The preparation of specifically iodine-125 (125I)-labeled peptides of high purity and specific activity represents a key tool for the detailed characterization of their binding properties in interaction with their binding partners. Early synthetic methods for the incorporation of iodine faced challenges such as harsh reaction conditions, the use of strong oxidants and low reproducibility. Herein, we review well-established radiolabeling strategies available to incorporate radionuclide into a protein of interest, and our long-term experience with a mild, simple and generally applicable technique of 125I late-stage-labeling of biomolecules using the Pierce iodination reagent for the direct solid-phase oxidation of radioactive iodide. General recommendations, tips, and details of optimized chromatographic conditions to isolate pure, specifically 125I-mono-labeled biomolecules are illustrated on a diverse series of (poly)peptides, ranging up to 7.6 kDa and 67 amino acids (aa). These series include peptides that contain at least one tyrosine or histidine residue, along with those featuring disulfide crosslinking or lipophilic derivatization. This mild and straightforward late-stage-labeling technique is easily applicable to longer and more sensitive proteins, as demonstrated in the cases of the insulin-like growth factor binding protein-3 (IGF-BP-3) (29 kDa and 264 aa) and the acid-labile subunit (ALS) (93 kDa and 578 aa).

• Keywords
• 125I-labeling of peptides, High specific activity, Intramolecular effect of 125I decay, Late-stage peptide labeling, Radiochemical stability, Radiohalogenated prosthetic groups, Site-specific labeling,
• Publication type
• Journal Article MeSH
• Review MeSH

OBJECTIVE: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. METHODS: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. RESULTS: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. CONCLUSIONS: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.

• Keywords
• Biased signaling, IGF-1, IGF1 receptor, IGF1 receptor inhibition, Insulin, Iridoviridae, Viral insulin/IGF-1 like peptides (VILPs),
• MeSH
• Cryoelectron Microscopy MeSH
• Gene Expression MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Phosphorylation MeSH
• Insulin-Like Growth Factor I * genetics   metabolism   MeSH
• Insulin metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Protein Isoforms metabolism   MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Receptor, IGF Type 1 * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphatidylinositol 3-Kinases MeSH
• IGF1R protein, human MeSH Browser
• Insulin-Like Growth Factor I * MeSH
• Insulin MeSH
• Protein Isoforms MeSH
• Proto-Oncogene Proteins c-akt MeSH
• Receptor, IGF Type 1 * MeSH

OBJECTIVE: Members of the insulin/insulin-like growth factor (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) for the first time. METHODS: The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. RESULTS: GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and 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 revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and 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 in WAT. CONCLUSIONS: Our 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 analogs that specifically target the tissues and provide new insights into their potential role in disease.

• Keywords
• Adipose tissue, GLUT4, Glucose metabolism, IGF-1, Insulin, VILPs, Viral insulin, Viral mimicry,
• MeSH
• Adipose Tissue, White metabolism   MeSH
• Cell Line MeSH
• Antigens, CD MeSH
• Phosphorylation MeSH
• Glucose metabolism   MeSH
• Adipose Tissue, Brown metabolism   MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Insulin genetics   metabolism   MeSH
• Insulins metabolism   MeSH
• Iridovirus genetics   MeSH
• Iridoviridae genetics   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Receptor, IGF Type 1 genetics   metabolism   MeSH
• Receptor, Insulin metabolism   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antigens, CD MeSH
• Glucose MeSH
• IGF1 protein, human MeSH Browser
• IGF1R protein, human MeSH Browser
• Igf1r protein, mouse MeSH Browser
• INSR protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin MeSH
• Insulins MeSH
• Receptor, IGF Type 1 MeSH
• Receptor, Insulin MeSH

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

• MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Insulin-Like Growth Factor II metabolism   MeSH
• Binding, Competitive MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Iodine Radioisotopes MeSH
• Radioligand Assay methods   MeSH
• Receptor, IGF Type 2 immunology   ultrastructure   MeSH
• Signal Transduction MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IGF1 protein, human MeSH Browser
• IGF2 protein, human MeSH Browser
• IGF2R protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin-Like Growth Factor II MeSH
• Iodine-125 MeSH Browser
• Iodine Radioisotopes MeSH
• Receptor, IGF Type 2 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.

• Keywords
• NMR structure, complex, hormone analog, insulin, insulin-like growth factor (IGF), molecular dynamics, mutagenesis, peptide hormone, receptor autophosphorylation, receptor binding, receptor tyrosine kinase, structural biology, structure-function,
• MeSH
• Insulin-Like Growth Factor I chemistry   genetics   MeSH
• Insulin-Like Growth Factor II chemistry   genetics   MeSH
• Insulin analogs & derivatives   chemical synthesis   chemistry   genetics   MeSH
• Humans MeSH
• Abnormalities, Multiple genetics   MeSH
• Mutation genetics   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Growth Disorders genetics   MeSH
• Protein Domains genetics   MeSH
• Receptor, IGF Type 1 chemistry   genetics   MeSH
• Receptor, Insulin chemistry   genetics   MeSH
• Amino Acid Sequence genetics   MeSH
• Protein Binding genetics   MeSH
• Binding Sites genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IGF1R protein, human MeSH Browser
• IGF2 protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin-Like Growth Factor II MeSH
• Insulin MeSH
• Receptor, IGF Type 1 MeSH
• Receptor, Insulin MeSH