Identification and Characterization of Potential Anti-Hypertensive Therapeutic Candidates in Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Axis

Abstract

Blood pressure (BP) is regulated by a variety of cellular mechanisms. We previously reported the regulation of endothelial nitric oxide (NO)-dependent vessel tone and BP through RhoBTB1-CUL3 mediated proteasomal turnover of Phosphodiesterase 5 (PDE5). We determined that the C-terminal half of RhoBTB1 consisting of two BTB-domains (B1 & B2) and the C-terminal (C) domain of RhoBTB1 can deliver PDE5 to proteasomal pathway. The cellular expression of RhoBTB1 and CUL3 is ubiquitous whereas expression of PDE5 is cell specific. Moreover, vessel tone is regulated through NO-independent mechanisms. Therefore, we hypothesized that RhoBTB1 may have other interacting partners, potentially functioning as regulators of vessel tone and BP in vascular smooth muscle (VSM) cells, through a similar mode of binding as PDE5. Therefore, we utilized ascorbate peroxidase (APEX2) proximity labelling system comprising APEX2 tagged B1B2C domain (B1B2C-APEX2) to identify unknown potential regulators of vasomotor tone in RhoBTB1-CUL3 axis in A7R5 VSM cells. The background of the proteomic screen was minimized by utilizing APEX2-B1B2 which lacks the final C-terminal region of the protein and cannot bind PDE5. We validated the interaction of several identified proteins with RhoBTB1 and the B1B2C domains using co-immunoprecipitation (Co-IP). Our Co-IP data corroborated the proteomic screen as several proteins demonstrated differential interaction with B1B2C and B1B2. Among them, we examined RbFox2, a regulator of mRNA splicing for further investigation. Further data established that RbFox2 is upregulated under the following conditions: 1) pharmacological inhibition of the proteasomal pathway, 2) in CRISPR edited CUL3KO HEK293 cells, 3) in aorta of VSM cell-specific CUL3 deleted mice, 4) by siRNA mediated knock-down of RhoBTB1 in SMCs, and 5) in Ang-II treated SMCs and aorta of Ang-II treated animals. We hypothesized that RbFox2 could modulate actin and actinin-dependent cytoskeletal dynamics, an important regulator of arterial stiffness. First, Co-IP data suggest an interaction between RbFox2 and SMC-specific alpha actinin. Next, siRNA-mediated knock down of RbFox2 altered the actin and actinin-dependent cytoskeletal dynamics in SMCs. Collectively, this establishes the potential regulation of RbFox2 through the RhoBTB1-CUL3 proteasomal pathway and that RbFox2 may play a mechanistic role in arterial stiffness in hypertension. Funding: NIH R35 HL144807 (to Dr. Curt D. Sigmund). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.