Parise, A. | bioRxiv Science

bioRxiv2026

Glutamine Tautomerization Drives RhoGAP-Aided GTP Hydrolysis in Small Rho GTPases

Rho GTPases promote GTP hydrolysis aided by specific GTPase-activating proteins (GAPs). By alternating between an active GTP-bound and an inactive GDP-bound state, Rho GTPases function as molecular switches regulating cytoskeletal dynamics and cell motility. Despite their biological relevance, the detailed molecular mechanism underlying Rho GTPases catalysis remains contentious. Here, using classical and hybrid quantum-classical molecular dynamics, we resolve the mechanism of GTP hydrolysis in the RhoGAP-RhoA complex. We reveal that GTP hydrolysis proceeds through a dissociative nucleophilic substitution mechanism, driven by an amide [->] imide tautomerization of Gln63, which aids in delivering a proton from the nucleophilic water to the leaving phosphate group. The Gln63 imide tautomer also loosens RhoGAP-RhoA interfacial contacts, allowing solvent molecules to enter and drive a water-mediated reverse tautomerization of Gln63 that restores the catalytically-competent configuration of the RhoA active site. Conservation of key interface residues across Rho/Rho GAP family members suggests that this mechanism may be shared by most Rho GTPases.

biophysics↗

bioRxiv2025

The Covalent Binding of Splicing Inhibitors occurs via a Zinc- Assisted Enzymatic-like mechanism

Branch-site recognition is a pivotal event in spliceosome assembly, and branch-site antagonists disrupt this process, inducing a splicing rewiring that underlies much of their antitumor activity. Antagonists from the spliceostatin family covalently bind the SF3b complex nearby the branch-site pocket, forming an adduct with Cys26 of the PHF5A zinc finger while remarkably preserving Zn2+ coordination - an unconventional mode of covalent inhibition. Yet the molecular features that activate this normally protected ZnCys4 cysteine and initiate spliceostatins epoxide ring opening have remained obscure. Here, we combine extensive classical and QM/MM molecular dynamics simulations to map the full reactive trajectory of spliceostatin attachment to PHF5A. We identify two alternative noncovalent spliceostatin binding poses and show that only its thermodynamically favoured conformation pre-organizes the epoxide for nucleophilic attack. The local distortion of the ZnCys4 coordination sphere weakens the Zn-Cys26 bond, enabling water-Cys26 exchange and generating a highly reactive nucleophilic thiolate. Covalent bond formation is subsequently accelerated by an Asp34-Lys29 proton relay that activates the epoxide leaving group. Free-energy calculations confirm that the overall reaction is fast and strongly exergonic. Together, these findings provide a complete mechanistic framework for zinc-assisted inhibition of early spliceosomes by branch-site antagonists and advance our fundamental understanding of zinc finger reactivity.

biochemistry↗

bioRxiv2022

Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Differentiation of Aortic Valve Cells Through STAT5

BackgroundCalcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in valve leaflet osteogenic reprogramming are not fully understood. As telomerase reverse transcriptase (TERT) overexpression primes mesenchymal stem cells to differentiate into osteoblasts, we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). MethodsHuman control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/-and Terc-/- mice were used for mechanistic studies. Calcification was assessed in Tert+/+ and Tert-/- mice ex vivo and in vivo. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. ResultsTERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic or inflammatory stimuli, and knock-down or genetic deletion of TERT prevented calcification in vitro and in vivo. Mechanistically, TERT was upregulated via NF-{kappa}B and required to initiate osteogenic reprogramming, independent of its canonical reverse transcriptase activity and the lncRNA TERC. TERT exerts non-canonical osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion or inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Lastly, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue compared to control tissue. ConclusionsTERTs non-canonical activity is required to initiate calcification. TERT is upregulated via inflammatory signaling pathways and partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification. Novelty and SignificanceWhat is known? Calcific aortic valve disease (CAVD) is the most prevalent form of aortic valve pathology. CAVD strongly correlates with age and leads to heart failure and a high risk of stroke. Currently, the only therapeutic option is valve replacement, which comes with significant healthcare costs and additional risks to patients. Runt-related transcription factor 2 (RUNX2) is the master transcription factor required for osteogenic differentiation of stem cells to osteoblasts and osteogenic reprogramming of cardiovascular cells. Yet, the early events driving its activity in aortic valve cells are poorly defined. In addition to its reverse transcriptase enzymatic activity, TERT exhibits non-canonical transcriptional regulatory functions and overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage. What new information does this article contribute? TERT protein levels in calcified aortic leaflets and valve interstitial cells, and its non-canonical osteogenic activity are independent of changes in telomere length and cell senescence. Genetic loss or depletion of TERT prevented calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells in vitro and the vasculature in vivo. Early in the osteogenic reprogramming inflammatory signaling promotes TERT to co-localize with SMARCA4 and STAT5, and this TERT-tethered STAT5 binds to the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs. STAT5 depletion and pharmacological inhibition prevent calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells. What are the clinical implications? We have identified TERT-STAT5 as a novel signaling axis that orchestrates the early steps in the osteogenic reprogramming of aortic valve cells. Inhibiting TERT/STAT5 interaction or their activity may be leveraged for the development of therapeutic strategies to halt or prevent calcification in the aortic valve, bioprosthetic valves, and or perhaps other cardiovascular tissues. Invasive and expensive surgical procedures are currently the only treatment option for patients with CAVD. The discovery and defining of the early events driving vascular calcification identifies novel and druggable targets for developing non-surgical therapies.

pathology↗