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Ubiquitinated sites on VPS34 by UBE3C LC-MSMS

ABSTRACT: Ubiquitinated sites on VPS34 identified by LC-MS/MS analysis of VPS34 derived from in vivo ubiquitination assays of UBE3C-transfected or -untransfected 293T cells.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yu-Hsuan Chen

LAB HEAD: Yu-Hsuan Chen

PROVIDER: PXD023959 | Pride | 2021-02-19

REPOSITORIES: Pride

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			Action	DRS
	Ctrl-Ub_sites_on_VPS34_in_cell_transfected_without_UBE3C.mgf	Mgf
	Ctrl-Ub_sites_on_VPS34_in_cell_transfected_without_UBE3C.raw	Raw
	Exp-Ub_sites_on_VPS34_in_cell_transfected_with_UBE3C.mgf	Mgf
	Exp-Ub_sites_on_VPS34_in_cell_transfected_with_UBE3C.raw	Raw
	checksum.txt	Txt

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Publications

VPS34 K29/K48 branched ubiquitination governed by UBE3C and TRABID regulates autophagy, proteostasis and liver metabolism.

Chen Yu-Hsuan YH Huang Tzu-Yu TY Lin Yu-Tung YT Lin Shu-Yu SY Li Wen-Hsin WH Hsiao Hsiang-Jung HJ Yan Ruei-Liang RL Tang Hong-Wen HW Shen Zhao-Qing ZQ Chen Guang-Chao GC Wu Kuen-Phon KP Tsai Ting-Fen TF Chen Ruey-Hwa RH

Nature communications 20210226 1

The ubiquitin-proteasome system (UPS) and autophagy are two major quality control processes whose impairment is linked to a wide variety of diseases. The coordination between UPS and autophagy remains incompletely understood. Here, we show that ubiquitin ligase UBE3C and deubiquitinating enzyme TRABID reciprocally regulate K29/K48-branched ubiquitination of VPS34. We find that this ubiquitination enhances the binding of VPS34 to proteasomes for degradation, thereby suppressing autophagosome form ...[more]

PMID: 33637724

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Ubiquitin chain types on ubiquitinated VPS34 in the presence or absence of UBE3C in vitro LC-MSMS

Project description:We identified VPS34 as the direct substrate of UBE3C E3 ligase in vivo and in vitro. Next, to analyze the ubiquitin chain on VPS34 that was assembled by UBE3C in vitro.

2021-02-17 | PXD023986 | Pride

Long noncoding RNA BCRP3 stimulates VPS34 and autophagy activities to promote protein homeostasis and cell survival

Project description:Background: Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored. Methods: Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA pull down and RNA immunoprecipitation were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas Mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes. Results: We identify lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 is mainly localized in the cytoplasm and binds VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 is upregulated to promote aggrephagy for the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity results in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromises cell proliferation and survival, which is mediated in part through the upregulation of TGF-/Smad2 pathway. Conclusions: Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.

2022-05-20 | PXD030756 | Pride

Identification of Ubiquitination site of PAICS by LC-MS/MS

Project description:Purines serve as the building blocks for DNA and RNA, confer cellular energy and signaling. Purines are generated by de novo synthesis pathway and salvage pathway. Under purine-depleted or other cellular stresses, enzymes in the purine de novo synthesis pathway form a dynamic and reversible condensate called purinosome, but the underlying mechanism of purinosome formation is unknown. In this thesis, we found that ASB11-based Cul5 E3 ligase promotes ubiquitination of PAICS, a purine de novo synthesis enzyme. This ubiquitination does not lead to PAICS degradation, but drives purinosome assembly. We provide evidence that purinosome assembly involves a liquid-liquid phase separation (LLPS) process and identify several ubiquitin binding proteins that may bind ubiquitinated PAICS through a multivalent mode to drive LLPS and purinosome assembly. Importantly, ASB11 is upregulated under the stresses that promote purinosome assembly, thus increasing the formation of ASB11/PAICS complex. Finally, we demonstrated that melanoma cells express a high level of ASB11 to confer a constitutive purinosome formation, which support their viability. In summary, our study identifies ASB11-mediated PAICS ubiquitination as a driving mechanism for purinosome assembly, the regulation of this mechanism under stressed conditions, and the importance of this regulation in cell viability.

2023-10-06 | PXD038411 | Pride

Identification of ubiquitin binding protein using TurboID analysis

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VPS34 regulates RAB7 activation and Retromer localization to autophagosomes and mitophagy in oocytes

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2023-09-01 | GSE216465 | GEO

Pro-oxidant therapy by a vitamin K precursor targets PI 3-Kinase VPS34 function

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2024-04-02 | GSE148011 | GEO

Antileukemic activity of the VPS34-IN1 inhibitor in acute myeloid leukemia

Project description:Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis. Vacuolar protein sorting 34 (VPS34) is a member of the phosphatidylinositol-3-kinase lipid kinase family that controls the canonical autophagy pathway and vesicular trafficking. Using a recently developed specific inhibitor (VPS34-IN1), we found that a VPS34 block induces apoptosis in AML cells but not in normal CD34+ hematopoietic cells. Complete and acute inhibition of VPS34 was required for the anti-leukemic activity of VPS34-IN1. This inhibitor also has pleiotropic effects against various cellular functions related to class III PI3K in AML cells that may explain their survival impairment. VPS34-IN1 inhibits basal and L-asparaginase-induced autophagy in AML cells. A synergistic cell death activity of this drug was also demonstrated. VPS34-IN1 was additionally found to impair vesicular trafficking and mTORC1 signaling. From an unbiased approach based on phosphoproteomic analysis, we identified that VPS34-IN1 specifically inhibits STAT5 phosphorylation downstream of FLT3-ITD signaling in AML. The identification of the mechanisms controlling FLT3-ITD signaling by VPS34 represents an important insight into the oncogenesis of AML and could lead to new therapeutic strategies.

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RBM7 activates P-TEFb for a pro-survival transcriptional response to DNA damage

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