The specificity of macrophage elastase on the insulin B-chain.
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ABSTRACT:
SUBMITTER: Kettner C
PROVIDER: S-EPMC1162899 | biostudies-other | 1981 May
REPOSITORIES: biostudies-other
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Project description:Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.
| S-EPMC4726324 | biostudies-literature
Project description:The specificity of action of the lysosomal elastase of human neutrophil leucocytes on the oxidized B chain of insulin is similar to that of pig pancreatic elastase, but is more directed towards valine than alanine as the residue contributing the carboxyl group of the cleaved bond. The most susceptible bonds are Val-12-Glu-13 and Val-18-Cys(O3H)-19. Other bonds hydrolysed are Ala-14-Leu-15, Ser-9-His-10 and Cys, (O3H3)-7-Gly-8. Tables listing amino acid composition, N-terminal residue, and yields of isolated peptides have been deposited as Supplementary Publication SUP 50 075 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161, 1.
| S-EPMC1164467 | biostudies-other
Project description:Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages and is implicated in several disease processes, including emphysema. Physiological functions for MMP12 have not been described. Here we show that Mmp12(-/-) mice exhibit impaired bacterial clearance and increased mortality when challenged with both gram-negative and gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed beta loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.
| S-EPMC2885871 | biostudies-literature
Project description:IntroductionElastase is a proteolytic pancreatic enzyme that passes through the gastrointestinal tract undergoing only limited degradation. ELISA tests to determine stool elastase concentrations have therefore been developed for the diagnosis of exocrine pancreatic insufficiency. Five different isoforms of pancreatic elastase (CELA1, CELA2A, CELA2B, CELA3A, CELA3B) are encoded in the human genome. We have investigated three different polyclonal antisera that are used in a commercial fecal elastase ELISA to determine their specificity for different pancreatic elastase isoforms.Material and methodsDifferent polyclonal rabbit antisera against human elastase peptides (BIOSERV Diagnostics GmbH, Germany) were tested by Western blot analysis of human pancreatic juice, in HEK-293 cells expressing Elastase constructs, and in the protein content of porcine pancreatin, used for treatment of exocrine pancreatic insufficiency.ResultsIn human pancreatic juice the polyclonal antisera detected proteins at the corresponding size of human pancreatic elastase isoforms (~29kDa). Transiently expressed GFP fusion protein of elastase isoform CELA3A (CELA3A-GFP), but not CELA2A (CELA2A-GFP) could be precipitated from HEK-293 cell lysates with the elastase antisera. We detected no cross-reactivity with elastases in the porcine pancreatic extracts (pancreatin) used for enzyme replacement therapy.ConclusionThe polyclonal antisera used in a commercial fecal elastase ELISA are specific for the human pancreatic elastase isoform CELA3 and do not cross-react with elastase contained in pig pancreatin. While pancreatic elastase 1 (CELA1) is not expressed in the adult human pancreas, possible differences between the other isoforms regarding their cellular expression, pathophysiological role and relevance in exocrine pancreatic insufficiency deserve further investigation.
| S-EPMC4961393 | biostudies-literature
Project description:An electrophoretically homogeneous elastase preparation free from tryptic and chymotryptic activities was obtained by chromatography on DEAE-Sephadex and CM-cellulose. This preparation exhibits a narrower specificity towards peptide bonds than that observed by Naughton & Sanger (1961). With oxidized insulin B chain as substrate, the fastest breaks occur between alanine-14 and leucine-15 and between valine-18 and cysteic acid-19. The bond between glycine-23 and phenylalanine-24 is also efficiently hydrolysed. Other bonds hydrolysed are that between valine-12 and glutamic acid-13 and that between serine-9 and histidine-10. Oxidized insulin A chain is hydrolysed only at one of two points, between alanine-8 and serine-9 or between serine-12 and leucine-13, and the rate of hydrolysis is very low.
| S-EPMC1184789 | biostudies-other
Project description:Neutrophil serine proteases (NSPs) in cytoplasmic granules of neutrophils are regarded as important antimicrobial defense weapons after engulfment and exposure of pathogens to the content of primary granules. Despite intensive studies on neutrophils during the last three decades, only three active serine proteases, neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) have been identified in these short-lived cells. Here, we report on the identification of a fourth serine protease (NSP4) with 39% identity to NE and PR3, but arginine specificity, yet sharing features like propeptide processing by dipeptidyl peptidase I, storage, and release as an active enzyme with the three active proteases. We established monoclonal antibodies against NSP4, excluded cross-reactivity to human granzymes, NE, CG, PR3, and azurocidin, and screened for NSP4 protein expression in various human tissues and blood leukocyte populations. Only granulocyte precursors and neutrophil populations from peripheral blood were positive. The content of NSP4 in neutrophil lysates, however, was about 20-fold lower compared with CG. Upon neutrophil activation, NSP4 was released into the supernatant. Profiling its specificity with peptide libraries from Escherichia coli revealed a preference for arginine in P1; it cleaved Tyr-Arg-Phe-Arg-AMC and Ala-Pro-Nva-thiobenzyl esters. NSP4 was inhibited by ?(1)-proteinase inhibitor (?(1)-antitrypsin), C1 inhibitor, and most efficiently by antithrombin-heparin, but not by elafin, secretory leukocyte protease inhibitor, ?(1)-antichymotrypsin, and monocyte-neutrophil elastase inhibitor. Functional specialization and preferred natural substrates of NSP4 remain to be determined to understand the biological interplay of all four NSPs during neutrophil responses.
| S-EPMC3341072 | biostudies-literature
Project description:Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.
| S-EPMC1162638 | biostudies-other
Project description:Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.
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Project description:Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.
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Project description:Macular subretinal fibrosis is the end-stage complication of neovascular age-related macular degeneration (nAMD). RPE/Choroid from control and subretinal fibrosis mice were collected and processed for RNA sequencing (RNA-seq) analysis to investigate the molecular mechanism of subretinal fibrosis.
2022-04-13 | GSE189555 | GEO