Faculty Collaboration Database

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination. Function (Oxf) 2023;4(5):zqad034

Date

08/14/2023

Pubmed ID

37575477

Pubmed Central ID

PMC10413933

DOI

10.1093/function/zqad034

Scopus ID

2-s2.0-85168062439 (requires institutional sign-in at Scopus site)   3 Citations

Abstract

We identified Rho-related BTB domain containing 1 (RhoBTB1) as a key regulator of phosphodiesterase 5 (PDE5) activity, and through PDE5, a regulator of vascular tone. We identified the binding interface for PDE5 on RhoBTB1 by truncating full-length RhoBTB1 into its component domains. Co-immunoprecipitation analyses revealed that the C-terminal half of RhoBTB1 containing its two BTB domains and the C-terminal domain (B1B2C) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via Cullin-3 (CUL3). The C-terminal domain was essential in recruiting PDE5 as constructs lacking this region could not participate in PDE5 binding or proteasomal degradation. We also identified Pro³⁵³ and Ser³⁶³ as key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of either of these residues exhibited impaired CUL3 binding and PDE5 degradation, although the binding to PDE5 was preserved. Finally, we employed ascorbate peroxidase 2 (APEX2) proximity labeling using a B1B2C-APEX2 fusion protein as bait to capture unknown RhoBTB1 binding partners. Among several B1B2C-binding proteins identified and validated, we focused on SET domain containing 2 (SETD2). SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to pharmacological inhibition of the proteasome or Cullin complex, CUL3 deletion, and RhoBTB1-inhibition with siRNA. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies will determine whether SETD2 plays a role in cardiovascular function.

Author List

Kumar G, Fang S, Golosova D, Lu KT, Brozoski DT, Vazirabad I, Sigmund CD

Authors

Curt Sigmund PhD Chair, Professor in the Physiology department at Medical College of Wisconsin
Ibrahim Y. Vazirabad Bioinformatics Analyst III in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Cullin Proteins
Proteasome Endopeptidase Complex
Substrate Specificity
Ubiquitination