Abstract P136: Exploring The Interactions Between Rho-related BTB Domain Containing 1 (RhoBTB1) And Phosphodiesterase 5 (PDE5) To Identify Novel Therapeutic Candidates To Counter Hypertension

Abstract

Protein-protein interaction studies have provided valuable insights to understand fundamental mechanisms in cells. We previously identified that PDE5 is a binding partner of RhoBTB1 and stringently regulates PDE5 activity in vascular smooth muscle (VSM). This in turn regulates the nitric oxide response of VSM and consequently blood pressure. We showed that RhoBTB1 can function as an adapter for Cullin-3 (CUL3) E3 ring ubiquitin ligase complex delivering PDE5 for ubiquitination and proteasomal degradation. In order to identify other RhoBTB1 binding partners, we assessed the binding interface of RhoBTB1 which binds to PDE5 with the notion that the same region will bind to other RhoBTB1-CUL3 substrates. Full-length RhoBTB1 was truncated into its component domains, separately (GTPase, BTB1, BTB2, and C-terminal (CT)) and in combination (e.g. B1B2C) domains. HEK293 cells were co-transfected with epitope-tagged PDE5 and RhoBTB1 domain constructs, and their interaction was analyzed by co-immunoprecipitation (Co-IP). Interestingly, the CT domain itself was essential for the interaction between RhoBTB1 and PDE5 but was not sufficient on its own to bind to PDE5. This was supported by our finding that the domains lacking CT region could not bind with PDE5. The Co-IP data was further corroborated by immunofluorescence and proximity labeling assay (PLA). Our data suggest that RhoBTB1 could bind to PDE5 utilizing its BTB1-BTB2-CT domains. Moreover, protein stability and ubiquitination studies revealed that the BTB1-BTB2-CT domain is sufficient for CUL3-dependent proteasomal degradation. Molecular approaches including site-directed mutagenesis are being explored to investigate potential “hotspots” underlying the RhoBTB1-PDE5 interaction. We are currently utilizing ascorbate peroxidase (APEX2) fusion system involving BTB1-BTB2-CT domain as “bait” to identify unknown interacting partner in RhoBTB1-Cullin3 axis. Preliminary experiments have been performed to validate the system. For example, BTB1-BTB2-CT-APEX2 can bind to PDE5. These studies are designed to assess if there are other proteins which are involved in the regulation of blood pressure in VSM which may become novel therapeutic candidates against hypertension.