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Intracellular Peptide Turnover: Properties and Physiological Significance of the Major Peptide Hydrolases of Brain Cytosol
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Prolyl oligopeptidase is widely distributed, and present at similar levels in all tissues studied4. A monospecific antiserum to the rat brain enzyme was used to demonstrate its widespread tissue distribution and to show that it is the only proline-specific endopeptidase in all rat tissues examined54. Northern blot analysis of its mRNA also reveals a similar level in all tissues studied55. Moreover sequences of cDNAs from several tissues are almost identical indicating conservation of structure55. Within the rabbit brain, the enzyme is also fairly evenly distributed with a threefold difference in specific activity from the highest (entorhinal cortex) to lowest (pons) regions48. In rat brain there is also a threefold separation of activity from highest to lowest regions56. Subcellular distribution studies confirm the cytosolic localization of prolyl oligopeptidase. The distribution of this enzyme in subcellular fractions closely follows that of the cytosolic marker enzyme lactate dehydrogenase38. Although a fraction of prolyl oligopeptidase was reported to be associated with rat brain synaptic membranes57, the cDNA does not reveal a membrane spanning domain, and it is unlikely that a true membrane-bound form exists. The cellular distribution also appears to be widespread. The enzyme was found in both cultured neurons and astrocytes58. A report on the neuronal localization of prolyl oligopeptidase must be treated with caution59. Neuronal localization was inferred by the observation of a marked decrease in enzymatic activity in caudate-putamen and substantia nigra following injection of kainate. However the substrate used Gly-Pro-Pro-MCA, is not specific for prolyl oligopeptidase. The fluorophore can be released by the concerted action of X-Pro-aminopeptidase (EC 3.4.11.9) and dipeptidyl peptidase IV as follows:
Peptidase PepP is a novel virulence factor of Campylobacter jejuni contributing to murine campylobacteriosis
Published in Gut Microbes, 2020
Markus M. Heimesaat, Anna-Maria Schmidt, Soraya Mousavi, Ulrike Escher, Nicole Tegtmeyer, Silja Wessler, Gabriele Gadermaier, Peter Briza, Dirk Hofreuter, Stefan Bereswill, Steffen Backert
To identify the major proteases produced by C. jejuni, total bacterial lysate of strain 81–176 was separated on a preparative SDS-PAGE gel containing embedded casein. After protein renaturation by removal of SDS, zymography demonstrated proteolytic activity of one major and several weaker protein bands (Figure 1a). The main band of 180 kDa corresponded to oligomeric forms of HtrA, as we had demonstrated previously.27 The weaker band with mobility of approximately 70 kDa was excised and following trypsin treatment peptides were analyzed by mass spectrometry (Figure 1b). Of the detected peptide sequences, nine were identified as identical to a peptidase of the M24 family described as UniProtKB protein A0A0M3VCY5 (locus WP_002854975) (Figure 1c). The corresponding gene in the genome sequence of C. jejuni 81–176 (NC_008787.1), with locus tag CJJ81176_0681, is annotated as ‘aminopeptidase P family protein’. The protein is highly conserved in other C. jejuni strains and contains three conserved Pfam domains, PF01321 und PF16189, typical for creatinase/prolidase activity, and PF16188, the C-terminal region of peptidase M24 (Supplementary Figure S1). Based on this information, the translated protein most likely represents a protease (E.C. 3.4.11.9) that catalyzes the release of any N-terminal amino acid that is linked with proline. This enzyme is a member of the MEROPS peptidase M24 family, and is possibly an Xaa-Pro aminopeptidase. The corresponding gene was named pepP. This gene is not only highly conserved in C. jejuni; a homolog was also identified in C. coli, C. hepaticus, C. upsaliensis, and C. helveticus, and a distant homolog was present in C. cuniculorum, but it was not detected in other Campylobacter species. The closest homolog in an alternative genus was identified in Helicobacter mesocricetorum as shown in a phylogenetic tree (Supplementary Figure S2).