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ASB11 controls purinosome formation through PAICS ubiquitination

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Fibroblast, Kidney

DISEASE(S): Kidney Cancer

SUBMITTER: Ming-Chieh CHOU

LAB HEAD: Ruey-Hwa Chen

PROVIDER: PXD025298 | Pride | 2023-10-06

REPOSITORIES: Pride

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			Action	DRS
	SILAC-Proteome_Repeat1_Fractionation1.raw	Raw
	SILAC-Proteome_Repeat1_Fractionation2.raw	Raw
	SILAC-Proteome_Repeat1_Fractionation3.raw	Raw
	SILAC-Proteome_Repeat1_Fractionation4.raw	Raw
	SILAC-Proteome_Repeat1_Fractionation5.raw	Raw

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Project description:Lung cancer is one of the most common and fatal cancer worldwide. There are two major types of lung cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Recently, gefitinib, a small molecule, which target tyrosine kinase, has been regarded as the first-line treatment in NSCLC patients. However, several patients have been observed tumor recurrence and eventually developed progressive outcomes after target therapy. Thus, an effective therapeutic approach need to be explored. Here, we found that ectopically expressed ATP synthase on plasma membrane exhibited gefitinib-resistance properties in lung cancer cell lines. Furthermore, we unraveled that citreoviridin, an ectopic ATP synthase inhibitor, suppressed the abilities of both proliferation and colony formation in lung cancer cell lines. To elucidate the comprehensive mechanism regulated by citreoviridin, we performed microarray analysis. The results indicated that not only mRNAs but also long non-coding RNAs (lncRNAs) are involved in citreoviridin-treated cell death. One of the well-known lncRNAs, growth arrest-specific transcript (GAS5), was robustly upregulated after citreoviridin treatment. In order to investigate the upstream modulator of GAS5, we utilized chromatin immunoprecipitation (ChIP) assay and revealed that E2F transcription factor 1 (E2F1) could bind to the promoter of GAS5. Consistently, both microarray and qPCR data showed the expression level of E2F1 was negatively correlated to GAS5 after citreoviridin treatment. The evidence suggests that E2F1 might be a potential repressor of GAS5. Furthermore, combining microarray and Gene Set Enrichment Analysis (GSEA) analysis as well as qPCR demonstrated that p53 pathway was activated. To further realize the GAS5-p53 regulating network, RNA-protein pull-down assay followed by LC-MS/MS will be utilized to dissect the GAS5-interacting proteomic profiling.From the results, we identified 107 GAS5-interacting proteins in common from A549 and H1975 cell lines. Our proteomics experiments identified topoisomerase 2-alpha (TOP2A) as the key protein involved in the citreoviridin-regulated gefitinib-resistance pathway, therefore, we picked out TOP2A for further study. Additionally, we further validated the interaction between TOP2A and GAS5 by western blot and RNA immunoprecipitation (RIP). Taken together, this study suggests targeting E2F1/GAS5/p53 axis is a potential therapeutic strategy for gefitinib-resistant lung cancer.

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ASB11 controls purinosome formation through PAICS ubiquitination

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