Biology subjects

Jiracek, J.

Publications and source records attributed to Jiracek, J..

6 recordsLinked to original sources

bioRxiv2026

Synthetic inhibitors of pregnancy-associated plasma protein-A regulate IGF-1 signaling in vitro and in vivo

Dysregulation of insulin-like growth factor (IGF) signaling contributes to diverse human diseases. Therapeutic strategies have largely targeted the IGF-1 receptor, but the complexity of the IGF system has limited their clinical efficacy. Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloproteinase that regulates local IGF signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs) and represents an attractive therapeutic target. Here, we report the first synthetic PAPP-A inhibitors designed to target the enzyme active site using an IGFBP-5-derived anchor sequence. Replacing the scissile bond with phosphinate or phosphonate moieties yielded potent and selective PAPP-A inhibitors. Cryo-electron microscopy revealed the molecular basis of inhibition and key active-site interactions. The compounds inhibited cleavage of IGFBP-4 and IGFBP-5, two physiological PAPP-A substrates. They suppressed IGF-1 signaling in A549 lung cancer cells, with efficacy comparable to the endogenous PAPP-A inhibitor stanniocalcin-1. They also inhibited Papp-aa-dependent IGF signaling and cell proliferation in wild-type and stc1a-/- mutant zebrafish in vivo in a dose-dependent manner. Together, these findings establish the first class of synthetic, selective PAPP-A inhibitors capable of modulating IGF signaling. They provide new chemical tools for studying and modulating pathological IGF signaling and offer new directions for developing PAPP-A-targeted therapeutic strategies.

biochemistry↗

bioRxiv2025

Structural basis of Drosophila insulin receptor activation by DILP2 hormone

Insulin-related hormones regulate key life processes in the animal kingdom, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin and insulin-like hormones signalling axis (IIS). In humans the IIS axis is controlled by insulin, two Insulin-like Growth Factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR) that follows the blueprint of hIR/hIGF-1R. This report describes two cryo-EM structures of the dmIR ectodomain dmIR-ECD:DILP2 complex, revealing their structural homology with dmIR:DILP5 complex. The high excess of DILP2 yielded two dmIR-ECD complexes in asymmetric conformations, similar to that observed in some complexes of hIR and in the dmIR-ECD:DILP5 complex. This stoichiometric and structural heterogeneity, yielding one- and two-DILP2:receptor complexes - were not observed in DILP5:dmIR-ECD assembly. Also, the resistance of dmIR-ECD to form more DILP2 saturated complexes, despite very high excess of this hormone, suggest that the specificities of DILPs may lie in their kon/koff kinetic parameters. This work expands understanding of the dmIR conformational flexibility, suggesting also that insect dmIR follows more hIR rather than hIGF-1R receptor signal transduction pattern induced by various DILPs.

biochemistry↗

bioRxiv2025

Antagonistic insulin mimetics lock the insulin receptor in an alternative apo-state

Despite significant advancements in high-resolution structural analysis of activated human insulin receptor (IR), the molecular mechanisms underlying its conformational plasticity that govern the transition from the apo state to the activated state are still not well understood. This leaves critical aspects of IR regulation unclear. Here, we reveal the mechanism by which the insulin mimetics Ada, Trim, and S661 fully inhibit the insulin receptor. The receptor is stabilized in a yet structurally un-described {cap}-shaped conformation which is induced by antagonist binding between the L1 and FnIII-1 domains. In contrast to insulin-bound IR structures, the -CT helix is not observable in the {cap} conformation, and the membrane-proximal regions of the FnIII-3 domains are >10 nm apart, which prohibits transmembrane signal transduction and kinase domain activation. Analysis of apo-IR electron cryo-microscopy data indicates that the {cap}-shaped state is one of several metastable apo-IR conformations. These findings underscore the intrinsic conformational dynamics of apo-IR and its role in integrating insulin binding and receptor activation.

molecular biology↗

bioRxiv2025

A novel screening assay for insulin receptor

Insulin is a key hormone in glucose homeostasis. Its lack causes severe health complications and has to be compensated by regular administration of insulin. Despite intense long-lasting research, a more stable and efficient substitute has yet to be discovered to alleviate patients issues. Here we report the development of a new assay for screening potential insulin receptor ligands based on the DNA-linked inhibitor antibody assay (DIANA). Our assay meets the need for a fast, sensitive, non-radioactive method as an alternative to the commonly used radioligand receptor binding assay.

biochemistry↗

bioRxiv2023

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults physiology. Incorrect processing of the IGF2 precursor, pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.

biochemistry↗

bioRxiv2020

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

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.

cell biology↗