Insulin Binding Sites Research Articles

Does insulin make breast cancer cells resistant to doxorubicin toxicity?

The effects of insulin on the doxorubicin (Dox) sensitivity of breast cancer cell line MCF-7 and its Dox-resistant counterpart MCF-7/Dox were studied and glucose metabolism, content of essential minerals, and the expression of several microRNAs in these cells upon exposure to insulin and Dox were compared. Cell viability colorimetric assay, colorimetric enzymatic technique, flow cytometry, immunocytochemical techniques, inductively-coupled plasma atomic emission spectroscopy, and quantitative polymerase chain reaction were used in the study. We found that insulin in high concentration significantly suppressed Dox toxicity, especially in parental MCF-7 cell line. The increase in proliferative activity triggered by insulin in MCF-7 but not MCF-7/Dox cells occurred in the setting of the increased level of specific binding sites for insulin and increased glucose uptake. Insulin treatment of MCF-7 cells in low and high concentrations resulted in the increase of Mg, Ca, and Zn content while in DOX-resistant cells, only Mg content increased upon exposure to insulin. High concentration of insulin increased the expression of kinase Akt1, P-glycoprotein 1 (P-gp1) and DNA excision repair protein ERCC-1 in MCF-7 cells, while in MCF-7/Dox cells, Akt1 expression decreased, and cytoplasmic expression of P-gp1 increased. In addition, insulin treatment affected expression of miR-122-5p, miR-133a-3p, miR-200b-3p, and miR-320a-3p. The decreased manifestation of biological effects of insulin in Dox-resistant cells could be partly explained by the different patterns of energy metabolism in MCF-7 cells and their Dox-resistant counterpart.

A proinsulin-dependent interaction between ENPL-1 and ASNA-1 in neurons is required to maintain insulin secretion in C. elegans.

Neuropeptides, including insulin, are important regulators of physiological functions of the organisms. Trafficking through the Golgi is crucial for the regulation of secretion of insulin-like peptides. ASNA-1 (TRC40) and ENPL-1 (GRP94) are conserved insulin secretion regulators in Caenorhabditis elegans (and mammals), and mouse Grp94 mutants display type 2 diabetes. ENPL-1/GRP94 binds proinsulin and regulates proinsulin levels in C. elegans and mammalian cells. Here, we have found that ASNA-1 and ENPL-1 cooperate to regulate insulin secretion in worms via a physical interaction that is independent of the insulin-binding site of ENPL-1. The interaction occurs in DAF-28/insulin-expressing neurons and is sensitive to changes in DAF-28 pro-peptide levels. Consistently, ASNA-1 acted in neurons to promote DAF-28/insulin secretion. The chaperone form of ASNA-1 was likely the interaction partner of ENPL-1. Loss of asna-1 disrupted Golgi trafficking pathways. ASNA-1 localization to the Golgi was affected in enpl-1 mutants and ENPL-1 overexpression partially bypassed the ASNA-1 requirement. Taken together, we find a functional interaction between ENPL-1 and ASNA-1 that is necessary to maintain proper insulin secretion in C. elegans and provides insights into how their loss might cause diabetes in mammals.

Bioinformatics and Molecular Modeling of the Camel Insulin Receptor

Camels are well known for their high blood glucose content and marked glucose tolerance. In order to understand the glycemic control in camels the role of insulin receptor was investigated. The camel insulin receptor sequence, structure model, and domain content were compared with those of humans and other vertebrates. The results indicated 100% identity rate in old world camels, 99.7% identity rate with new world camels and 96% with humans. There was a high identity rate among insulin receptors in domestic animals (82.3–100%). Despite the conserved features of the insulin 1 binding site (the main functional insulin molecules), the second insulin-binding site in camel insulin receptors showed interesting differences. Most of the sequence differences between human and camel insulin receptors were concentrated in the insertion domains (ID), particularly the ID-β loop. ID-α′∼αCT′∼ID-β, which is important for insulin receptor signal transduction, showed a greater positive electrostatic potential in camels. Such differences might be associated with the noticed hyperglycemia and insulin resistance in camels by affecting the movement of the α-CT helix which lies between the IDs and signifcantly affects the main insulin molecules, lowering the affnity at insulin site 1 and by affecting the transmission of the insulin signal to the intracellular domain.

Effects of tauroursodeoxycholic acid on glucose homeostasis: Potential binding of this bile acid with the insulin receptor

AimsThe bile acid (BA), tauroursodeoxycholic acid (TUDCA) regulates glucose homeostasis; however, it is not clear whether its effects on insulin signaling are due to its direct interaction with the insulin receptor (IR) or through activation of the G-coupled BA receptor, TGR5. We, herein, investigated whether the actions of TUDCA on glucose homeostasis occur via IR or TGR5 activation. Main methodsGlucose homeostasis was evaluated in high-fat diet (HFD)-obese or control (CTL) mice, after 30 days or one intraperitoneal (ip) injection of 300 mg/kg TUDCA, respectively. Molecular docking was performed to investigate the potential binding of TUDCA on the IR and TGR5. Key findingsAfter 30 days of TUDCA treatment, HFD mice exhibited improvements in glucose tolerance and insulin sensitivity, which were abolished when these rodents received the IR antagonist, S961. Molecular docking experiments showed that TUDCA demonstrates high binding affinity for TGR5 and IR and strongly interacts with the insulin binding sites 1 and 2 of the IR. Consistent with this potential agonist activity of TUDCA on IR, CTL mice displayed increased hepatic phosphorylation of AKT after an ip injection of TUDCA. This effect was not associated with altered glycemia in CTL mice and was dependent on IR activation, as S961 prevented hepatic AKT activation by TUDCA. Furthermore, TUDCA activated the hepatic protein kinase A (PKA) and cAMP response element-binding protein (CREB) pathway in CTL mice, even after the administration of S961. SignificanceWe provide novel evidence that TUDCA may be an agonist of the IR, in turn activating AKT and contributing, at least in part, to its beneficial effects upon glucose homeostasis.

Genotype-Structure-Phenotype Correlations of Disease-Associated IGF1R Variants and Similarities to Those of INSR Variants.

We previously reported genotype-phenotype correlations in 12 missense variants causing severe insulin resistance, located in the second and third fibronectin type III (FnIII) domains of the insulin receptor (INSR), containing the α-β cleavage and part of insulin-binding sites. This study aimed to identify genotype-phenotype correlations in FnIII domain variants of IGF1R, a structurally related homolog of INSR, which may be associated with growth retardation, using the recently reported crystal structures of IGF1R. A structural bioinformatics analysis of five previously reported disease-associated heterozygous missense variants and a likely benign variant in the FnIII domains of IGF1R predicted that the disease-associated variants would severely impair the hydrophobic core formation and stability of the FnIII domains or affect the α-β cleavage site, while the likely benign variant would not affect the folding of the domains. A functional analysis of these variants in CHO cells showed impaired receptor processing and autophosphorylation in cells expressing the disease-associated variants but not in those expressing the wild-type form or the likely benign variant. These results demonstrated genotype-phenotype correlations in the FnIII domain variants of IGF1R, which are presumably consistent with those of INSR and would help in the early diagnosis of patients with disease-associated IGF1R variants.

Roles of interstitial fluid pH and weak organic acids in development and amelioration of insulin resistance.

Type 2 diabetes mellitus (T2DM) is one of the most common lifestyle-related diseases (metabolic disorders) due to hyperphagia and/or hypokinesia. Hyperglycemia is the most well-known symptom occurring in T2DM patients. Insulin resistance is also one of the most important symptoms, however, it is still unclear how insulin resistance develops in T2DM. Detailed understanding of the pathogenesis primarily causing insulin resistance is essential for developing new therapies for T2DM. Insulin receptors are located at the plasma membrane of the insulin-targeted cells such as myocytes, adipocytes, etc., and insulin binds to the extracellular site of its receptor facing the interstitial fluid. Thus, changes in interstitial fluid microenvironments, specially pH, affect the insulin-binding affinity to its receptor. The most well-known clinical condition regarding pH is systemic acidosis (arterial blood pH < 7.35) frequently observed in severe T2DM associated with insulin resistance. Because the insulin-binding site of its receptor faces the interstitial fluid, we should recognize the interstitial fluid pH value, one of the most important factors influencing the insulin-binding affinity. It is notable that the interstitial fluid pH is unstable compared with the arterial blood pH even under conditions that the arterial blood pH stays within the normal range, 7.35-7.45. This review article introduces molecular mechanisms on unstable interstitial fluid pH value influencing the insulin action via changes in insulin-binding affinity and ameliorating actions of weak organic acids on insulin resistance via their characteristics as bases after absorption into the body even with sour taste at the tongue.

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain.

Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.

Effect of biomolecule chemical structure on the synthesis of surface magnetic molecularly imprinted polymer in aqueous solution using various monomers for high-capacity selective recognition of human insulin

Magnetic molecular imprinting technique was used for the detection of human insulin. The structure and binding sites of insulin determined by the use of Cn3D software were considered in the selection of functional monomers types and ratios. Methacrylic acid (MAA) was chosen as the electrostatic functional monomer and acrylamide (AAm) as the neutral monomer with a cross-linker of methylenebisacrylamide (MBAAm) for surface imprinting onto vinyl-functionalized Fe3O4 nanoparticles (MNPs). The effect of factors including the monomers' molar ratio, pH of the synthesis media, template concentration, type of eluent solution, and elution time was investigated on the characteristics and performance of molecularly imprinted polymers along with the molecular analysis of the system at the elution stage. The results revealed the importance of considering the template structure. The best result was obtained with an imprinting factor equal to 2.2 by magnetic molecularly imprinted polymers at the molar ratio of 20:100:5 for MAA:AAm:MBAAm (on the basis of 1 M of insulin), synthesized in phosphate buffer (pH = 6) and eluted with methanol/acetic acid (9/1, v/v) solution for 72 h. Its high selective recognition to insulin in the presence of a competitor demonstrated the separation ability of the imprinted polymer to the template molecule.

Structure of the insulin receptor-insulin complex by single-particle cryo-EM analysis.

SummaryThe insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

Structural Basis and Genotype-Phenotype Correlations of INSR Mutations Causing Severe Insulin Resistance.

The insulin receptor (INSR) gene was analyzed in four patients with severe insulin resistance, revealing five novel mutations and a deletion that removed exon 2. A patient with Donohue syndrome (DS) had a novel p.V657F mutation in the second fibronectin type III domain (FnIII-2), which contains the α-β cleavage site and part of the insulin-binding site. The mutant INSR was expressed in Chinese hamster ovary cells, revealing that it reduced insulin proreceptor processing and impaired activation of downstream signaling cascades. Using online databases, we analyzed 82 INSR missense mutations and demonstrated that mutations causing DS were more frequently located in the FnIII domains than those causing the milder type A insulin resistance (P = 0.016). In silico structural analysis revealed that missense mutations predicted to severely impair hydrophobic core formation and stability of the FnIII domains all caused DS, whereas those predicted to produce localized destabilization and to not affect folding of the FnIII domains all caused the less severe Rabson-Mendenhall syndrome. These results suggest the importance of the FnIII domains, provide insight into the molecular mechanism of severe insulin resistance, will aid early diagnosis, and will provide potential novel targets for treating extreme insulin resistance.

Insulin binding characteristics in canine muscle tissue: effects of the estrous cycle phases

Abstract: Hormonal fluctuations during the different estrous cycle are a well-recognized cause of insulin resistance in bitches, and little is known about insulin receptor binding or post-binding defects associated with insulin resistance in dogs. To evaluate insulin binding characteristics in muscle tissue of bitches during the estrous cycle, 17 owned bitches were used in the study (six in anestrus, five in estrus, and six in diestrus). An intravenous glucose tolerance test (IVGTT) was performed in all patients by means of injection of 1mL/kg of a glucose 50% solution (500mg/kg), with blood sample collection for glucose determination at 0, 3, 5, 7, 15, 30, 45 and 60 minutes after glucose infusion. Muscle samples, taken after spaying surgery, were immediately frozen in liquid nitrogen and then stored at -80 ºC until the membranes were prepared by sequential centrifugation after being homogenized. For binding studies, membranes were incubated in the presence of 20,000cpm of human 125I-insulin and in increasing concentrations of unlabeled human regular insulin for cold saturation. The IVGTT showed no differences among bitches during the estrous cycle regarding baseline glycemia or glycemic response after glucose infusion. Two insulin binding sites - high-affinity and low-affinity ones - were detected by Scatchard analysis, and significant statistical differences were observed in the dissociation constant (Kd1) and maximum binding capacity (Bmax1) of the high-affinity binding sites. The Kd1 for the anestrus group (6.54±2.77nM/mg of protein) was smaller (P&lt;0.001) than for the estrus (28.54±6.94nM/mg of protein) and diestrus (15.56±3.88nM/mg of protein) groups. Bmax1 in the estrus (0.83±0.42nM/mg of protein) and diestrus (1.24±0.24nM/mg of protein) groups were also higher (P&lt;0.001) than the values observed in anestrus (0.35±0.06nM/mg of protein). These results indicate modulation of insulin binding characteristics during different phases of the estrous cycle in dogs, showing that muscle insulin binding affinity for its receptor is reduced during estrus and diestrus. However, this poor hormone-receptor affinity is compensated for by a greater total binding capacity, once there is no difference in patients' glycemic response after an intravenous glucose load.

Identification of Host Insulin Binding Sites on Schistosoma japonicum Insulin Receptors

Schistosoma japonicum insulin receptors (SjIRs) have been identified as encouraging vaccine candidates. Interrupting or blocking the binding between host insulin and the schistosome insulin receptors (IRs) may result in reduced glucose uptake leading to starvation and stunting of worms with a reduction in egg output. To further understand how schistosomes are able to exploit host insulin for development and growth, and whether these parasites and their mammalian hosts compete for the same insulin source, we identified insulin binding sites on the SjIRs. Based on sequence analysis and the predicted antigenic structure of the primary sequences of the SjIRs, we designed nine and eleven peptide analogues from SjIR-1 and SjIR-2, respectively. Using the Octet RED system, we identified analogues derived from SjIR-1 (10) and SjIR-2 (20, 21 and 22) with insulin-binding sequences specific for S. japonicum. Nevertheless, the human insulin receptor (HIR) may compete with the SjIRs in binding human insulin in other positions which are important for HIR binding to insulin. However, no binding occurred between insulin and parasite analogues derived from SjIR-1 (2, 7 and 8) and SjIR-2 (14, 16 and 18) at the same locations as HIR sequences which have been shown to have strong insulin binding affinities. Importantly, we found two analogues (1 and 3), derived from SjIR-1, and two analogues (13 and 15) derived from SjIR-2, were responsible for the major insulin binding affinity in S. japonicum. These peptide analogues were shown to have more than 10 times (in KD value) stronger binding capacity for human insulin compared with peptides derived from the HIR in the same sequence positions. Paradoxically, analogues 1, 3, 13 and 15 do not appear to contain major antigenic determinants which resulted in poor antibody responses to native S. japonicum protein. This argues against their future development as peptide-vaccine candidates.

Harnessing Structural Data of Insulin and Insulin Receptor for Therapeutic Designs

To lower blood glucose concentration, insulin binds to insulin receptor (IR) that possesses two distinct insulin binding sites to trigger downstream signaling events leading to an increased uptake of glucose into muscle and fat cells. Comprehensive understandings of structural and dynamic mechanisms of insulin and its receptor are essential to design therapeutic agents for treating and delaying the onset of diabetes that affects over 347 million people worldwide. No full-length IR structure is available hitherto. Harnessing the currently available and state-of-the-art sequence and structural data, we have reviewed the insulin, IR, its extracellular domains and transmembrane domain, to derive structure-based clues to regulate aberrant insulin and its receptor. To propose testable hypotheses and future experiments, we have performed literature review, text mining, multiple structural clustering and normal mode analysis on insulin and its receptor. It appears that insulin-receptor interaction involves allostery and conformational changes (including rotation and tilting) to overcome steric clashes. To target a particular aberrant isoform of IRs, we need to identify the subtle yet distinct differences between IR isoforms. To improve the life quality of diabetics, better structure-based designs of insulin mimetics, formulation and nanotechnology-based delivery are required; efforts to bring them to patients necessitate thorough structural understandings of insulin and its receptor. J Endocrinol Metab. 2015;5(5):273-283 doi: http://dx.doi.org/10.14740/jem302w

Theoretical investigation on insulin dimer-β-cyclodextrin interactions using docking and molecular dynamics simulation

In our study, molecular docking and molecular dynamics (MD) simulations were performed in order to explore the interactions between human insulin and β-cyclodextrin (β-CD). Molecular docking study was performed using the Autodock v4.2 program to determine the number of β-CD molecules that adhere to the binding sites of insulin. A random structure docking approach using an initial ratio of 1:1 insulin-β-CD was conducted and from these, additional β-CDs were added. Molecular docking results revealed that a maximum of four β-CDs are able to bind to the insulin structure with the 1:3 insulin-β-CD ratio producing the lowest binding free energy. The docked conformations showed that hydrophobic interactions played a crucial role in insulin-β-CD conformational stability in addition to the formation of hydrogen bonds. A 50 ns MD simulation was further conducted using an NPT ensemble to verify the results obtained by molecular docking. The analysis of the MD simulation results of the 1:3 insulin-β-CD formation system conclude that a good interaction exists between insulin and β-CDs and the RMSD value obtained was 4.00 ± 0.50 Å. The RMSF profiles of insulin in the 1:3 insulin-β-CD formation also show reduced amino acid residues flexibility as compared to the free insulin system. The theoretical results indicated the presence of significant interactions between insulin and β-CD which could provide interesting insights into an insulin formulation.

Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation.

Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.

A novel insulin receptor-binding protein from Momordica charantia enhances glucose uptake and glucose clearance in vitro and in vivo through triggering insulin receptor signaling pathway.

Diabetes, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principal mediator of glucose homeostasis. In a previous study, we identified a trypsin inhibitor, named Momordica charantia insulin receptor (IR)-binding protein (mcIRBP) in this study, that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-cross-linking coupled with mass spectrometry showed that three regions (17-21, 34-40, and 59-66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cysteine-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by (5.87 ± 0.45)-fold, increased the amount of phospho-IR protein by (1.31 ± 0.03)-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3-L1 cells by (1.36 ± 0.12)-fold. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9 ± 3.2% and 10.8 ± 3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggest that mcIRBP is a novel IRBP that binds to sites different from the insulin-binding sites on IR and stimulates both the glucose uptake in cells and the glucose clearance in mice.

In Memoriam: Guy Dodson (1937–2012)

In Memoriam: Guy Dodson (1937–2012)

How Insulin Binds: From the Co-Crystal Structure of a Model Hormone-Receptor Complex to Novel Therapeutics

Recent advances in the crystallographic analysis of the insulin receptor and its mode of activation by insulin provide a novel foundation for the design of insulin analogues for the treatment of diabetes mellitus. The structure of the receptor ectodomain, elucidated by M. Lawrence, C. Ward, and their colleagues in Melbourne, Australia, revealed an inverted-V dimer in which the primary insulin-binding site comprises the N-terminal beta-helix of one alpha-subunit and a nascent C-terminal alpha-helix from the other. Contact sites and evidence for bidirectional induced fit have been obtained from cross-linking studies employing synthetic photo-reactive insulin derivatives or biosynthetic photo-reactive receptor variants engineered by expanded genetic-code technology. Such basic advances have enabled translational progress toward the development of ultra-concentrated and rapid-acting insulin analogs designed to address unmet clinical needs of minority populations, including African-American women and indigenous Americans with diabetes complicated by obesity and marked insulin resistance. Together, our biochemical, biophysical, and structural studies illustrate the potential value of basic science to multi-disciplinary approaches to health-care disparities among disadvantaged populations.

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K D of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K D of 0.14 nM and for the high-affinity insulin binding sites with K D of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min −1 and 0.013 min −1, respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells.

PH-Dependent Interactions of Human Islet Amyloid Polypeptide Segments with Insulin Studied by Replica Exchange Molecular Dynamics Simulations

Amyloidogenesis of human islet amyloid polypeptide (hIAPP) within or surrounding secretory beta-cells of pancreas has long been related to the pathology of type II diabetes. Insulin, coexpressed and cosecreted with hIAPP in vivo, has the capacity of interacting with hIAPP and further inhibiting the amyloid deposition of the peptide. On the basis of the reported experimental data, we conducted replica exchange molecular dynamics simulations on the complexes of insulin and the binding segment 9-20 of rat/human IAPP under different pH values. The deprotonation of H18 at neutral pH reduces the possibility of polar interactions at position 18 as well as the flanking positions. Moreover, it destabilizes the helical motif of insulin-bound hIAPP. From several perspectives, hIAPP with charged H18 bears more resemblance to rat IAPP (rIAPP) with a point mutation of R18 than its neutral version does. It is likely that the positively charged residue at position 18 is critical to insulin binding. At neutral pH, three loosely binding sites can be recognized on the insulin surface, while at acidic pH one dominant binding site is resolved, which is similar to the previously identified rIAPP-insulin binding site. Several critical intermolecular contacts, including salt-bridges and pi-stacking interactions, identified in the current study, could be used as the starting point for devising a potent peptide inhibitor of hIAPP amyloidogenesis.