PXD031703

PXD031703 is an original dataset announced via ProteomeXchange.

Title	Enzymatic Analysis of WWP2 E3 Ubiquitin Ligase Using Protein Microarrays Identifies Autophagy-Related Substrates
Description	WWP2 is a HECT E3 ligase that targets protein Lys residues for ubiquitination. As a member of the NEDD4 family of E3 ligases, WWP2 is comprised of an N-terminal C2 domain, four central WW domains, and a C-terminal catalytic HECT domain. The peptide segment between the middle WW domains, the 2,3-linker, can autoinhibit the catalytic domain and this can be relieved by phosphorylation at Tyr369. Several protein substrates of WWP2 have been identified, including the tumor suppressor lipid phosphatase PTEN, but the full substrate landscape and biological functions of WWP2 remain to be elucidated. Here we use protein microarray technology and the activated enzyme phosphomimetic mutant WWP2Y369E to identify potential WWP2 substrates. We identified 31 substrate hits for WWP2Y369E using protein microarrays of which three are the autophagy receptors, NDP52, OPTN, and SQSTM1. These three hits were validated with in vitro and cell-based transfection assays and the Lys ubiquitination sites on these proteins were mapped by mass spectrometry. Among the mapped ubiquitin sites on these autophagy receptors, many were previously identified in studies on the endogenous proteins. WWP2 knockout SH-SH5Y neuroblastoma cells using CRISPR-Cas9 showed a defect in mitophagy in a fashion that could be rescued by WWP2Y369E transfection. These studies suggest that WWP2-mediated ubiquitination of the autophagy receptors NDP52, OPTN, and SQSTM1 may positively contribute to the regulation of autophagy.
HostingRepository	PRIDE
AnnounceDate	2022-05-20
AnnouncementXML	Submission_2022-05-20_06:26:00.117.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD031703
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Hanjie Jiang
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562;
ModificationList	ubiquitination signature dipeptidyl lysine
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2022-02-16 02:45:41	ID requested
⏵ 1	2022-05-20 06:26:00	announced

Jiang H, Chiang CY, Chen Z, Nathan S, D'Agostino G, Paulo JA, Song G, Zhu H, Gabelli SB, Cole PA, Enzymatic analysis of WWP2 E3 ubiquitin ligase using protein microarrays identifies autophagy-related substrates. J Biol Chem, 298(5):101854(2022) [pubmed]

submitter keyword: LC-MSMS, protein microarray, WWP2, Ubiquitination, autophagy receptor

Philip A. Cole
contact affiliation	Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts, 02115, USA
contact email	pacole@bwh.harvard.edu
lab head
Hanjie Jiang
contact affiliation	Brigham and Women's Hospital
contact email	hjiang9@bwh.harvard.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD031703
     1. Label: PRIDE project
     2. Name: Enzymatic Analysis of WWP2 E3 Ubiquitin Ligase Using Protein Microarrays Identifies Autophagy-Related Substrates