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Deubiquitinating Enzyme USP12 Regulates the Pro-Apoptosis Protein Bax.

ABSTRACT: The Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family, involved in inducing apoptosis at the mitochondrial level. Regulating the protein levels of Bax is essential to enhancing apoptosis. In the current study, we ascertained the presence of deubiquitinating enzymes (DUBs) associated with Bax by performing the yeast two-hybrid screening (Y2H). We determined that ubiquitin-specific protease 12 (USP12), one of the DUBs, is associated with Bax. The binding of USP12 to Bax shows the interaction as a DUB, which regulates ubiquitination on Bax. Taken together, we believe that USP12 regulates Bax by detaching ubiquitin on K63-linked chains, indicating that USP12 affects the cellular functions of Bax, but it is not related with proteasomal degradation. The half-life of the Bax protein was determined by performing the site-directed mutagenesis of putative ubiquitination sites on Bax (K128R, K189R, and K190R). Of these, Bax (K128R and K190R) showed less ubiquitination; therefore, we compared the half-life of Bax (WT) and Bax K mutant forms in vitro. Interestingly, Bax (K189R) showed a higher ubiquitination level and shorter half-life than Bax (WT), and the (K128R and K190R) mutant form has a longer half-life than Bax (WT).

SUBMITTER: Choi HS

PROVIDER: S-EPMC9657785 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Deubiquitinating Enzyme USP12 Regulates the Pro-Apoptosis Protein Bax.

Choi Hae-Seul HS Lim Eun-Su ES Baek Kwang-Hyun KH

International journal of molecular sciences 20221028 21

The Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family, involved in inducing apoptosis at the mitochondrial level. Regulating the protein levels of Bax is essential to enhancing apoptosis. In the current study, we ascertained the presence of deubiquitinating enzymes (DUBs) associated with Bax by performing the yeast two-hybrid screening (Y2H). We determined that ubiquitin-specific protease 12 (USP12), one of the DUBs, is associated with Bax. The binding of USP12 to Bax shows th ...[more]

PMID: 36361894

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Project description:Background:Esophageal cancer is one of the most common cancers worldwide with poor prognosis and high mortality. The transcription factor SNAI1, encoding Snail1, is important for metastatic progression in esophageal cancer whereas the microRNA (miRNA)-203 has been shown to function as an inhibitor of metastasis in EC. The Snail1 protein is stabilized in EC partially by the deubiquitinating enzyme USP26; however, how USP26 is regulated is not completely known. Methods:Expression of SNAI1 and USP26 messenger RNA (mRNA) and miR-203 was performed in datasets within The Cancer Genome Atlas and Gene Expression Omnibus, respectively. Expression of Snail1 and USP26 protein and miR-203 was determined in the normal esophageal cell line HET-1A and EC cell lines Kyse150 and TE-1 using western blot and quantitative polymerase chain reaction, respectively. TargetScan was used for in situ prediction of miR-203 targets and in vitro heterologous reporter assays using the wild-type and miR-203 seed mutant of the 3' Untranslated region (UTR) of USP26 were used to investigate whether USP26 is a target of miR-203. Effects of increasing miR-203 using MIR203A/5P mimic on USP26 and Snail1 in the HET-1A, Kyse150 and TE-1 cell lines were performed using western blot and cycloheximide-based protein stability analysis. Effects of modulating miR-203 in Kyse150 and TE-1 cell lines on in vitro pro-metastatic effects were analyzed by invasion assay, scratch wound-healing assay, and chemosensitivity to 5-fluoruracil (5-FU). In vivo lung metastasis assay was used to study the effect of modulating miR-203 in Kyse150 cells. Results:SNAI1 mRNA and HSA/MIR203 was higher and lower, respectively, in EC patients compared to tumor-adjacent normal tissues. No changes in expression of USP26 mRNA were observed in these datasets. MIR/203 expression was downregulated whereas protein expression of both Snail1 and USP26 were higher in EC cell lines Kyse150 and TE-1 compared to normal esophageal cell line HET-1A. USP26 was predicted as a potential target of miR-203 by TargetScan Release 2.0. Reporter assays confirmed USP26 as a target of miR-203 in the EC cell lines. Transfection of EC cell lines with MIR203 mimic decreased USP26 protein expression and Snail1 protein stability indicating the ability of miR-203 to regulate Snail1 protein levels via USP26. Exogenous increase in miR-203 in the EC cell lines significantly inhibited Snail-1 mediated in vitro pro-metastatic function of invasion, wound-healing, and increased chemosensitivity to 5-FU. Finally, overexpression of miR-203 inhibited in vivo lung metastasis of Kyse150 cells, which was reversed following overexpression of USP26, indicating a direct role of miR-203-mediated regulation of USP26 in metastatic progression of EC. Conclusions:Cumulatively, these results establish an important mechanism by which decrease in miR-203 expression potentiates metastatic progression in EC via USP26-mediated stabilization of Snail1. Hence, miR-203 can serve as a biomarker of metastasis in EC and is a potential target for therapeutic intervention in EC.

Project description:Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs) are the primary mediators for inter-neuronal communication and play a crucial role in higher brain functions including learning and memory. Our previous work demonstrated that AMPARs are subject to ubiquitination by the E3 ligase Nedd4, resulting in EPS15-mediated receptor internalization and Ubiquitin (Ub)-proteasome pathway (UPP)-dependent degradation. Protein ubiquitination is a highly dynamic and reversible process, achieved via the balance between ubiquitination and deubiquitination. However, deubiquitination of mammalian AMPARs and the responsible deubiquitinating enzymes remain elusive. In this study, we identify USP46 as the deubiquitinating enzyme for AMPARs. We find that AMPARs are subject to K63 type ubiquitination, and USP46 is able to deubiquitinate AMPARs in vivo and in vitro. In heterologous cells and neurons, expression of USP46 results in a significant reduction in AMPAR ubiquitination, accompanied by a reduced rate in AMPAR degradation and an increase in surface AMPAR accumulation. By contrast, knockdown of USP46 by RNAi leads to elevated AMPAR ubiquitination and a reduction in surface AMPARs at synapses in neurons. Consistently, miniature excitatory postsynaptic currents recordings show reduced synaptic strength in neurons expressing USP46-selective RNAi. These results demonstrate USP46-mediated regulation of AMPAR ubiquitination and turnover, which may play an important role in synaptic plasticity and brain function. Protein ubiquitination is a highly dynamic and reversible process, achieved via the balance between ubiquitination and deubiquitination. The glutamatergic AMPARs, which mediate most of the excitatory synaptic transmission in the brain, are known to be subjected to Nedd4-mediated ubiquitination; however, the deubiquitination process and the responsible deubiquitinating enzymes (DUBs) for mammalian AMPARs remain elusive. We find that AMPARs are subject to K63-type ubiquitination, and identify USP46 as the DUB for AMPARs. USP46 deubiquitinates AMPARs in vitro and in vivo. Up- or down-regulation of USP46 leads to changes in AMPAR ubiquitination, surface expression, and trafficking, as well as the strength of synaptic transmission. USP46-mediated regulation of AMPAR ubiquitination and turnover may play an important role in synaptic plasticity and brain function.

Dataset Information

Deubiquitinating Enzyme USP12 Regulates the Pro-Apoptosis Protein Bax.

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Deubiquitinating Enzyme USP12 Regulates the Pro-Apoptosis Protein Bax.

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