Proteomic approach to explore the role of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin-3 (CUL-3) pathway in identifying potential therapeutic candidates in cardioprotection

Abstract

Protein-protein interaction studies have contributed enormously to understand the fundamentals of human impairments including cardiovascular diseases. We previously identified Phosphodiesterase 5 (PDE5) as an interacting partner of RhoBTB1. The latter can function as an adapter for CUL-3 E3 ring ubiquitin ligase complex delivering PDE5 for ubiquitination and proteasomal degradation. This in turn regulates the nitric oxide response of vascular smooth muscle (VSM) and consequently blood pressure. Ubiquitous expression of RhoBTB1, its link with CUL-3, and PDE5 suggests that RhoBTB1 may have other binding partners in VSM and other cells. The binding sites of these partners on RhoBTB1 might be similar to PDE5. With the notion that PDE5 binding region on RhoBTB1 could be responsible for binding of unknown partners in RhoBTB1-CUL-3 pathway, we truncated full-length RhoBTB1 into its component domains, individually (GTPase, BTB1, BTB2, and C-terminal (CT)) and in combination (e.g. B1B2C, B2C, and PB1B2). HEK293 cells were used to express epitope-tagged RhoBTB1 constructs and PDE5. The interaction between different constructs of RhoBTB1 and PDE5 was determined by co-immunoprecipitation (Co-IP). We showed that RhoBTB1 could bind to PDE5 utilizing the combination of its B1B2C domains. Our data also suggest that the CT domain itself was essential for the interaction between RhoBTB1 and PDE5 but was not sufficient on its own to bind PDE5. The Co-IP data was further corroborated with the findings of immunofluorescence and proximity labeling assay (PLA). Subsequent protein stability and ubiquitination studies revealed that B1B2C is catalytically sufficient for CUL3-dependent proteasomal degradation like full-length RhoBTB1. Furthermore, macromolecular protein-protein docking revealed the binding interface between RhoBTB1 and CUL-3 comprising of 24 amino acids. Additional Co-IP data suggested that Ser363 and Pro353 on RhoBTB1 domain are required for binding to CUL-3 as mutants of RhoBTB1 (Ser363Ala and Pro353Ala) could not deliver PDE5 for proteasomal degradation. To further identify unknown interacting partners in RhoBTB1-CUL-3 axis, we utilized the ascorbate peroxidase (APEX2) proximity labelling system involving B1B2C as “bait.” Proteomic profiling of the biotinylated proteome through mass spectrometry has identified several unknown interacting partners involved in RhoBTB1-CUL-3 pathway. Some of these have demonstrated upregulation on pharmacological inhibition of proteasome and CUL-3 which establishes their potential involvement in RhoBTB1-CUL-3 pathway. Exploring the involvement of such interacting partners in RhoBTB1-CUL-3 axis may open novel therapeutic avenues in cardioprotection. NIH R35 HL144807 (to Dr. Curt D. Sigmund) This is the full abstract presented at the American Physiology Summit 2023 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.