Differential rates of conversion of rat proinsulins I and II. Evidence for slow cleavage at the B-chain/C-peptide junction of proinsulin II.
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ABSTRACT:
SUBMITTER: Sizonenko SV
PROVIDER: S-EPMC1151392 | biostudies-other | 1991 Sep
REPOSITORIES: biostudies-other
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Project description:Proinsulin is converted into insulin by the action of two endoproteases. Type I (PC1/PC3) is thought to cleave between the B-chain and the connecting peptide (C-peptide) and type II (PC2) between the C-peptide and the A-chain. An acidic region immediately C-terminal to the point of cleavage at the B-chain/C-peptide junction is well conserved throughout evolution and has been suggested to be important for proinsulin conversion [Gross, Villa-Komaroff, Kahn, Weir and Halban (1989) J. Biol. Chem. 264, 21486-21490]. We have here compared the precise role of this region as a whole and just the first acidic residue C-terminal to the point of cleavage in processing of proinsulin by PC3. To this end, several mutations were introduced in this region of human proinsulin (native sequence, B-chain RREAEDL C-peptide): RRPAEDL (C1Pro mutant); RRLAEDL (C1Leu mutant); RRL (C1-C4del mutant); RRE (del-C1Glu mutant). Mutant and native cDNAs were stably transfected into AtT20 (pituitary corticotroph) cells, in which PC3 is known to be the major conversion endoprotease, and kinetics of proinsulin conversion were studied (pulse-chase/HPLC analysis of proinsulin-related peptides). The results show that the acidic region following the B-chain/C-peptide junction is indeed important for PC3 cleavage at this site, and that the reduced cleavage observed for the C1-C4del mutant proinsulin can be partially overcome by replacing the acidic region with a single acidic residue (del-C1Glu mutant). Replacing only the first residue of the acidic region with leucine (C1Leu mutant) has no impact on conversion, whereas its replacement with proline (C1Pro mutant) almost completely abolishes cleavage at the B-chain/C-peptide junction without affecting that at the C-peptide/A-chain junction.
| S-EPMC1135976 | biostudies-other
Project description:Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.
| S-EPMC8922542 | biostudies-literature
Project description:Due to its potential as an antibiotic target, E. coli peptide deformylase (PDF(Ec)) serves as a model enzyme system for inhibitor design. While investigating the structural-functional and inhibitory features of this enzyme, we unexpectedly discovered that 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) served as a slow-binding inhibitor of PDF(Ec) when the above compound was dissolved only in dimethylformamide (DMF), but not in any other solvent, and allowed to age. The time dependent inhibitory potency of the DMF-dissolved AMT was correlated with the broadening of the inhibitor's 295 nm spectral band toward the visible region, concomitant with the increase in the mass of the parent compound by about 2-fold. These data led to the suggestion that DMF facilitated the slow dimerization of AMT (via the formation of a disulfide bond), and that the dimeric form of AMT served as an inhibitor for PDF(Ec). The latter is not caused by the simple oxidation of sulfhydryl groups by oxidizing agents such as H(2)O(2). Newly synthesized dimeric/dithiolated form of AMT ("bis-AMT") exhibited similar spectral and inhibitory features as given by the parent compound when incubated with DMF. The computer graphic modeling data revealed that bis-AMT could be reliably accommodated within the active site pocket of PDF(Ec), and the above enzyme-ligand interaction involves coordination with the enzyme resident Ni(2+) cofactor. The mechanism of the DMF-assisted activation of AMT (generating bis-AMT), the overall microscopic pathway for the slow-binding inhibition of PDF(Ec) by bis-AMT, and the potential of bis-AMT to serve as a new class of antibiotic agent are presented.
| S-EPMC2855385 | biostudies-literature
Project description:BackgroundHeat shock proteins (Hsp) act as intracellular chaperones and in addition are used as adjuvant in vaccines of peptides complexed with recombinant Hsp. By interacting with autologous peptides, Hsp may promote the induction of autoimmune reactivity.ObjectiveHere, we analysed whether the effect of Hsp on macrophages is modulated by insulin peptides known to interact with Hsp.ResultsCombinations of the 70 kDa Hsp DnaK with peptide B11-23 from the core region of the proinsulin B-chain induced the release of the inflammatory mediators interleukin-6, tumor necrosis factor α, and interleukin-1β from cells of human and murine macrophage lines. In parallel, there was high-affinity binding of B11-23 to DnaK. DnaK mixed with peptides from other regions of the insulin molecule did not stimulate cytokine secretion. DnaK alone induced little cytokine production, and peptides alone induced none.ConclusionThe macrophage-stimulating potential of Hsp70 family proteins when combined with the proinsulin B-chain peptide B11-23 may contribute to the immunodominance of this peptide in the development of beta cell-directed autoimmunity in type 1 diabetes.
| S-EPMC6304834 | biostudies-literature
Project description:Proinsulin C-peptide (C-peptide) has drawn much research attention. Even if the peptide has turned out not to be important in the treatment of diabetes, every phase of C-peptide research has changed our view on insulin and peptide hormone biology. The first phase revealed that peptide hormones can be subject to processing, and that their pro-forms may involve regulatory stages. The second phase revealed the possibility that one prohormone could harbor more than one activity, and that the additional activities should be taken into account in the development of hormone-based therapies. In the third phase, a combined view of the evolutionary patterns in hormone biology allowed an assessment of C-peptide´s role in physiology, and of how biological activities and physiological functions are shaped by evolutionary processes. In addition to this distinction, C-peptide research has produced further advances. For example, C-peptide fragments are successfully administered in immunotherapy of type I diabetes, and plasma C-peptide levels remain a standard for measurement of beta cell activity in patients. Even if the concept of C-peptide as a hormone is presently not supported, some of its bioactivities continue to influence our understanding of evolutionary changes of also other peptides.
| S-EPMC7897624 | biostudies-literature
Project description:In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific CD8⁺ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8⁺ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8⁺ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8⁺ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.
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Project description:BACKGROUND:Prohormone convertase 1 is involved in maturation of peptides. Rare mutations in gene PCSK1, encoding this enzyme, cause childhood obesity and abnormal glucose homeostasis with elevated proinsulin concentrations. Common single nucleotide polymorphisms (SNPs) within this gene, rs6232 and rs6235, are associated with obesity. We studied whether these SNPs influence the prediabetic traits insulin resistance, beta-cell dysfunction, or glucose intolerance. METHODS:We genotyped 1498 German subjects for SNPs rs6232 and rs6235 within PCSK1. The subjects were metabolically characterized by oral glucose tolerance test with glucose, insulin, proinsulin, and C-peptide measurements. A subgroup of 512 subjects underwent a hyperinsulinemic-euglycemic clamp. RESULTS:The minor allele frequencies were 25.8% for SNP rs6235 and 6.0% for rs6232. After adjustment for sex and age, we found no association of SNPs rs6235 and rs6232 with BMI or other weight-related traits (all p >or= 0.07). Both minor alleles, adjusted for sex, age, BMI and insulin sensitivity were associated with elevated AUCproinsulin and AUCproinsulin/AUCinsulin (rs6235: p(additive) model <or= 0.009, effect sizes 8/8%, rs6232: pdominant model <or= 0.01, effect sizes 10/21%). Insulin secretion was not affected by the variants (different secretion parameters, all p >or= 0.08). The minor allele of SNP rs6232 was additionally associated with 15% higher OGTT-derived and 19% higher clamp-derived insulin sensitivity (pdom <or= 0.0047), 4.5% lower HOMAIR (pdom = 0.02) and 3.5% lower 120-min glucose (pdom = 0.0003) independently of BMI and proinsulin conversion. SNP rs6235 was not associated with parameters of glucose metabolism. CONCLUSIONS:Like rare mutations in PCSK1, the more common variants tested determine glucose-stimulated proinsulin conversion, but not insulin secretion. In addition, rs6232, encoding the amino acid exchange N221D, influences insulin sensitivity and glucose homeostasis.
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Project description:INTRODUCTION: Recent meta-analyses of genome-wide association studies revealed new genetic loci associated with fasting glycemia. For several of these loci, the mechanism of action in glucose homeostasis is unclear. The objective of the study was to establish metabolic phenotypes for these genetic variants to deliver clues to their pathomechanism. METHODS: In this cross-sectional study 1782 non-diabetic volunteers at increased risk for type 2 diabetes underwent an oral glucose tolerance test. Insulin, C-peptide and proinsulin were measured and genotyping was performed for 12 single nucleotide polymorphisms (SNP) in or near the genes GCK (rs4607517), DGKB (rs2191349), GCKR (rs780094), ADCY5 (rs11708067), MADD (rs7944584), ADRA2A (rs10885122), FADS1 (rs174550), CRY2 (rs11605924), SLC2A2 (rs11920090), PROX1 (rs340874), GLIS3 (rs7034200) and C2CD4B (rs11071657). Parameters of insulin secretion (AUC Insulin(0-30)/AUC Glucose(0-30), AUC C-peptide(0-120)/AUC Glucose(0-120)), proinsulin-to-insulin conversion (fasting proinsulin, fasting proinsulin/insulin, AUC Proinsulin(0-120)/AUCInsulin(0-120)) and insulin resistance (HOMA-IR, Matsuda-Index) were assessed. RESULTS: After adjustment for confounding variables, the effect alleles of the ADCY5 and MADD SNPs were associated with an impaired proinsulin-to-insulin conversion (p?=?0.002 and p?=?0.0001, respectively). GLIS3 was nominally associated with impaired proinsulin-to-insulin conversion and insulin secretion. The diabetogenic alleles of DGKB and PROX1 were nominally associated with reduced insulin secretion. Nominally significant effects on insulin sensitivity could be found for MADD and PROX1. DISCUSSION: By examining parameters of glucose-stimulated proinsulin-to-insulin conversion during an OGTT, we show that the SNP in ADCY5 is implicated in defective proinsulin-to-insulin conversion. In addition, we confirmed previous findings on the role of a genetic variant in MADD on proinsulin-to-insulin conversion. These effects may also be related to neighboring regions of the genome.
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