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HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases.

ABSTRACT: The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica, hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in Escherichia coli, Salmonella, and Yersinia pestis than in Y. enterocolitica. Analysis of the distribution of hreP between different Yersinia isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of Anabaena variabilis, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His(6) tag fusion protein in E. coli revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein.

SUBMITTER: Heusipp G

PROVIDER: S-EPMC95231 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases.

Heusipp G G Young G M GM Miller V L VL

Journal of bacteriology 20010601 12

The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enter ...[more]

PMID: 11371518

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Project description:We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.

Project description:Proprotein convertase subtilisin/kexins (PCSKs) constitute a family of nine related proteases: PCSK1-7, MBTPS1, and PCSK9. Apart from PCSK9, little is known about PCSKs in cardiovascular disease. Here, we aimed to investigate the expression landscape and druggability potential of the entire PCSK family for CVD. We applied an integrative approach, combining genetic, transcriptomic and proteomic data from three vascular biobanks comprising carotid atherosclerosis, thoracic and abdominal aneurysms, with patient clinical parameters and immunohistochemistry of vascular biopsies. Apart from PCSK4, all PCSK family members lie in genetic regions containing variants associated with human cardiovascular traits. Transcriptomic analyses revealed that FURIN, PCSK5, MBTPS1 were downregulated, while PCSK6/7 were upregulated in plaques vs. control arteries. In abdominal aneurysms, FURIN, PCSK5, PCSK7, MBTPS1 were downregulated, while PCSK6 was enriched in diseased media. In thoracic aneurysms, only FURIN was significantly upregulated. Network analyses of the upstream and downstream pathways related to PCSKs were performed on the omics data from vascular biopsies, revealing mechanistic relationships between this protein family and disease. Cell type correlation analyses and immunohistochemistry showed that PCSK transcripts and protein levels parallel each other, except for PCSK9 where transcript was not detected, while protein was abundant in vascular biopsies. Correlations to clinical parameters revealed a positive association between FURIN plaque levels and serum LDL, while PCSK6 was negatively associated with Hb. PCSK5/6/7 were all positively associated with adverse cardiovascular events. Our results show that PCSK6 is abundant in plaques and abdominal aneurysms, while FURIN upregulation is characteristic for thoracic aneurysms. PCSK9 protein, but not the transcript, was present in vascular lesions, suggesting its accumulation from circulation. Integrating our results lead to the development of a novel 'molecular' 5D framework. Here, we conducted the first integrative study of the proprotein convertase family in this context. Our results using this translational pipeline, revealed primarily PCSK6, followed by PCSK5, PCSK7 and FURIN, as proprotein convertases with the highest novel therapeutic potential.

Dataset Information

HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases.

Publications

HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases.

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