Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit.
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ABSTRACT:
SUBMITTER: Tisljar U
PROVIDER: S-EPMC1131321 | biostudies-other | 1990 Apr
REPOSITORIES: biostudies-other
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Project description:Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.
| S-EPMC1149619 | biostudies-other
Project description:1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000-96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn(2+) was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn(2+)- activated endopeptidase to be characterized.
| S-EPMC1166148 | biostudies-other
Project description:Accumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties.
2019-02-25 | GSE117353 | GEO
Project description:An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.
| S-EPMC179000 | biostudies-other
Project description:Accumulation of subcutaneous white adipose tissue (WAT) is associated with increased insulin sensitivity, low levels of inflammation and a generally metabolically-healthy state, whereas accumulation of visceral adipose tissue is associated with insulin resistance, adipose tissue inflammation and metabolic syndrome. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. Here, we show that MME in white preadipocytes is differently expressed in subcutaneous vs visceral WAT, and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in preadipocytes increases the inflammatory response to substance P and amyloidβ aggregates. This is associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor. Thus, MME is a novel regulator of the insulin receptor in adipose tissue and may serve as a therapeutic target to increase insulin sensitivity and decrease inflammatory susceptibility.
2019-02-25 | GSE117352 | GEO
Project description:Accumulation of subcutaneous white adipose tissue (WAT) is associated with increased insulin sensitivity, low levels of inflammation and a generally metabolically-healthy state, whereas accumulation of visceral adipose tissue is associated with insulin resistance, adipose tissue inflammation and metabolic syndrome. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. Here, we show that MME in white preadipocytes is differently expressed in subcutaneous vs visceral WAT, and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in preadipocytes increases the inflammatory response to substance P and amyloidβ aggregates. This is associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor. Thus, MME is a novel regulator of the insulin receptor in adipose tissue and may serve as a therapeutic target to increase insulin sensitivity and decrease inflammatory susceptibility.
2019-02-25 | GSE117270 | GEO
Project description:Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.
| S-EPMC6782589 | biostudies-literature
Project description:ObjectiveAccumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties.MethodsWe performed RNA sequencing on human subcutaneous and visceral preadipocytes and array gene expression profiling in murine subcutaneous and visceral preadipocytes. We conducted several insulin signaling and inflammatory response experiments on different cellular states of MME expression.ResultsMME in white preadipocytes is expressed at a higher level in subcutaneous compared to visceral WAT and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in subcutaneous preadipocytes increased the inflammatory response to substance P and amyloid β aggregates. This associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor.ConclusionMME is a novel regulator of the insulin receptor in adipose tissue. Given the clinical significance of both chronic inflammation and insulin sensitivity in metabolic disease, these results show a potentially new target to increase insulin sensitivity and decrease inflammatory susceptibility.
| S-EPMC6437599 | biostudies-literature
Project description:BACKGROUND:Sedolisins are acid proteases that are related to the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both TriPeptidyl Peptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies. RESULTS:We studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows one major clade containing non-fungal sequences only and two major as well as two minor clades containing only fungal sequences. One of the major fungal clades contains all known TPPs whereas the other contains characterized endosedolisins. We identified four Cluster Specific Inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried ?-helix that induces a conformational change near the binding pocket. We also identified a total of 15 specificity determining positions (SDPs) of which five, identified in two independent analyses, form highly connected SDP sub-networks. Modeling of virtual mutants suggests a key role for the W307A or F307A substitution. The remaining four key SDPs physically interact at the interface of the catalytic domain and the enzyme's prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. One of the two small fungal clades concerns a subfamily that contains 213 sequences, is mostly similar to the major TPP subfamily but differs, interestingly, in position 307, showing mostly isoleucine and threonine. CONCLUSIONS:Analysis confirms there are at least two sedolisin subfamilies in fungi: TPPs and endopeptidases, and suggests a third subfamily with unknown characteristics. Sequence and functional diversification was centered around buried SDP307 and resulted in a conformational change of the pocket. Mutual Information network analysis forms a useful instrument in the corroboration of predicted SDPs.
| S-EPMC6278154 | biostudies-literature
Project description:Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides.
| S-EPMC3092155 | biostudies-literature