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Identification of ubiquitin binding protein using TurboID analysis

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Ming-Chieh CHOU

LAB HEAD: Ruey-Hwa Chen

PROVIDER: PXD027946 | Pride | 2023-10-06

REPOSITORIES: Pride

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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.

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Project description:Cell cycle and metabolism are two major outputs controlled by circadian rhythm in many organisms. Here we show that the three processes were linked through inosine 5'-phosphate dehydrogenase (impdh), a rate-limiting enzyme in de novo purine synthesis. We using adult zebrafish as a model system,we applied a genome-wide transcriptome approach that allowed us to investigate circadian gene expression. The whole-genome transcriptome profiles of adult brain in time-series were assayed on Agilent zebrafish microarrays. We used a similar statistical method to identify zebrafish circadian genes (ZCOG) as our previous study in larval zebrafish. Three isoforms of impdh show strong circadian oscillations in different tissues of zebrafish. impdh1a contributes to the ocular development and pigment synthesis, impdh2 promotes and impdh1b delays the development. By limiting the GTP required by DNA synthesis, impdh2 contributes to the daily rhythm of S phase in cell cycle. Multiple enzymes in the de novo purine synthesis pathway show the same circadian oscillations with peaks similar to impdh2. The circadian expression of this pathway is conserved in mouse liver. In summary, we show that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is critical for gating cell cycle in circadian rhythm. Adult zebrafish were sacrificed and dissected at 4h intervals starting at CT0 in both LD and DD conditions for 12 time points. Total RNA of individual zebrafish brain was extracted using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturerM-bM-^@M-^Ys instruction. Microarrays were manufactured by Agilent Technologies (Agilent Technologies, Palo Alto, CA), containing 43,603 probes for zebrafish whole-genome transcriptional profiling.

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ASB11 controls purinosome formation through PAICS ubiquitination

Project description:Purine act as building block for DNA and RNA, provide cellular energy and signaling, and can generate cofactors such as NADH and coenzyme A. Purine de novo synthesis pathway begins with amino acids, ribose 5-phosphate, CO2 and NH3 with 6 enzymes and 10 enzymatic steps to convert PRPP to IMP. In previous study, under purine-depleted or other stimuli the six enzymes will form dynamic and reversible complex called purinosome to enhance the pathway flux. However, the mechanism of purinosome formation is unknown. Phosphoribosyl aminoimidazole succinocarboxamide synthetase (PAICS), an enzyme involved in the step7 and 8 of purine biosynthesis. In our lab, we used interactome and ubiquiylome to identify PAICS, which is a substrate of ASB11. ASB11 is a substrate adaptor of cullin 5 ubiquitin ligase complex and is able to bind PAICS for ubiquitination. Here, we found that ASB11 could increase purinosome formation without any stimuli. This effect depends on ASB11 E3 function, which can ubiquitinate PAICS on K74 site. We generate K74R mutation of PAICS so that ASB11 couldn’t ubiquitinate it. Interestingly, this mutant reduces the formation of purinosomes under purine-depleted and other stressed conditions. After further research, we found ASB11 expression level will increase under some specific conditions. Moreover, we analyzed TCGA database and found that the expression of ASB11 in melanoma cell is higher than normal cell. Consistent with our finding, melanoma cell has partially formed purinosome under basal condition because of the higher expression of ASB11. We guess that ASB11 may plays an important role in cancer cell.

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