Serine-type Endopeptidase Research Articles

Serpin Peptidase Inhibitor Clade A Member 1 as a Potential Marker for Malignancy in Insulinomas

The biological behavior of insulinomas cannot be predicted based on histopathologic criteria in which the diagnosis of malignancy is confirmed by the presence of metastases. In this study, microarray and quantitative real-time reverse transcription-PCR were applied to identify differentially expressed genes between malignant and nonmalignant insulinomas to search for useful biomarkers to recognize the metastatic potential of insulinomas. Code Link human bioarrays were used to analyze differences in approximately 20,000 genes between six well-differentiated endocrine tumors of benign behavior compared with one well-differentiated endocrine carcinoma (WDEC) and three metastases of endocrine carcinomas (MEC). Quantitative real-time reverse transcription-PCR was used to validate differential expressions of five genes in a series of 35 sporadic insulinomas. Serpin peptidase inhibitor clade A member 1 (SERPINA1; alpha-1-antitrypsin) expression, identified as up-regulated in malignant insulinomas, was also evaluated by immunohistochemistry. Analysis of microarray data resulted in 230 differentially expressed genes. Gene Ontology analysis identified serine-type endopeptidase activity and serine-type endopeptidase inhibitor activity as pathways presenting significant differential expression. Protease serine 2 and complement factor B (from serine-type endopeptidase activity pathway) were respectively confirmed as up-regulated in well-differentiated endocrine tumors of benign behavior (WDET) and in WDEC/MEC. Angiotensinogen and SERPINA1 (from serine-type endopeptidase inhibitor activity pathway) were confirmed as up-regulated in WDEC/MEC. SERPINA1 was shown to be expressed in 85.7% of malignant versus 14.3% of nonmalignant insulinomas by immunohistochemistry. Our data are consistent to the possibility that SERPINA1 is a marker of malignancy in insulinomas. Given the widespread availability of antibody anti-alpha-1-antitrypsin in pathology services, SERPINA1 expression evaluation might be of clinical utility in recognizing patients more likely to develop an aggressive presentation.

Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL

HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acute-phase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

SERPINA1 (ALPHA-1-ANTITRYPSIN) AS A POTENTIAL MARKER FOR MALIGNANCY OF INSULINOMAS

Background/Aim: The biological behavior of insulinomas can not be predicted based on histopathological criteria and the diagnosis of malignancy is made in the presence of metastases. Microarray and RT-qPCR were applied in this study to identify differentially expressed genes between malignant and non-malignant insulinomas with the aim of searching for useful biomarkers to recognize the metastatic potential of insulinomas. Methods: We have used CodeLink human bioarrays to analyze differences in ~20,000 genes between 6 well-differentiated endocrine tumors (WDET) of benign behavior (BB) compared to one well-differentiated endocrine carcinoma (WDEC) and 3 metastases of endocrine carcinomas (MEC). RT-qPCR was employed to validate the differential expression of 4 genes in 35 sporadic insulinomas. SERPINA1 (alpha-1-antitrypsin), identified as up-regulated in malignant insulinomas, was also evaluated by immunohistochemistry. Results: Analysis of microarray data resulted in 310 differentially expressed genes. Gene Ontology analysis identified Serine-type endopeptidase activity and Serine-type endopeptidase inhibitor activity as pathways presenting significant differential expression. PRSS2 and CFB (from Serine-type endopeptidase activity pathway) were respectively confirmed as up-regulated in WDET and in WDEC/MEC. AGT and SERPINA1 (from Serine-type endopeptidase inhibitor activity pathway) were confirmed as up-regulated in WDEC/MEC. By immunohistochemistry, SERPINA1 was expressed in 85.7% malignant versus 14.3 % non-malignant insulinomas. Conclusion: Our data are consistent with the possibility that SERPINA1 is a marker of malignancy in insulinomas. Given the widespread availability of antiserum anti alpha-1-antitrypsin in pathology services, SERPINA1 evaluation might be of clinical utility in recognizing patients more likely to develop aggressive disease.

Molecular Diversity of a Putative Virulence Factor: Purification and Characterization of Isoforms of an Extracellular Serine Glutamyl Endopeptidase of Enterococcus faecalis with Different Enzymatic Activities

A previously identified gene sprE of Enterococcus faecalis strain OG1 was shown to encode an extracellular serine protease that appears to belong to the glutamyl endopeptidase I staphylococcal group. A single form of SprE with a molecular mass of 25 kDa and a pH optimum between 7.0 and 7.5 was isolated from culture supernatant of wild-type E. faecalis strain OG1RF (TX4002); this form was apparently generated by cleavage of the Ser-1-Leu1 and Arg230-Leu231 peptide bonds of the secreted zymogen. In contrast, the culture supernatant of the gelatinase-null mutant, TX5264, with a nonpolar deletion of gelE which encodes the E. faecalis gelatinase, was found to contain several forms of SprE proteolytically processed on both the N and C termini; in addition to a full-length zymogen and a truncated zymogen, three mature forms of the SprE proteinase, Leu1-Ala237, Ser-1-Glu227, and Leu1-Glu227, were identified. As with the V8 proteinase of Staphylococcus aureus, the closest homologue of SprE, all of the active forms cleaved specifically Glu-Xaa peptide bonds but with substantially different efficiencies, while none was able to hydrolyze peptide bonds with Asp in the P1 position. The most active of all these enzyme forms against several substrates, including human fibrinogen and beta-chain insulin, was the Ser-1-Glu227 (-1S-SprE) isolated from TX5264; -1S-SprE, in contrast to other forms of SprE, was unstable at 37 degrees C, apparently due to autodegradation. In conclusion, our results demonstrate that sprE encodes a highly specific serine-type glutamyl endopeptidase, the maturation of which is dependent on the presence of gelatinase. In the absence of gelatinase activity, the aberrant processing of pro-SprE results in the appearance of a "superactive" form of the enzyme, -1S-SprE.

The isolated D1/D2/cyt b-559 reaction centre complex of Photosystem II possesses a serine-type endopeptidase activity

The isolated D1/D2/cyt b-559 reaction centre complex of Photosystem II has been shown to possess an endopeptidase activity which has been investigated using synthetic substrates and proteinase inhibitors. Substrate specificity and inhibition by serine type proteinase inhibitors suggest that the endopeptidase is of a serine type. Divalent cations, especially calcium, enhanced the activity of the intrinsic peptidase which also showed pH and temperature optima of 8.2 and 37°C respectively. Treatment with strong light had little or no effect on the enzyme activity.

Arginine carboxypeptidase activity in the male reproductive glands of the silkworm, Bombyx mori

A carboxypeptidase that liberates free arginine from peptides split from proteins by the prostatic endopeptidase, initiatorin, and which serves to supply this amino acid in the spermatophore, was found in the male reproductive system of Bombyx mori. It was consistently detected in the glandula (g.) prostatica at the pH optimum 7.8. Unlike porcine pancreatic carboxypeptidase B, the Bombyx enzyme hydrolyzed hippuryl- l-arginine, but not hippuryl- l-lysine. In extracts of three other glands, the vesicula (v.) seminalis, g. lacteola and g. spermatophorae, the enzyme activity was detected only after their treatment with a serine-type endopeptidase, such as bovine trypsin or endoproteinase Arg-C from mouse submaxillary gland. The carboxypeptidase detected after this treatment showed the same substrate specificity as that of the prostatic enzyme. After proteinase treatment, the extract of the v. seminalis had the highest carboxypeptidase activity, followed by those of the g. prostatica, g. lacteola and g. spermatophorae. These findings indicate that the carboxypeptidase is present in the male reproductive glands as an inactive zymogen, and that it is activated by initiatorin in the spermatophore built during mating.