Degradation Of Proinsulin Research Articles

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.

UDP-glucose, cereblon-dependent proinsulin degrader

Insulin secretion is regulated in multiple steps, and one of the main steps is in the endoplasmic reticulum (ER). Here, we show that UDP-glucose induces proinsulin ubiquitination by cereblon, and uridine binds and competes for proinsulin degradation and behaves as sustainable insulin secretagogue. Using insulin mutagenesis of neonatal diabetes variant-C43G and maturity-onset diabetes of the young 10 (MODY10) variant-R46Q, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) protects cereblon-dependent proinsulin ubiquitination in the ER. Cereblon is a ligand-inducible E3 ubiquitin ligase, and we found that UDP-glucose is the first identified endogenous proinsulin protein degrader. Uridine-containing compounds, such as uridine, UMP, UTP, and UDP-galactose, inhibit cereblon-dependent proinsulin degradation and stimulate insulin secretion from 3 to 24 h after administration in β-cell lines as well as mice. This late and long-term insulin secretion stimulation is designated a day sustainable insulin secretion stimulation. Uridine-containing compounds are designated as proinsulin degradation regulators.

Proteasomal degradation of WT proinsulin in pancreatic beta cells

Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta-cell gene expression that ultimately lead to beta-cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta-cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently synthesized WT INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing endoplasmic reticulum–associated protein degradation. However, we found that within 60 min after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eukaryotic initiation factor 2 alpha, which can be inhibited by blocking the general control nonderepressible 2 kinase. Together, these data demonstrate that a meaningful subfraction of newly synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing general control nonderepressible 2 kinase activity to maintain ongoing proinsulin biosynthesis.

Fasting Proinsulin Independently Predicts Incident Type 2 Diabetes in the General Population.

Fasting proinsulin levels may serve as a marker of β-cell dysfunction and predict type 2 diabetes (T2D) development. Kidneys have been found to be a major site for the degradation of proinsulin. We aimed to evaluate the predictive value of proinsulin for the risk of incident T2D added to a base model of clinical predictors and examined potential effect modification by variables related to kidney function. Proinsulin was measured in plasma with U-PLEX platform using ELISA immunoassay. We included 5001 participants without T2D at baseline and during a median follow up of 7.2 years; 271 participants developed T2D. Higher levels of proinsulin were associated with increased risk of T2D independent of glucose, insulin, C-peptide, and other clinical factors (hazard ratio (HR): 1.28; per 1 SD increase 95% confidence interval (CI): 1.08–1.52). Harrell’s C-index for the Framingham offspring risk score was improved with the addition of proinsulin (p = 0.019). Furthermore, we found effect modification by hypertension (p = 0.019), eGFR (p = 0.020) and urinary albumin excretion (p = 0.034), consistent with an association only present in participants with hypertension or kidney dysfunction. Higher fasting proinsulin level is an independent predictor of incident T2D in the general population, particularly in participants with hypertension or kidney dysfunction.

PGRMC1 acts as a size-selective cargo receptor to drive ER-phagic clearance of mutant prohormones

The reticulon-3 (RTN3)-driven targeting complex promotes clearance of misfolded prohormones from the endoplasmic reticulum (ER) for lysosomal destruction by ER-phagy. Because RTN3 resides in the cytosolic leaflet of the ER bilayer, the mechanism of selecting misfolded prohormones as ER-phagy cargo on the luminal side of the ER membrane remains unknown. Here we identify the ER transmembrane protein PGRMC1 as an RTN3-binding partner. Via its luminal domain, PGRMC1 captures misfolded prohormones, targeting them for RTN3-dependent ER-phagy. PGRMC1 selects cargos that are smaller than the large size of other reported ER-phagy substrates. Cargos for PGRMC1 include mutant proinsulins that block secretion of wildtype proinsulin through dominant-negative interactions within the ER, causing insulin-deficiency. Chemical perturbation of PGRMC1 partially restores WT insulin storage by preventing ER-phagic degradation of WT and mutant proinsulin. Thus, PGRMC1 acts as a size-selective cargo receptor during RTN3-dependent ER-phagy, and is a potential therapeutic target for diabetes.

The inducible β5i proteasome subunit contributes to proinsulin degradation in GRP94-deficient β-cells and is overexpressed in type 2 diabetes pancreatic islets

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when β-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible β5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of β5i-containing intermediate proteasomes was significantly increased in these cells, as was β5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon β5i small interfering RNA-mediated knockdown. Finally, the fraction of β-cells expressing the β5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that β5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.

Calreticulin protects insulin against reductive stress in vitro and in MIN6 cells

Oxidative folding of proinsulin in the endoplasmic reticulum (ER) is critical for the proper sorting and secretion of insulin from pancreatic β-cells. Here, by using non-cell-based insulin aggregation assays and mouse insulinoma-derived MIN6 cells, we searched for a candidate molecular chaperone for (pro)insulin when its oxidative folding is compromised. We found that interaction between insulin and calreticulin (CRT), a lectin that acts as an ER-resident chaperone, was enhanced by reductive stress in MIN6 cells. Co-incubation of insulin with recombinant CRT prevented reductant-induced aggregation of insulin. Furthermore, lysosomal degradation of proinsulin, which was facilitated by dithiothreitol-induced reductive stress, depended on CRT in MIN6 cells. Together, our results suggest that CRT may be a protective molecule against (pro)insulin aggregation when oxidative folding is defective, e.g. under reductive stress conditions, in vitro and in cultured cells. Because CRT acts as a molecular chaperone for not only glycosylated proteins but also non-glycosylated polypeptides, we also propose that (pro)insulin is a novel candidate client of the chaperone function of CRT.

RILP Restricts Insulin Secretion Through Mediating Lysosomal Degradation of Proinsulin.

Insulin secretion is tightly regulated by membrane trafficking. RILP (Rab7 interacting lysosomal protein) regulates the endocytic trafficking, but its role in insulin secretion has not been investigated. In this study, we found that overexpression of RILP inhibited insulin secretion in both the β-cell lines and freshly isolated islets. Consequently, the expression of RILP in islets suppressed the ability to recover the glucose homeostasis in type 1 diabetes mice upon transplantation. Of physiological relevance is that RILP expression was upregulated in the diabetic mouse islets. Mechanistically, overexpression of RILP induced insulin granule clustering, decreased the number of proinsulin-containing granules in β-cells, and significantly promoted proinsulin degradation. Conversely, RILP depletion sustained proinsulin and increased insulin secretion. The proinsulin degradation induced by RILP expression was inhibited by lysosomal inhibitors and was Rab7-dependent. Finally, we showed that RILP interacts with insulin granule-associated Rab26 to restrict insulin secretion. This study presents a new pathway regulating insulin secretion and mechanically demonstrates a novel function of RILP in modulating insulin secretion through mediating the lysosomal degradation of proinsulin.

A prominent role of PDIA6 in processing of misfolded proinsulin

Despite its critical role in maintaining glucose homeostasis, surprisingly little is known about proinsulin folding in the endoplasmic reticulum. In this study we aimed to understand the chaperones involved in the maturation and degradation of proinsulin. We generated pancreatic beta cell lines expressing FLAG-tagged proinsulin. Several chaperones (including BiP, PDIA6, calnexin, calreticulin, GRP170, Erdj3 and ribophorin II) co-immunoprecipitated with proinsulin suggesting a role for these proteins in folding. To investigate the chaperones responsible for targeting misfolded proinsulin for degradation, we also created a beta cell line expressing FLAG-tagged proinsulin carrying the Akita mutation (Cys96Tyr). All chaperones found to be associated with wild type proinsulin also co-immunoprecipitated with Akita proinsulin. However, one additional protein, namely P58IPK, specifically precipitated with Akita proinsulin and approximately ten fold more PDIA6, but not other PDI family members, was bound to Akita proinsulin. The latter suggests that PDIA6 may act as a key reductase and target misfolded proinsulin to the ER-degradation pathway. The preferential association of PDIA6 to Akita proinsulin was also confirmed in another beta cell line (βTC-6). Furthermore, for the first time, a physiologically relevant substrate for PDIA6 has been evidenced. Thus, this study has identified several chaperones/foldases that associated with wild type proinsulin and has also provided a comprehensive interactome for Akita misfolded proinsulin.

Autophagy is a major regulator of beta cell insulin homeostasis.

We studied the role of protein degradation pathways in the regulation of insulin production and secretion and hypothesised that autophagy regulates proinsulin degradation, thereby modulating beta cell function. Proinsulin localisation in autophagosomes was demonstrated by confocal and electron microscopy. Autophagy was inhibited by knockdown of autophagy-related (ATG) proteins and using the H(+)-ATPase inhibitor bafilomycin-A1. Proinsulin and insulin content and secretion were assessed in static incubations by ELISA and RIA. Confocal and electron microscopy showed proinsulin localised in autophagosomes and lysosomes. Beta-Atg7 (-/-) mice had proinsulin-containing sequestosome 1 (p62 [also known as SQSTM1])(+) aggregates in beta cells, indicating proinsulin is regulated by autophagy in vivo. Short-term bafilomycin-A1 treatment and ATG5/7 knockdown increased steady-state proinsulin and hormone precursor chromogranin A content. ATG5/7 knockdown also increased glucose- and non-fuel-stimulated insulin secretion. Finally, mutated forms of proinsulin that are irreparably misfolded and trapped in the endoplasmic reticulum are more resistant to degradation by autophagy. In the beta cell, transport-competent secretory peptide precursors, including proinsulin, are regulated by autophagy, whereas efficient clearance of transport-incompetent mutated forms of proinsulin by alternative degradative pathways may be necessary to avoid beta cell proteotoxicity. Reduction of autophagic degradation of proinsulin increases its residency in the secretory pathway, followed by enhanced secretion in response to stimuli.

Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97.

Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8+ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8+ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.

Endoplasmic reticulum targeting alters regulation of expression and antigen presentation of proinsulin.

Peptide ligands presented by MHC class I (MHC-I) molecules are produced by degradation of cytosolic and nuclear, but also endoplasmic reticulum (ER)-resident, proteins by the proteasome. However, Ag processing of ER proteins remains little characterized. Studying processing and presentation of proinsulin, which plays a pivotal role in autoimmune diabetes, we found that targeting to the ER has profound effects not only on how proinsulin is degraded, but also on regulation of its cellular levels. While proteasome inhibition inhibited degradation and presentation of cytosolic proinsulin, as expected, it reduced the abundance of ER-targeted proinsulin. This targeting and protein modifications modifying protein half-life also had profound effects on MHC-I presentation and proteolytic processing of proinsulin. Thus, presentation of stable luminal forms was inefficient but enhanced by proteasome inhibition, whereas that of unstable luminal forms and of a cytosolic form were more efficient and compromised by proteasome inhibitors. Distinct stability of peptide MHC complexes produced from cytosolic and luminal proinsulin suggests that different proteolytic activities process the two Ag forms. Thus, both structural features and subcellular targeting of Ags can have strong effects on the processing pathways engaged by MHC-I-restricted Ags, and on the efficiency and regulation of their presentation.

Stromal Cell-Derived Factor 2 Like-1 (SDF2L1) Associates with the Endoplasmic Reticulum-Associated Degradation (ERAD) Machinery and Retards the Degradation of Mutant Proinsulin in Pancreatic β-Cells

Stromal cell-derived factor 2-like 1 (SDF2L1) is an endoplasmic reticulum (ER)-localized protein whose function is undefined. Here we show that SDF2L1 protein levels are increased in response to ER stress-inducing compounds, but not other cell stressors that we tested in insulinoma cell lines. SDF2L1 protein levels were also induced by expression of misfolded proinsulin in insulinoma cells and in islets from diabetic mice. Immunoprecipitation and binding assays demonstrated that SDF2L1 interacts with the ER chaperone GRP78/BiP, the ER-associated degradation (ERAD) machinery and with misfolded proinsulin. Unexpectedly, knockdown of SDF2L1 in INS-1 (insulin 2 C96Y-GFP) cells increased the degradation kinetics of mutant proinsulin, suggesting that SDF2L1 regulates substrate availability for the ERAD system. We suggest that SDF2L1 increases the time that misfolded proteins have to achieve a correctly folded conformation and thus that SDF2L1 can act as a buffer for substrate availability for ERAD in pancreatic β-cells.

Hyperproinsulinemia segregates young adult patients with newly diagnosed autoimmune (type 1) and non‐autoimmune (type 2) diabetes

ObjectiveTo investigate whether measurements of proinsulin and/or intermediate proinsulin degradation products could be used to differentiate between autoimmune (type 1) and non‐autoimmune (type 2) diabetes in young adults. Material and methods Total proinsulin, intact proinsulin and 32,33 split proinsulin concentrations were measured in 25 patients aged 15–34 years with type 1 diabetes, as defined by the presence of at least two positive islet autoantibodies, and in 23 antibody‐negative patients of similar age with type 2 diabetes, at the time of clinical onset of diabetes and at 3–4 months thereafter. Comparisons were made with data from 25 healthy subjects matched for gender and age. Results Plasma levels of total proinsulin, intact proinsulin and 32,33 split proinsulin were significantly increased 2–3‐fold in the patients with newly diagnosed type 2 diabetes as compared with the controls, both in absolute terms (p<0.0001) and when related to circulating insulin (p<0.01–0.0002). In contrast, absolute proinsulin and 32,33 split proinsulin concentrations were significantly lower in patients with onset of type 1 diabetes than in controls. When proinsulin and split proinsulin release were related to plasma insulin, however, similar ratios were found in the type 1 diabetes patients and in controls. Using the 90th percentile for total proinsulin in the control group as the cut‐off, the sensitivity and specificity for differentiation between autoimmune and non‐autoimmune diabetes were 87% and 92%, respectively. At 3–4 months after clinical onset of diabetes, proinsulin secretion was still 2–3 times higher in type 2 than in type 1 diabetes patients (p<0.001). Conclusions. Young adult patients with newly diagnosed type 2 diabetes display disproportionate hyperproinsulinemia, whereas proinsulin secretion appears to be normal in patients with clinical onset of type 1 diabetes. Evaluation of proinsulin and 32,33 split proinsulin concentrations may be useful as a diagnostic tool in differentiating between autoimmune and non‐autoimmune diabetes in young adults, particularly in those lacking islet autoantibodies at diagnosis.

Cell itinerary and metabolic fate of proinsulin in rat liver: in vivo and in vitro studies.

Proinsulin, the insulin precursor in pancreatic beta-cells, displays a slower hepatic clearance than insulin and exerts a more prolonged metabolic effect on liver in vivo. To elucidate the mechanisms underlying these differences, the cellular itinerary and processing of proinsulin and insulin in rat liver have been comparatively studied using cell fractionation. As [125I]-insulin, [125I]-proinsulin taken up into liver in vivo was internalized and accumulated in endosomes, in which it underwent dissociation from the insulin receptor and degradation in a pH- and ATP-dependent manner. However, relative to [125I]-insulin, [125I]-proinsulin showed a delayed and prolonged in vivo association with endosomes, a slower in vivo and cell-free endosomal processing, and a higher cell-free endosome-lysosome transfer. Endosomal extracts degraded to a lesser extent proinsulin than insulin at acidic pH; so did, and even proportionally less, at neutral pH, plasma membrane and cytosolic fractions. Proinsulin degradation products generated by soluble endosomal extracts were isolated by HPLC and characterized by mass spectrometry. Under conditions resulting in multiple cleavages in insulin, proinsulin was cleaved at eight bonds in the C peptide but only at the Phe24-Phe25 bond in the insulin moiety. As native insulin, native proinsulin induced a dose- and time-dependent endocytosis and tyrosine phosphorylation of the insulin receptor; but at an inframaximal dose, proinsulin effects on these processes were of longer duration. We conclude that a reduced proteolysis of proinsulin in endosomes, and probably also at the plasma membrane, accounts for its slower hepatic clearance and prolonged effects on insulin receptor endocytosis and tyrosine phosphorylation.

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells. Implications for the prolonged serum half-life of proinsulin.

Recent studies have reported that elevated proinsulin levels are indicative of an increased cardiovascular risk. Renal proximal tubular cells represent a major site for the metabolism of insulin-like hormones after glomerular filtration into the tubular lumen. To determine the binding and degradation of proinsulin in comparison with insulin and insulin-like growth factor-1 (IGF-1), we have used a rabbit proximal tubular cell line (PT-1). As confirmed by electron microscopy. PT-1 cells exhibit bipolar differentiation, demonstrating apical microvilli and invaginations of the basolateral membrane. To allow selective incubation of both compartments, cells were grown on filter membranes. Performing equilibrium binding assays with 125I-labelled hormones, severalfold higher binding was found at the apical than at the basolateral cell membrane, with the capacity range IGF-1 > insulin > proinsulin. Half-maximal displacement of 125I-labelled insulin and IGF-1 was observed at 0.6 and 2 nM, respectively, while crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Half-maximal displacement of 125I-proinsulin binding was obtained at approx. 8 nM proinsulin and insulin, whereas IGF-1 was 10-fold less potent. The relative degradation of specifically bound tracer was lowest for proinsulin (apical 10%, basolateral: 13%). IGF-1 was degraded by 20% at the apical cell membrane, and up to 78% at the basolateral membrane. In contrast, almost the total amount of insulin bound was degraded at both membrane sites (apical 99%, basolateral: 83%). These results suggest separate insulin and IGF-1 receptors while proinsulin binds with high affinity to a third insulin-like receptor on the apical membrane of PT-1 cells.

A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex

The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins.

Degradation of porcine relaxin by glutathione-insulin transhydrogenase and a neutral peptidase

The susceptibility of porcine relaxin and 125I-polytyrosyl-porcine relaxin to degradation by 3 purified senzymes involved in the degradation of insulin and proinsulin was examined. Rat liver glutathione-insulin transhydrogenase (GIT), which cleaves disulfide bonds in insulin, catalyzed a time- and concentration-dependent increase in trichloroacetic acid (TCA)-soluble radioactivity of relaxin. The Sephadex G-50 profile of the reaction products revealed conversion to the A- and B-chains. Relaxin competitively inhibited the degradation of insulin by GIT; however, kinetic analysis revealed insulin to be preferred over relaxin as a substrate. Rat liver cytosol neutral thiol peptidase (NTP) catalyzed a time- and concentration-dependent increase in the TCA solubility of relaxin and a shift in the Sephadex G-50 radioactivity profile to low molecular weight products. Kinetic analysis revealed that insulin and B-chain are preferred over relaxin as substrates for NTP. A third enzyme, rat kidney neutral metalloendopeptidase, which degrades proinsulin and insulin C-peptide but not insulin, also did not degrade porcine relaxin.

Degradation of proinsulin and isolated C-peptide by rat kidney neutral metallo-endopeptidase

Previous studies have shown that a neutral metallo-endopeptidase purified from rat kidney degrades the B chain of insulin, glucagon, ACTH and, at a markedly slower rate, the A chain of insulin. In contrast the enzyme does not attack native insulin, oxytocin, vasopressin, ribonuclease, albumin or denatured hemoglobin. The current studies demonstrate that the neutral peptidase also degrades the isolated C-peptide of proinsulin and cleaves certain peptide bonds in and near the C-peptide moiety of native proinsulin. Time courses of the formation of fluorescamine-reactive material during digestion of proinsulin and isolated C-peptide with the peptidase were identical. However, structural analysis of the peptidase-digested proinsulin showed that the enzyme does not convert proinsulin to insulin but that the peptidase cleaves one bond, Tyr 26-Thr 27, in the B chain moiety and five bonds in the C-peptide moiety, producing four split proinsulins. One of the split proinsulins is des-octacosapeptide (27–54) porcine proinsulin or des-tetracosapeptide (27–50) bovine proinsulin. Each is a derivative of the insulin molecule having an extension of nine residues (ten residues in the case of the derivative from bovine proinsulin) at the N-terminus of A chain and lacking four residues at the C-terminus of B chain. This two-chain derivative retains full immunoreactivity with insulin antibodies and exhibits 2.4-times more biological activity (promotion of glycogenesis in primary cultured hepatocytes) than proinsulin and about two-thirds the activity of insulin.