Panek, D. | bioRxiv Science

bioRxiv2023

Topical siRNA therapy of diabetic-like wound healing

Non-healing wounds are a serious complication in diabetic patients. One of the detrimental factors contributing to limited wound healing is the accumulation of metalloproteinase-9 (MMP-9) in the wound. Selective inhibition of MMP-9 is one of the established therapeutic targets for diabetic wound healing and is therefore of great interest. Here we focused on development of gene silencing system for localized delivery of antisense siRNA against MMP-9 into the wound. We have developed a functional and biocompatible wound dressing allowing controlled release of a traceable vector loaded with the target siRNA. Specifically, the dressing consists of a degradable scaffold of polymer nanofibers embedded with the vector nanosystem, polymer-coated fluorescent nanodiamonds (FNDs). The biocompatible cationic polymer shell on FNDs was designed and optimized for binding of siRNA and formation of colloidally stable FND-siRNA complexes in physiological environment. A hybrid nanofiber scaffold consisting of poly(vinyl alcohol) and poly(caprolactone) ensured continuous release of FND-siRNA complexes from the dressing. The photostable luminescence of FNDs allowed us to monitor the vector system in the wound. Our dressing was tested on murine fibroblasts and also applied to wounds in a diabetic murine model to evaluate its suitability in terms of in vivo toxicity, biological efficacy and manipulation. The treatment resulted in significant local inhibition of MMP-9 and reduction of the wound healing time. The scar formation for treated diabetic-like animals became comparable with non-treated diabetes-free mice. Our results suggest that the application of our biocompatible dressing loaded with a non-toxic vector nanosystem is an effective and promising approach in gene therapy of non-healing wounds.

bioengineering↗

bioRxiv2022

Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase

The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signaling hub in a variety of cellular processes.

cell biology↗