Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation

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

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain : Chemical Synthesis and Biological Activities

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications

Characterization of Viral Insulins Reveals White Adipose Tissue Specific Effects in Mice [preprint]

Members of the insulin/IGF superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single chain (sc), i.e. IGF-1-like, forms of the viral insulin/IGF-1 like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e. more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1). GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A, IR-B) and to the IGF1R, and for the latter show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments in awake mice revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75nmol/kg/min) stimulated a comparable glucose uptake in heart, skeletal muscle and brown adipose tissue, GIV dcVILP stimulated ~2 fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin. Taken together, these results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogues that specifically target the tissues, and provide new insights into their potential role in disease

Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone : Receptor Binding Specificity

Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain

Interaction of a viral insulin-like peptide with the IGF-1 receptor produces a natural antagonist

Lymphocystis disease virus-1 (LCDV-1) and several other Iridoviridae encode viral insulin/IGF-1 like peptides (VILPs) with high homology to human insulin and IGFs. Here we show that while single-chain (sc) and double-chain (dc) LCDV1-VILPs have very low affinity for the insulin receptor, scLCDV1-VILP has high affinity for IGF1R where it can antagonize human IGF-1 signaling, without altering insulin signaling. Consequently, scLCDV1-VILP inhibits IGF-1 induced cell proliferation and growth hormone/IGF-1 induced growth of mice in vivo. Cryo-electron microscopy reveals that scLCDV1-VILP engages IGF1R in a unique manner, inducing changes in IGF1R conformation that led to separation, rather than juxtaposition, of the transmembrane segments and hence inactivation of the receptor. Thus, scLCDV1-VILP is a natural peptide with specific antagonist properties on IGF1R signaling and may provide a new tool to guide development of hormonal analogues to treat cancers or metabolic disorders sensitive to IGF-1 without affecting glucose metabolism. The authors previously identified a family of viral insulin-like peptides (VILPs) with high homology to human insulin/IGF−1. Here, they report that one of these VILPs exhibits antagonist properties associated with a unique conformation of the IGF1R

The efficiency of insulin production and its content in insulin-expressing model β-cells correlate with their Zn 2+ levels : Zn 2+ levels in β-cells

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn 2+ -stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn 2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn 2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage

Interaction studies of insulin, IGF-1/2 and IGF-1 analogue with insulin and IGF-1 receptors

Insulin-like growth factors 1 and 2 (IGF-1/2) are single-chain peptides exerting homology (in both amino-acid sequence and tertiary structure) to insulin. The main function of these peptides is promoting celular growth, proliferation and differentiation. Both insulin and insulin-like growth factors mediate their function through membrane receptors - insulin receptor (isoforms A and B) and IGF-1 receptor. All these receptors are members of the tyrosinkinase family of receptors and they exert the same subunit and domain composition. The activation of insulin and IGF-1 receptors is tightly associated with activation of two intracellular signaling pathways. The PI3-K/Akt pathway is involved in the glucose transport to the cell, induction of proliferation or inhibition of apoptosis, while the Ras/MAPK pathway is involved mainly in the induction of cell growth and differentiation. Due to the structure similarity in both the ligands and receptors, every ligand can activate different receptors (with different potency) and the signaling pathways associated with these receptors. Thus, the functions of IGFs and insulin, the same as their receptors, are overlapping. The distinct function of the concrete ligand can be distinguished by the different tissue distribution of both isoforms of insulin receptor and..

Studium aktivačních vlastností analogů insulinu, IGF-1 a IGF-2 vůči receptorům pro insulin a IGF-1

Systém insulinu a insulinu podobných růstových faktorů (IGF) je velmi komplexní soustava podílející se na regulaci metabolismu a buněčného a tělesného růstu. Součástí tohoto systému jsou tři strukturně příbuzné peptidové hormony: insulin a dva insulinu podobné růstové faktory (IGF-1 a IGF-2) a jejich membránové receptory: dvě isoformy insulinového receptoru (IR-A a IR-B) a IGF-1R receptor (IGF-1R). Zatímco insulin se podílí převážně na regulaci metabolismu, IGF-1 a IGF-2 se účastní hlavně regulace buněčného růstu, proliferace a diferenciace. Vzhledem ke strukturní podobnosti jak hormonů, tak jejich receptorů, mají všechny hormony schopnost vázat se ke všem třem receptorům a aktivovat je, i když v různé míře. Jak dochází k odlišení "metabolických" a "mitogenních" buněčných odpovědí po stimulaci receptorů jednotlivými ligandy nebylo stále zcela objasněno, stejně jako klíčové strukturní faktory podílející se na rozdílných afinitách jednotlivých ligandů k IR-A, IR-B a IGF-1R. Nerovnováha v celém systému vede k řadě patologických stavů, z nichž nejvýznamnější jsou diabetes mellitus a rakovina. Porozumění celému systému na strukturní úrovni je tedy zásadní pro vývoj analogů se žáoucími vlastnostmi, uplatnitelných v klinické praxi. V rámci této práce byla nejprve optimalizována metodologie vhodná pro...The insulin/insulin-like growth factor signaling axis is a complex system that is involved in the regulation of metabolism and body growth. It includes three related peptide hormones: insulin and two insulin-like growth factors (IGF-1 and IGF-2), and their receptors: two isoforms of insulin receptor (IR-A and IR-B), and IGF-1 receptor (IGF-1R). Whereas insulin is involved predominantly in regulation of the metabolism, IGFs participate mainly in the regulation of cell growth, proliferation and differentiation. Due to the similarities in the structures of both the hormones and the receptors, all the hormones can bind and activate all the receptors, although with different potencies. At which point and how are the "metabolic" and "mitogenic" functional outcomes of the system diversified, as well as the structural determinants responsible for the different affinities of the hormones to the individual receptors, is still not completely understood. Deregulation of the insulin/IGF system can result in many types of pathological states, mainly diabetes mellitus and cancer. Therefore, to deeply understand the functioning of the insulin/IGF system to the structural details is crucial for development of analogs with desirable properties, that are of great clinical interest. First of all, we developed a...Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Study of activation properties of insulin, IGF-1 and IGF-2 analogs towards the receptors for insulin and IGF-1

The insulin/insulin-like growth factor signaling axis is a complex system that is involved in the regulation of metabolism and body growth. It includes three related peptide hormones: insulin and two insulin-like growth factors (IGF-1 and IGF-2), and their receptors: two isoforms of insulin receptor (IR-A and IR-B), and IGF-1 receptor (IGF-1R). Whereas insulin is involved predominantly in regulation of the metabolism, IGFs participate mainly in the regulation of cell growth, proliferation and differentiation. Due to the similarities in the structures of both the hormones and the receptors, all the hormones can bind and activate all the receptors, although with different potencies. At which point and how are the "metabolic" and "mitogenic" functional outcomes of the system diversified, as well as the structural determinants responsible for the different affinities of the hormones to the individual receptors, is still not completely understood. Deregulation of the insulin/IGF system can result in many types of pathological states, mainly diabetes mellitus and cancer. Therefore, to deeply understand the functioning of the insulin/IGF system to the structural details is crucial for development of analogs with desirable properties, that are of great clinical interest. First of all, we developed a..

2-DE analysis of hepatocyte secretome under hyperhomocysteinemia

Homocysteine is a plasmatic, sulphur-containing amino acid derived from methionin. It is an amino acid not used in protein synthesis and it's role is to serve as an intermediate in numerous metabolic pathaways. Elevated concentrations of homocysteine in blood are associated with many pathologies, as cardiovascular and neurodegenerative diseases, diabetes or osteoporosis, thus homocysteine has been a common subject of many biochemical experiments in last two decades. For this bachelor thesis, the proteomic comparison of proteins secreted by primary human hepatocytes under 2mM homocysteine and primary human hepatocytes with no homocysteine added to the incubation medium was made, using large-gel two-dimensional electrophoresis. Hepatocytes were incubated in serum free medium for 48 hours. Proteins secreted to medium were precipitated by acetonitrile. Two dimensional electrophoresis was made. Protein maps were analysed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (Bio-Rad, USA). Search criteria for differentially secreted proteins were three-fold change at the signicance level 95 %, Student's t-test. These proteins were cut off the preparative gels and identified by tandem mass spectrometry. In total, there were 17 spots equal to 15 proteins found as differentially exprimed proteins. Among these..