Insulysin and pitrilysin: insulin-degrading enzymes of mammals and bacteria.
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Animals↗
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Overexpression of insulin degrading enzyme: cellular localization and effects on insulin signaling.
To investigate the role of insulin degrading enzyme (insulysin, EC 3.4.24.56) in insulin signaling, Chinese hamster ovary cells overexpressing the human insulin receptor were genetically engineered to also stably overexpress the rat insulin degrading enzyme. In comparison to the parental cells, these cells expressed 2.7-fold elevated levels of enzyme and insulin degradation was also increased 2-fold. These cells also exhibited a more rapid decrease in receptor tyrosine phosphorylation after removal of insulin. Moreover, low concentrations of insulin were less effective at stimulating proliferation of the cells overexpressing the enzyme. Finally, a fraction of the overexpressed enzyme as well a fraction of the endogenous enzyme could be detected on the plasma membrane surface of these cells. These results support the hypothesis that this enzyme may function in insulin signaling by degrading the insulin molecule.
Animals↗
Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.
The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.
Amino Acid Sequence↗
NRD convertase and aminopeptidase B: two processing metallopeptidases with a selectivity for basic residues.
An endoprotease and an aminopeptidase B were isolated from rat testis and characterized. The first one is a metalloendopeptidase of 1161 residues which contains a canonical HXXEHX76E Zn(2+)-binding site and an acidic stretch of 71 amino acids containing 79% of Glu and Asp. It exhibits an in vitro selectivity for peptide bonds at the N-terminus of Arg (R) moieties in dibasic sites and was thus called NRD convertase (Nardilysin: EC 3.4.24.61). It belongs to the pitrilysin family and shows 24 and 34% identity with E. coli protease III (EC 3.4.24.54) and insulysin (EC 3.4.24.55) respectively. The aminopeptidase B component is a 72 kDa metalloexopeptidase which is able to remove Lys and Arg residues from naphtylamide derivatives and from the N-terminus of various peptide substrates. A combination of biochemical and immunochemical studies revealed its ubiquitous character. In the testis, both enzymes are highly expressed at late stages of spermatogenesis and NRD convertase expression is exclusively restricted to the germ cells. The subcellular localization of both enzymes supports the involvement of aminopeptidase B in processing events associated with the secretory pathway but led to new hypothesis on the possible physiological role(s) of NRD convertase.
Amino Acid Sequence↗
Differentiation of BC3H1 and primary skeletal muscle cells and the activity of their endogenous insulin-degrading enzyme are inhibited by the same metalloendoprotease inhibitors.
Upon reduction of serum in their media, mouse BC3H1 muscle cells withdraw from the cell cycle and begin to differentiate. In differentiating cells, the induction of muscle-specific genes is accompanied by a distinct morphological chance. However, differentiated BC3H1 cells do not fuse with each other; they remain mononucleated. Metalloendoprotease inhibitors selectively block the differentiation of BC3H1 cells while inhibitors of other protease types are ineffective. In these cells, the degradation of the internalized insulin is initiated by a 110 kDa, non-lysosomal protease known as the insulin-degrading enzyme. The same metalloendoprotease inhibitors that block BC3H1 differentiation also inhibit, with a similar specificity and potency, the in vitro and the in vivo degradation of insulin by the insulin-degrading enzyme. When the serum in the medium is reduced, the activity of the insulin-degrading enzyme in the cell cytoplasm increases rapidly. This increase precedes any detectable change in the differentiation state of these cells by about 12 hours. These results, together with very similar ones obtained with primary rat skeletal muscle cells, support our earlier proposal that the insulin-degrading enzyme is the metalloendoprotease involved in the initiation of the morphological and biochemical differentiation of muscle cells in culture.
Animals↗
An evolutionarily conserved TGF-alpha/insulin-degrading enzyme.
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Amino Acid Sequence↗
Facile identification by electrospray mass spectrometry of the insulin fragment A14-21-B17-30 produced by insulin proteinase.
We confirm the cleavage at position B16-17 of porcine insulin which occurs during in vitro digestion by insulin proteinase. The fragment A14-21-B17-30 was purified by reversed-phase high performance liquid chromatography and characterized by electrospray ionization mass spectrometry. Fast-atom bombardment mass spectrometry, on the other hand, failed to detect the presence of this fragment.
Animals↗
Short-term insulin-induced glycogen formation in primary hepatocytes as a screening bioassay for insulin action.
We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1-13B1-9, A1-14B1-9,and A14-21B14-30 showed no measurable activity. Although preparations of fragment A14-21B10-30 displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.
Animals↗
Identification and isolation of a cytosolic proteolytic complex containing insulin degrading enzyme and the multicatalytic proteinase.
The insulin degrading enzyme (IDE) is the first recognized member of a new class of metalloproteinases. Studies on the purification and the properties of this enzyme have led to divergent results and conclusions from different laboratories. The present manuscript suggests that many of the divergent results may be due to the interaction of this enzyme with other proteins as part of a proteolytic complex. IDE co-isolates with the multicatalytic proteinase (MCP) during a wide variety of purification approaches including affinity chromatography and conventional purification approaches. Ion exchange chromatography will partially or completely separate IDE and MCP. The SDS-PAGE protein bands at various purification steps suggest the presence of a cytosolic proteolytic complex containing IDE, MCP and other unidentified components and raise the possibility of a functional interaction among these proteins.
Amino Acid Sequence↗
Dexamethasone inhibits insulin binding to insulin-degrading enzyme and cytosolic insulin-binding protein p82.
We recently demonstrated that insulin specifically binds to several cytosolic insulin-binding proteins (CIBPs) including insulin-degrading enzyme (IDE) and CIBP p82 in cytosol isolated from H35 rat hepatoma cells. Insulin binding to these CIBPs was regulated by culture conditions, such as serum or insulin. In the present study, we examined the effect of dexamethasone on insulin binding to CIBPs in H35 cells. When the cells were treated with 100 nM dexamethasone for 24 hrs, insulin binding to IDE and CIBP p82 was decreased by about 50% without decreasing the expression level of IDE. Insulin added with the dexamethasone prevented the steroid's effect. Furthermore, dexamethasone directly blocked insulin binding to CIBPs in isolated cytosol. These results suggest that dexamethasone, directly or as a complex with other proteins, binds to IDE and CIBP p82 and changes their ability to bind insulin, possibly by inducing a conformational change or by blocking insulin binding sites. IDE was recently identified as a receptor accessory factor for androgen and glucocorticoid receptors and plays an important role in the regulation of gene transcriptional responses. Combined with previous reports, our findings suggest IDE and other CIBPs such as CIBP p82 may play a role in the cross-talk between insulin and the signal transduction pathways of steroid hormones.
Animals↗
The expression of and insulin binding to cellular thyroid hormone binding protein, but not insulin degrading enzyme, is increased during 3T3-L1 adipocytes differentiation.
Specific insulin binding to several proteins (cytosolic insulin binding proteins; CIBPs) in the isolated cytoplasm of numerous cell types has been demonstrated. CIBPs include insulin degrading enzyme (IDE), CIBP p55 (identified as cellular thyroid hormone binding protein (CTHBP), which is also known as protein disulfide isomerase, or glutathione insulin transhydrogenase). To assess the possible role of CIBP p55/CTHBP in insulin action, we compared 125I-insulin binding to CIBP in cytosol isolated from 3T3-L1 cells at various time points during differentiation of the adipocytes. Insulin did not bind to CTHBP in fibroblasts, but the labeling was markedly increased during adipocyte differentiation. In contrast, insulin binding to IDE did not change during differentiation. Protein expression level of CTHBP in the cytosol fraction increased gradually during the differentiation of adipocytes, whereas that of IDE did not change throughout the period. These data indicate that CTHBP, but not IDE, was up-regulated during differentiation of the adipocytes, suggesting that CIBP p55/CTHBP may play a role in regulating some insulin action, especially the counter regulation between insulin and other hormones during adipocyte differentiation.
3T3 Cells↗
Insulin acts intracellularly on proteasomes through insulin-degrading enzyme.
Insulin decreases cellular protein degradation, but the mechanism of this action is poorly understood. We have shown that insulin can have an inhibitory effect on the action of the proteasome in vitro, which requires the presence of insulin degrading enzyme (IDE). In this study we have used an antibody which inhibits the activity of IDE to show that IDE is required for insulin inhibition of protein degradation in intact cells. The anti-IDE antibody blocked the insulin effect on cellular degradation of proteins prelabeled with radioactive amino acids. The anti-IDE antibody also decreased insulin inhibition of proteasome degradation of a specific substrate in intact cells. These data suggest that insulin works intracellularly via IDE to inhibit protein degradation by the proteasome. Thus, IDE may function as an intracellular mediator for insulin effects on protein degradation. This is a novel signal transduction mechanism for peptide hormones.
Animals↗
Purification of nonantibiotic insulinase inhibitors from bacitracin.
Bacitracin is commonly used in metabolic studies as an insulinase inhibitor. The many isoforms of the commercial preparation were fractionated by charge and size in order to find the most active rat-muscle insulinase inhibitors. CM-Sepharose chromatography revealed that most of the inhibitory activity was contained in a fraction (CM-Inh) that amounted to less than 5% of the mixture. The CM-Inh fraction could be further separated by size on Bio-Gel P4 columns. Six subgroups, each with characteristic specific activity, were isolated. The most potent inhibitor fractions have no antibiotic activity and have molecular weights about twice that of bacitracin A. The peaks isolated by means of Bio-Gel P4 chromatography can be further fractionated by reversed phase HPLC on a C8 column, and by electrophoresis on nonreducing acrylamide gels.
Amino Acids↗
Functional human insulin-degrading enzyme can be expressed in bacteria.
Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE. Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins.
Amino Acid Sequence↗
125I-labelled insulin degradation by isolated rat hepatocytes: the roles of glutathione-insulin transhydrogenase and insulin-specific protease.
Isolated rat hepatocytes degraded 125I-insulin with a Km of 150 nmol/l. Degradation was stimulated by the addition of glutathione and dithiothreitol. In cells incubated with diamide, glutathione was oxidised to the disulphide. Regeneration of reduced glutathione commenced after a further 30 min incubation at 37 degrees C. Diamide (1 mmol/l) significantly inhibited insulin degradation by hepatocytes (p less than 0.001). The 'apparent Vmax' for insulin degradation was decreased tenfold and the Km decreased to 25 nmol/l. The diamide-insensitive degrading activity was cell-associated and produced an intermediate of hormone degradation that was apparently of a higher molecular weight than insulin A chain. The biological activity of the intermediate was 0.03% of that of insulin. The diamide-insensitive activity was not due to release of protease into the medium by cell lysis. We conclude that there are at least two pathways capable of degrading insulin existing in rat hepatocytes.
Animals↗
The renal metabolism of insulin.
The kidney plays a pivotal role in the clearance and degradation of circulating insulin and is also an important site of insulin action. The kidney clears insulin via two distinct routes. The first route entails glomerular filtration and subsequent luminal reabsorption of insulin by proximal tubular cells by means of endocytosis. The second involves diffusion of insulin from peritubular capillaries and subsequent binding of insulin to the contraluminal membranes of tubular cells, especially those lining the distal half of the nephron. Insulin delivered to the latter sites stimulates several important processes, including reabsorption of sodium, phosphate, and glucose. In contrast, insulin delivered to proximal tubular cells is degraded to oligopeptides and amino-acids by one of two poorly delineated enzymatic pathways. One pathway probably involves the sequential action of insulin protease and either GIT or non-specific proteases; the other probably involves the sequential action of GIT and lysosomal proteases. The products of insulin degradation are reabsorbed into the peritubular capillaries, apparently via simple diffusion. Impairment of the renal clearance of insulin prolongs the half-life of circulating insulin by a number of mechanisms and often results in a decrease in the insulin requirement of diabetic patients. Much needs to be learned about these metabolic events at the subcellular level and how they are affected by disease states. Owing to the heterogeneity of cell types within the kidney and to their anatomical and functional polarity, investigation of these areas will be challenging indeed.
Animals↗
Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process.
In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage.
Animals↗
Possible role of cell surface insulin degrading enzyme in cultured human lymphocytes.
The kinetic changes of insulin receptors and cell surface insulin degrading enzyme were examined in Bri-7 cultured human lymphocytes after preincubation with or without insulin. The concentration of cell surface insulin degrading enzyme was determined by immunoenzymatic labeling method using a polyclonal antiserum to insulin degrading enzyme. In Bri-7 cells preincubated with 10(-10) to 10(-5) mol/l insulin for 18 h, the surface insulin receptors and insulin degrading enzyme decreased progressively as a function of the concentration of insulin in the preincubation medium. The surface insulin receptors and insulin degrading enzyme of cells preincubated with 10(-6) mol/l insulin were decreased to 25 and 35% of the control respectively. In Bri-7 cells preincubated with 10(-6) mol/l insulin for 30 min to 18 h, the loss of surface insulin degrading enzyme was slightly slower than that of the receptors; however, the curves were essentially parallel to each other. Thus, the treatment of Bri-7 cells with insulin caused down-regulation of insulin receptors in a dose- and time-dependent manner. Cell surface insulin degrading enzyme also decreased simultaneously. A combination of several insulin degradation assays (trichloroacetic acid precipitation, gel filtration and receptor rebinding) demonstrated that cell surface bound insulin remained intact, and that the degradation in Bri-7 cells seemed to be a limiting proteolysis of insulin. Furthermore, by the receptor rebinding method insulin degrading activity in cells after preincubation with 10(-6) mol/l insulin (19.6 +/- 4.6%) was decreased, although not significantly, as compared with cells after preincubation without insulin (24.6 +/- 4.8%).(ABSTRACT TRUNCATED AT 250 WORDS)
B-Lymphocytes↗