Preptin is a 34-amino-acid-long peptide derived from the E-domain of a precursor of insulin-like growth factor 2 (pro-IGF2) with bone-anabolic and insulin secretion amplifying properties. Here, we describe the synthesis, structures, and biological activities of six shortened analogues of human preptin. Eight- and nine-amino-acid-long peptide amides corresponding to the C-terminal part of human preptin were stabilised by two types of staples to induce a higher proportion of helicity in their secondary structure. We monitored the secondary structure of the stapled peptides using circular dichroism. The biological effect of the structural changes was determined afterwards by the ability of peptides to stimulate the release of intracellular calcium ions. We confirmed the previous observation that the stabilisation of the disordered conformation of human preptin has a deleterious effect on biological potency. However, surprisingly, one of our preptin analogues, a nonapeptide stabilised by olefin metathesis between positions 3 and 7 of the amino acid chain, had a similar ability to stimulate calcium ions' release to the full-length human preptin. Our findings could open up new ways to design new preptin analogues, which may have potential as drugs for the treatment of diabetes and osteoporosis.
- MeSH
- insulinu podobný růstový faktor II * chemie MeSH
- kosti a kostní tkáň MeSH
- lidé MeSH
- peptidové fragmenty chemie MeSH
- peptidy MeSH
- vápník * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58-IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.
- MeSH
- insulinu podobný růstový faktor I chemie genetika MeSH
- insulinu podobný růstový faktor II chemie genetika MeSH
- inzulin analogy a deriváty chemická syntéza chemie genetika MeSH
- lidé MeSH
- mnohočetné abnormality genetika MeSH
- mutace genetika MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- poruchy růstu genetika MeSH
- proteinové domény genetika MeSH
- receptor IGF typ 1 chemie genetika MeSH
- receptor inzulinu chemie genetika MeSH
- sekvence aminokyselin genetika MeSH
- vazba proteinů genetika MeSH
- vazebná místa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2', which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications.
- MeSH
- fosforylace MeSH
- insulinu podobný růstový faktor I chemie genetika MeSH
- insulinu podobný růstový faktor II chemie genetika MeSH
- inzulin chemie metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární evoluce MeSH
- mutace MeSH
- protein - isoformy MeSH
- receptor inzulinu chemie metabolismus MeSH
- receptory somatomedinů chemie genetika MeSH
- signální transdukce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.
- MeSH
- CD antigeny chemie genetika metabolismus MeSH
- insulinu podobný růstový faktor II chemie genetika metabolismus MeSH
- lidé MeSH
- missense mutace MeSH
- protein - isoformy chemie genetika metabolismus MeSH
- proteinové domény MeSH
- receptor IGF typ 1 chemie genetika metabolismus MeSH
- receptor inzulinu chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- substituce aminokyselin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.
- MeSH
- antitumorózní látky terapeutické užití MeSH
- cetuximab MeSH
- chemorezistence MeSH
- erbB receptory chemie MeSH
- exony MeSH
- humanizované monoklonální protilátky terapeutické užití MeSH
- imunohistochemie MeSH
- insulinu podobný růstový faktor II chemie MeSH
- klinické zkoušky jako téma MeSH
- kolorektální nádory farmakoterapie metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- mutace MeSH
- myši MeSH
- nádorové biomarkery metabolismus MeSH
- transplantace nádorů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Until recently, despite decades of intensive research, the exact mode of interaction of insulin with its membrane receptor remained elusive. However, recent advances achieved with insulin analogues and crystallographic studies with the insulin receptor provided a first insight into the interaction of insulin with its primary binding site on the insulin receptor. This review describes the story of insulin research at the above Institute.
- MeSH
- diabetes mellitus * farmakoterapie MeSH
- dimerizace MeSH
- farmacie MeSH
- insulinu podobný růstový faktor I farmakokinetika farmakologie chemie škodlivé účinky terapeutické užití MeSH
- insulinu podobný růstový faktor II farmakokinetika farmakologie chemie škodlivé účinky terapeutické užití MeSH
- inzulin * analogy a deriváty chemická syntéza chemie MeSH
- lidé MeSH
- molekulární konformace MeSH
- receptor inzulinu chemie MeSH
- výzkum MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
