PXD043245

PXD043245 is an original dataset announced via ProteomeXchange.

Title	RhoBTB1 C-Terminal Binding Proteins
Description	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 the 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 if SETD2 plays a role in cardiovascular function.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_09:04:44.449.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD043245
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Curt Sigmund
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2023-06-23 08:16:21	ID requested
1	2023-08-21 10:08:48	announced
⏵ 2	2023-11-14 09:04:45	announced	2023-11-14: Updated project metadata.

Kumar G, Fang S, Golosova D, Lu KT, Brozoski DT, Vazirabad I, Sigmund CD, Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination. Function (Oxf), 4(5):zqad034(2023) [pubmed]

10.6019/PXD043245;

submitter keyword: mass spec,proteomics, protein:protein interactions

Curt D. Sigmund
contact affiliation	Department of Physiology Medical College of Wisconsin
contact email	csigmund@mcw.edu
lab head
Curt Sigmund
contact affiliation	Department of Physiology Medical College of Wisconsin
contact email	csigmund@mcw.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD043245
     1. Label: PRIDE project
     2. Name: RhoBTB1 C-Terminal Binding Proteins