Kretschmer, M. | bioRxiv Science

bioRxiv2023

Harnessing PROTAC technology to combat stress hormone receptor activation

Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, novel catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GRs genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in-vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.

molecular biology↗

bioRxiv2022

Matrix stiffness regulates Notch signaling activity in endothelial cells

The Notch signaling pathway plays a critical role in many developmental and disease related processes. It is widely accepted that Notch has a mechano-transduction module that regulates cleavage of the receptor. However, the role of biomechanical properties of the cellular environment on this module and on Notch signaling in general is still poorly understood. During angiogenesis, differentiation into tip and stalk cells is regulated by Notch. The endothelial cells in this process respond to biochemical and mechanical cues triggered by local stiffening of the ECM. Here, we investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness tuned PDMS substrates we show that Notch signaling pathway activity inversely correlates with the physiologically relevant substrate stiffness, with increased Notch activity on softer substrates. We show that trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness. Furthermore, we could show that integrin cell-matrix connections are both stiffness-dependent and influenced by Notch. Cadherin mediated cell-cell adhesion and Notch, however, influence each other in that basal Notch signaling is cell-cell contact dependent, but inhibition of the Notch signaling pathway also results in a reduction of VE-cadherin levels. We conclude that mechano-transduction of Notch activation depends on substrate stiffness highlighting the role of substrate rigidity as a modulator of Notch signaling. This may have important implications in pathological situations, such as tumor growth, associated with stiffening of the extracellular matrix.

cell biology↗