMMS aims to make the work more efficient and effective, especially in matters relating to maintenance. That as a benchmark to measure the performance of CMMs for spare parts inventory control is a key performance indicator (KPI). This study aims to evaluate and compare key performance indicators before and after implementation of CMMS for the implementation of MRO activities. Another goal to be achieved is to determine the factors that affect the value of the inventory of spare parts. Based on the results of data processing and analysis can be seen that by looking at a comparison of before and after implementation of CMMS on key performance indicators, it appears that there is an improvement in every parameter, one of which is the value of the inventory prior to the implementation of CMMS for $34,790,000.00 compared to after the application of the inventory value fell to$ 19,632,000.00. Factors that affect the value of the inventory is the inventory of spare parts, maintenance, procurement, and human resources

MHJ-0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

© 2015 The Authors. Published. Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ-0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ-0461 (rMHJ-0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ-0461 resides on the surface of M. hyopneumoniae. rMHJ-0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ-0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ-0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

© 2016 The Authors. Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endopro-teolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity

The Imprint of Galaxy Formation on X-ray Clusters

It is widely believed that structure in the Universe evolves hierarchically, as primordial density fluctuations, amplified by gravity, collapse and merge to form progressively larger systems. The structure and evolution of X-ray clusters, however, seems at odds with this hierarchical scenario for structure formation. Poor clusters and groups, as well as most distant clusters detected to date, are substantially fainter than expected from the tight relations between luminosity, temperature and redshift predicted by these models. Here we show that these discrepancies arise because, near the centre, the entropy of the hot, diffuse intracluster medium (ICM) is higher tha$achievable through gravitational collapse, indicating substantial non-gravitational heating of the ICM. We estimate this excess entropy for the first time, and argue that it represents a relic of the energetic winds through which forming galaxies polluted the ICM with metals. Energetically, this is onl$ possible if the ICM is heated at modest redshift (z \ltsim 2) but prior to cluster collapse, indicating that the formation of galaxies precedes that of clusters and that most clusters have been assembled very recently.Comment: 5 pages, plus 2 postscript figures (one in colour), accepted for publication in Natur

Elongation factor Tu is a multifunctional and processed moonlighting protein

© 2017 The Author(s). Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs

A Systematic Review of Online Sex Addiction and Clinical Treatments Using CONSORT Evaluation

Researchers have suggested that the advances of the Internet over the past two decades have gradually eliminated traditional offline methods of obtaining sexual material. Additionally, research on cybersex and/or online sex addictions has increased alongside the development of online technology. The present study extended the findings from Griffiths’ (2012) systematic empirical review of online sex addiction by additionally investigating empirical studies that implemented and/or documented clinical treatments for online sex addiction in adults. A total of nine studies were identified and then each underwent a CONSORT evaluation. The main findings of the present review provide some evidence to suggest that some treatments (both psychological and/or pharmacological) provide positive outcomes among those experiencing difficulties with online sex addiction. Similar to Griffiths’ original review, this study recommends that further research is warranted to establish the efficacy of empirically driven treatments for online sex addiction