Biology subjects

Cash, J.

Publications and source records attributed to Cash, J..

3 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of the sub-50 kD PDZ-RhoGEF catalytic core-RhoA complex reveals an allosteric mechanism for GEF switch

PDZ-RhoGEF (PRG) drives cellular processes including migration through activation of the GTPase RhoA. The GEF switch (GS) is an accessory region N-terminal to the PRG catalytic core that significantly enhances its activity against RhoA, but its mechanism is unknown. We take an integrative structural biology approach, including cryogenic electron microscopy, to deduce the GS mechanism. Enzymatic assays define interactions between the GS and PRG catalytic core that are required for enhancement of activity. While SEC-SAXS data suggest that the GS does not alter the global structure of the catalytic core-RhoA complex, HDX-MS data reveal that the GS induces widespread stabilization, particularly in the RhoA switch regions. We determined a sub-4 angstrom resolution structure of 46 kD of the complex between the catalytic core and RhoA, revealing that Switch I is more flexible without the GS. Collectively, these data suggest both direct and allosteric factors in the mechanism of the GS.

biochemistry↗

bioRxiv2026

P-Rex2 exhibits unique structural features and regulatory mechanisms distinct from the closely related RhoGEF P-Rex1

Rho guanine-nucleotide exchange factors (RhoGEFs) activate small GTPases to drive cytoskeletal rearrangement, cell motility, and proliferation. The phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) subfamily of RhoGEFs includes P-Rex1 and P-Rex2 which, when misregulated, contribute to cancer progression and metastasis. P-Rex activity is controlled by accessory domains that maintain the protein in a cytosolic, autoinhibited state until activated by the lipid PIP3 and G protein {beta}{gamma} subunits. While P-Rex1 autoinhibition has been structurally and biochemically characterized, P-Rex2 has remained largely unexplored. Furthermore, despite high sequence similarity and domain conservation, P-Rex homologs differ in substrate specificity and regulatory interactions, and the molecular basis for these divergences is unknown. Here, we have taken an integrative structural biology approach to investigate these gaps. Using cryo-EM, we determined the first structure of full-length P-Rex2 to moderate resolution, revealing that, while the overall structure closely resembles that of P-Rex1, there is a substantial repositioning of the N-terminal module relative to the C-terminal core. This may play a key role in precluding the intramolecular interactions between the N- and C-terminal domains that are observed in autoinhibited P-Rex1. Hydrogen-deuterium exchange mass spectrometry revealed that, unlike P-Rex1, P-Rex2 dynamics are unaffected by IP4, the headgroup of PIP3. SEC-SAXS data support that the N-terminal module itself is less dynamic, and biochemical assays show that P-Rex2 may be more tightly regulated by autoinhibition, likely through a mechanism different from P-Rex1. These findings uncover unique features in the molecular mechanisms of P-Rex2 regulation.

biochemistry↗

bioRxiv2017

Phosphatidylserine Exposure after Vascular Injury-Platelet or Endothelial

BackgroundPlatelets membranes are considered the paramount site for the assembly of tenase and prothrombinase complex and are key players in localising coagulation to wound sites. However, the endothelium is also known to express phosphatidylserine (PS) and support the binding of recombinant FVa/FXa even beyond the site of injury. It thus remains unclear, what cell type play the preeminent role in the cellular control of coagulation after vascular injury.\n\nApproachTo address this question, we utilised a model of haemostasis (full thickness 1mm excisional skin wounds) as well as tissues after injury in laser and ferric chloride models of thrombosis. Damage to the endothelium was assessed by the combined methods of picrosirius red staining, immunofluorescence and electron microscopy. Using multiphoton microscopy, we then compared the spatial distribution of PS on platelets and the endothelium.\n\nResultsPlatelets and detectable PS significantly co-localised compared with similar analysis of endothelial cell and exposed PS on wounded carotids arteries which was not significant. Point injury by laser induced restricted damage of the endothelium which was associated with limited platelets recruitment. In consistence with platelets response after FeCl3 injury, platelets exposed most of the PS detected at the wound edge where skin vessels were transected in our haemostasis model (Correlation Coeff. 0.78 +/- 0.12 vs 0.35 +/- 0.23).\n\nConclusionsWe surmised that data from the different models support a paradigm of graded haemostatic response to vascular injury, in which full platelets response is limited to wound sites exposing the sub-endothelial matrix.

physiology↗