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通讯作者:

曹新,E-mail:caoxin@njmu.edu.cn

中图分类号:R393

文献标识码:A

文章编号:1007-4368(2022)06-759-09

DOI:10.7655/NYDXBNS20220601

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目录contents

    摘要

    目的:研究泛素特异性蛋白酶14(ubiquitin⁃specific peptidase 14,USP14)通过调控氧化固醇结合样蛋白2(oxysterol binding protein⁃like 2,OSBPL2)的泛素化水平稳定OSBPL2表达的分子机制。方法:以HeLa细胞为工具细胞,通过对USP14过表达、敲降以及活性抑制,检测OSBPL2的表达水平;采用体外过表达实验考察USP14对OSBPL2的泛素信号和泛素化水平的调控;采用免疫共沉淀(co⁃immunoprecipitation,Co⁃IP)阐明USP14与OSBPL2相互作用的具体结构域、不同结构域在蛋白相互作用中的作用以及OSBPL2的关键泛素化位点。结果:USP14与OSBPL2存在相互作用关系并稳定OSBPL2的表达而不改变其转录水平;体外过表达和Co⁃IP实验结果表明,USP14催化(catalytic,CAT)结构域与OSBPL2氧固醇结合蛋白相关结构域(OSBP ⁃related domain,ORD)的相互作用降低OSBPL2的泛素化水平;类泛素(ubiquitin⁃like,UBL)结构域在USP14与OSBPL2相互作用中起到了促进作用;Lys209和Lys361是OSBPL2泛素化和去泛素化的关键位点。结论:USP14通过调控OSBPL2蛋白Lys209 和Lys361两个位点的泛素化进而调控其泛素化水平,稳定OSBPL2蛋白的表达。

    Abstract

    Objective:This study aims to explore the molecular mechanism of ubiquitin⁃specific peptidase 14(USP14)instabilizing oxysterol binding protein⁃like 2(OSBPL2)expression by regulating the ubiquitination level of OSBPL2. Methods:HeLa cells was used as tool cells to detect the expression of OSBPL2 by overexpression,knockdown and activity inhibition of USP14. The regulation of USP14 on ubiquitin signal and ubiquitination level of OSBPL2 was investigated in vitro. The specific domain of USP14 interacting with OSBPL2,the interactional role of different domains and the key ubiquitination sites of OSBPL2 were determined by Co ⁃ immunoprecipitation(Co⁃IP). Results:USP14 interacted with OSBPL2 and stabilized the expression of OSBPL2 without changing its transcriptional level. In vitro ubiquitination experiment and Co⁃IP showed that the catalytic(CAT)domain of USP14 interacted with the OSBP ⁃ related domain(ORD)of OSBPL2,which reduced the ubiquitination level of OSBPL2. The ubiquitin ⁃ like(UBL)domain of USP14 could promote the interaction between USP14 and OSBPL2. In addition,Lys209 and Lys361 could be the key sites for OSBPL2 ubiquitination and deubiquitination. Conclusion:USP14 modulated the ubiquitination level of OSBPL2 by regulating the ubiquitination of Lys209 and Lys361 and further stabilized OSBPL2 expression.

  • 氧化固醇结合蛋白(oxysterol binding protein, OSBP)和氧化固醇结合蛋白样蛋白(OSBP⁃related protein,ORP)是一类与脂质代谢相关的蛋白,进化上高度保守,在大多数真核生物中广泛表达。人类ORP家族中包含12个成员,它们参与了囊泡运输、脂质代谢、信号转导和泛素化修饰等生物过程[1-3]。氧化固醇结合蛋白样蛋白2(oxsterol binding protein⁃like2,OSBPL2/ORP2)是OSBP/ORP家族成员,与其他ORP成员相比,仅含FFAT(phenylalanines in an acidic tract)和OSBP相关配体结合结构域(OSBP⁃re⁃ lated ligand binding domain,ORD)[4-5]。本课题组前期研究结果显示,OSBPL2突变是人常染色体显性遗传性耳聋(DFNA67)的分子病因,OSBPL2缺失与胆固醇代谢紊乱和肥胖表型相关[5-8]。本课题组在前期研究中发现,OSBPL2通过与其他效应蛋白的互作介导了细胞内胆固醇合成、脂滴分解和脂肪前体细胞发育与分化等过程[7-9]。其中,去泛素化酶泛素特异性蛋白酶14(ubiquitin⁃specific peptidase14,USP14)也是OSBPL2的效应蛋白之一。

  • 泛素化修饰是真核生物中蛋白修饰的一种重要方式,80%~90%的蛋白通过泛素化途径降解[10-11]。去泛素化是泛素化的逆向过程,通过去泛素化酶发挥作用,泛素化与去泛素化之间呈现出可逆的动态平衡[12]。哺乳动物体内包含6个家族共约100种去泛素化酶,其中USP14是泛素特异性蛋白酶(ubiquitin⁃ specific peptidase,USP)家族中唯一一个与蛋白酶体可逆结合的去泛素化酶,由类泛素(ubiguitin⁃like, UBL)结构域和催化(catalytic,CAT)结构域构成。其中UBL结构域通过与蛋白酶体的19S亚基结合,抑制蛋白酶体活性,从而使靶蛋白免于降解;而CAT结构域主要发挥去泛素化作用,通过与泛素化蛋白结合,移除泛素链,使之不能被蛋白酶体识别和降解,进而达到稳定蛋白表达的目的[12-15]。此外, USP14在线粒体自噬、内质网应激、DNA损伤修复以及炎症发生中具有重要作用[16-19]。目前已有关于OSBP/ORP家族某些成员泛素化研究的报道[20-21],但关于OSBPL2泛素化和去泛素化的研究尚未见报道,本研究将探索USP14调控OSBPL2去泛素化的作用机制,为从OSBPL2泛素化与去泛素化修饰水平探索OSBPL2生物学功能提供理论依据和新思路。

  • 1 材料和方法

  • 1.1 材料

  • HeLa细胞和293Ta细胞由本实验室保存; pXJ40载体由本实验室保存;E.coli DH5ɑ化学感受态细胞(南京诺唯赞公司);pCDNA3.1⁃3×HA⁃UBB、 pCDNA3.1⁃HA⁃K48、pCDNA3.1⁃HA⁃K63(武汉淼灵质粒平台)。

  • ANTI ⁃FLAG® M2Affinity Gel(A2220,Sigma公司,美国)、Western及IP细胞裂解液(P0013)、RIPA裂解液(P0013B)(杭州碧云天公司),GAPDH(5174S)(CST公司,美国)、OSBPL2(A14199)(武汉爱博泰克生物科技有限公司)、放线菌酮(cyclohexi⁃ mide,CHX,S7418)(上海Selleck公司)、免疫(共)沉淀(Co⁃immunoprecipitation,Co⁃IP)试剂盒(abs955) (北京爱必信生物技术有限公司),HA⁃Tag(3724S)、 Ubiquitin抗体(3936)(CST公司,美国),K48抗体 (A18163)(武汉爱博泰克生物科技有限公司)、K63抗体(ab179434)(Abcam公司,美国)、Flag抗体 (14793)(CST公司,美国)、乳胞素(XY ⁃LC26246) (上海熹垣生物科技有限公司)、PSMD2抗体 (A1999)(武汉爱博泰克生物科技有限公司)、 MG132(HY⁃13259)(上海MCE公司);Lipofectamine3000转染试剂盒(L3000150)(ThermoFisher公司,美国)、X ⁃ tremeGENE siRNA Transfection Reagent (04476093001)(Roche公司,瑞士);定量PCR引物、过表达载体构建引物、截短体构建引物和点突变载体构建引物均由北京擎科生物科技有限公司合成,siR⁃ NA序列由上海吉玛公司合成。

  • 1.2 方法

  • 1.2.1 质粒转染

  • 过表达实验:转染前1天细胞种板,过夜培养,第2天待细胞密度达到70%~80%后,使用Lipo⁃ fectamine3000转染试剂盒,用Opti转染试剂分别稀释Lipo3000、p3000和质粒,分别静置5min,混合后再静置20min。将混合液加入细胞中,6h后换液, 48~72h收集细胞,提取蛋白。

  • siRNA敲降实验:转染前1天细胞种板,过夜培养,第2天待细胞密度达到30%~50%后,使用X ⁃ tremeGENE siRNA Transfection Reagent试剂盒,用Opti转染试剂稀释小干扰试剂和siRNA,混合静置15~20min,将混合液加入到完全培养基中,24~48h收集细胞提取RNA,48~72h收集细胞提取蛋白。

  • 1.2.2 Co⁃IP试验

  • 293Ta细胞共转目的质粒48h后,加入IP裂解液提取蛋白,取150 μL为对照(Input),剩余裂解液中加入proteinA/G和目标抗体原液,置于颠倒混匀器上摇晃过夜,第2天对样品进行洗脱之后,加入蛋白Loading变性,金属浴100℃ 10min,-20℃保存。

  • 1.2.3 蛋白质免疫印迹

  • 取培养细胞加入RIPA裂解液提取总蛋白,使用BCA试剂进行蛋白定量。取等量的蛋白加入10%SDS⁃PAGE电泳胶中,分离并转膜至硝酸纤维膜上。 5%脱脂奶粉溶液封闭2h,加入目标一抗稀释液4℃ 孵育过夜。次日回收一抗,加入相应二抗室温孵育2h。然后用ECL发光液孵育,凝胶成像系统曝光。

  • 1.2.4 实时定量PCR

  • TRIzol提取细胞总RNA,测定提取的RNA浓度,取1 μg总RNA反转录成cDNA,然后进行实时荧光定量PCR(real⁃time quantitative PCR,qRT⁃PCR)检测。采用20 μL扩增体系:SYBR®Green Master Mix (2×)10 μL,cDNA 1 μL,上游引物0.5 μL,下游引物0.5 μL,ddH2O 8 μL。扩增程序如下:预变性95℃ 5min;变性95℃ 10s,退火/延伸60℃ 30s,共40个循环,目标基因CT值使用GAPDH基因CT值校正。引物序列见表1。

  • 表1 实时定量PCR引物

  • Table1 Primers for qRT⁃RCR

  • 1.2.5 过表达质粒构建

  • 采用https://www.uniprot.org/在线软件分别查询USP14的UBL结构域和CAT结构域以及OSBPL2的ORD结构域的氨基酸范围,采用http://plmd.bio⁃ cuckoo.org/在线数据库预测OSBPL2的泛素化位点,在截短体和突变位点附近设计截短引物和点突变引物,使用Mut Express ⅡFast Mutagenesis Kit V2试剂盒进行目的片段的扩增和消化,然后切胶回收,产物进行重组转化,将转化产物均匀涂布在相应抗性的LB平板上,平板倒置于37℃培养箱中培养12~16h。挑板、摇菌,菌液送公司测序,比对测序结果,找出序列正确的菌斑,扩增保存菌液,用于后续实验。USP14过表达与截短体载体构建引物见表2,点突变载体构建引物见表3。USP14⁃siRNA序列: Forward⁃5′⁃GCCUCGCAGAGUUGAAAUATT⁃3′,Re⁃ verse⁃5′⁃UAUUUCAACUCUGCGAGGCTT⁃3′。

  • 1.2.6 泛素化信号检测及OSBPL2泛素化水平检测

  • 泛素化信号检测:在细胞中过表达目的蛋白Flag⁃OSBPL2、Myc⁃USP14以及HA⁃Ub质粒,48~72h收取细胞RIPA裂解液,提取蛋白,以标签定量目的蛋白,以GAPDH定量总体泛素化,Western blot检测目的蛋白泛素信号的改变。

  • OSBPL2泛素化水平检测:293Ta细胞中共转染目的蛋白Flag⁃OSBPL2、Myc⁃USP14以及HA⁃UBB、 HA⁃K48或者HA⁃K63质粒,使用ANTI⁃FLAG® M2Affinity Gel富集目的蛋白,以Flag标签定量目的蛋白,检测目的蛋白的泛素化水平。

  • 1.3 统计学方法

  • 采用GraphPad Prism 7.0进行统计分析。两独立样本采取 t 检验,定量结果和灰度扫描等数据采用均数±标准差(x-±s)表示,P< 0.05为差异有统计学意义。

  • 2 结果

  • 2.1 OSBPL2与USP14的相互作用验证

  • 外源性过表达和Co⁃IP实验结果显示,OSBPL2与USP14之间存在相互作用(图1A)。通过构建Flag⁃OSBPL2和HA⁃USP14的截短蛋白(图1B)进一步阐明两者互作的关键结构域,Co⁃IP检测结果发现,USP14的CAT结构域与OSBPL2的ORD结构域存在相互作用关系,而UBL结构域不与ORD结构域相互作用(图1C)。这一结果提示,CAT是OSBPL2与USP14互作的关键结构域。

  • 表2 USP14过表达与截短体载体构建引物

  • Table2 Primers for USP14⁃overexpression and truncated vector

  • 加粗字体表示同源重组连接的同源臂,加粗小写字体表示KpnⅠ和Bam HⅠ酶切位点。

  • 表3 突变体载体构建引物

  • Table3 Primers for mutant vector

  • 黑色小写加粗字体表示点突变的碱基。

  • 2.2 USP14参与OSBPL2的翻译后修饰过程

  • 过表达USP14的HeLa细胞中OSBPL2的蛋白水平增加,而转录水平没有改变(图2A)。敲降USP14的HeLa细胞中OSBPL2的蛋白表达水平降低,而转录水平并未改变(图2B)。采用USP14活性抑制剂IU1处理细胞之后,USP14表达量并没有改变,OSBPL2的表达量降低,并且有剂量依赖趋势(图2C)。用CHX处理过表达USP14的细胞,OSBPL2蛋白的周转速率降低(图2D)。综合以上结果说明, USP14通过与OSBPL2相互作用参与OSBPL2的翻译后修饰过程,从而稳定OSBPL2蛋白的表达。

  • 2.3 USP14 通过抑制OSBPL2 的K48 泛素链降低OSBPL2的泛素化水平

  • 体外过表达实验的结果表明USP14能够识别并切割多泛素基团,尤其是Ub4⁃8泛素基团的信号明显减弱(图3A)。采用Anti⁃Flag富集OSBPL2,Western blot检测结果显示USP14可以明显降低OSBPL2的泛素化水平(图3B)。进一步探究USP14抑制的泛素链类型可知,USP14抑制了OSBPL2偶联的K48链泛素化水平(图3C)而不影响K63链泛素化水平(图3D)。综合以上结果说明,USP14通过抑制OSBPL2的K48链泛素化进而调控OSBPL2的泛素化水平。

  • 图1 OSBPL2与USP14的相互作用验证以及关键结构域

  • Fig.1 Interaction between USP14and OSBPL2and the key interactional domains

  • 图2 USP14参与OSBPL2的翻译后修饰过程

  • Fig.2 USP14involved in the post⁃translational modification of OSBPL2

  • 2.4 蛋白酶体抑制或UBL结构域突变诱导USP14 与OSBPL2互作增强

  • UBL结构域是USP14的另一个结构域,介导USP14与蛋白酶体的结合。乳胞素(Lactacysin)是一种蛋白酶体抑制剂,可以特异性不可逆地从蛋白酶体上解离USP14。在乳胞素处理的HeLa细胞中, USP14与非ATP酶蛋白酶体26S亚基2(PSMD2)的相互作用明显减弱(图4A)。UBL结构域中第54~60位的氨基酸高度保守,所以将第58位的色氨酸突变为丙氨酸(W58A),构建UBL结构域突变的质粒,以模拟乳胞素抑制蛋白酶体的效果,Co⁃IP检测结果显示,USP14与PSMD2的结合明显减弱(图4B),与乳胞素处理所得结果相同,进一步确定了USP14通过UBL结构域与蛋白酶体相结合。HeLa细胞转染过表达野生型USP14质粒和UBL结构域突变的USP14质粒,Co⁃IP检测结果表明,当USP14的UBL结构域突变之后,USP14与OSBPL2的结合会明显增强(图4C)。当加入MG132抑制蛋白酶体活性之后,与对照组相比,USP14与OSBPL2的相互作用也有所增强,趋势与UBL结构域突变之后的结果一致(图4D)。综合以上结果可知,当抑制蛋白酶体活性或者突变UBL结构域之后,都会导致USP14与OSBPL2相互作用增强,进而稳定OSBPL2蛋白的表达。

  • 2.5 Lys209 和Lys361 位点参与OSBPL2 去泛素化调控

  • 为了进一步探究OSBPL2上K48泛素链可能的连接位点,通过蛋白质赖氨酸修饰数据库(Protein⁃ Lysine Modifications Database,PLMD,http://plmd.biocuckoo.org/)预测,提示OSBPL2有5个泛素化位点且都在ORD结构域中(表4)。根据预测结果,构建5个位点的突变质粒(图5A)。Co⁃IP检测结果发现,当Lys209和Lys361位点突变后,OSBPL2与USP14的相互作用减弱(图5B、C),OSBPL2的泛素化水平未受影响(图5D)。此结果说明OSBPL2的Lys209和Lys361很可能是K48泛素链连接OSBPL2并被USP14去泛素化修饰的关键位点。

  • 3 讨论

  • 本课题组在国际上最先定位和克隆了人常染色体显性遗传性耳聋的致病基因OSBPL2,但OSBPL2突变致聋的分子发病机制迄今仍未阐明。该基因编码的蛋白属于OSBP/ORP家族成员,ORP作为一类重要的固醇感受器,参与了脂质代谢与转运、囊泡运输和信号转导等过程[1-2]。此外,研究发现一些OSBP/ORP成员通过泛素化修饰参与一系列生物学过程,如在探索镉元素(cadmium,Cd)对胆固醇代谢影响的研究中发现,Cd能够诱导OSBPL蛋白泛素化而降解,进而导致胆固醇代谢紊乱[20]。OSBP/ORP家族的另一个成员OSBPL8是USP5的新底物,USP5使OSBPL8去泛素化并在结直肠癌细胞内累积,进而导致内质网应激产生[21]。这些结果均提示了OSBP/ORP家族成员具有泛素化修饰的可能性。

  • 图3 USP14通过抑制OSBPL2的K48链泛素化降低OSBPL2的泛素化水平

  • Fig.3 USP14reduced the ubiquitination level of OSBPL2by inhibiting K48ubiquitin chain of OSBPL2

  • 图4 蛋白酶体抑制或UBL结构域突变诱导USP14与OSBPL2互作增强

  • Fig.4 Proteasome inhibition or UBL domain mutation induced enhanced⁃interaction between USP14and OSBPL2

  • 表4 OSBPL2蛋白泛素化位点预测

  • Table4 Prediction of ubiquitination sites of OSBPL2pro⁃ tein

  • 红色字体为PLMD数据库预测的OSBPL2泛素化位点。

  • 目前关于OSBPL2泛素化与去泛素化的相关研究还鲜见报道。基于PLMD在线软件的预测结果提示,OSBPL2的ORD结构域有5个泛素化位点,并且实验结果显示OSBPL2能够与泛素Ub和K48泛素链结合,提示存在OSBPL2翻译后去泛素化修饰的可能。本研究进一步证实USP14与OSBPL2之间具有相互作用,USP14过表达或者敲降对OSBPL2mRNA水平无明显影响,而对其蛋白表达影响显著,因此认为USP14通过与OSBPL2互作参与OSBPL2的翻译后修饰过程,从而稳定OSBPL2蛋白的表达。USP14作为去泛素化酶,其作用就是结合并切割靶蛋白的泛素链,避免靶蛋白的降解而维持蛋白表达的稳定[22]。因此,如同USP14在FASN、RIG⁃I等蛋白表达中的作用[23-24],USP14可能通过翻译后去泛素化修饰稳定OSBPL2表达。

  • 图5 Lys209和Lys361位点参与OSBPL2去泛素化调控

  • Fig.5 Lys209and lys361sites involved in the regulation of OSBPL2deubiquitination

  • 本研究进一步验证了USP14对OSBPL2的去泛素化修饰作用,这与文献报道的USP14参与cGAS等的生理调控功能一致[25-26]。USP14通过CAT结构域发挥去泛素化功能,CAT结构域包含两个保守基序:Cys和His盒,这也是USP家族共有的保守基序,催化残基就位于这段基序中。研究结果表明USP14的CAT结构域能够与OSBPL2结合,因此USP14通过CAT结构域切割OSBPL2泛素链,从而实现OSB⁃ PL2去泛素化,进而稳定OSBPL2表达,这与文献报道的CAT结构域能够稳定Tau蛋白的表达一致[13]。本研究还进一步证实USP14通过UBL结构域与蛋白酶体相结合,并调控OSBPL2的表达。

  • Kim等[27] 研究显示USP14的UBL结构域通过与泛素链结合,能展现出一些共有的特性。例如,UBL可以刺激19S蛋白酶体的ATPases,刺激内在26S蛋白酶体中无序蛋白Sic1的降解,增加内源性蛋白的降解速率等。这些研究提示,UBL结构域在调控蛋白酶体活性以及蛋白降解方面的作用也非常重要。本研究表明UBL结构域突变导致USP14与蛋白酶体结合减弱,这与蛋白酶体抑制剂的效应一致。此外,UBL结构域突变或抑制蛋白酶体活性都能够增强USP14与OSBPL2的互作,且UBL突变的USP14与OSBPL2的结合能力远大于加入抑制剂之后的结合能力,这可能是由于USP14与蛋白酶体结合抑制了蛋白酶体活性的同时提高了自身活性[28]

  • 近年来,USP14引起了研究者们的广泛关注。综合现有的关于USP14去泛素化底物的研究发现, USP14广泛参与了机体各种生理过程,并且与多种疾病密切相关,其中约61%的癌症都与USP14有关[29]。USP14主要通过去泛素化Cyclin B1等细胞周期蛋白,抑制细胞的侵袭和迁移。USP14还参与了自噬的调控,如USP14通过与RNF168和Beclin 1等蛋白的互作调控DNA损伤修复过程,进而调控自噬[1830]。在炎症反应中,USP14可通过去泛素化RIG⁃I耦连的K63泛素链介导免疫应答等[24]。此外,USP14与脂质代谢关系密切,如脂肪酸合酶 (synthase,FASN)是USP14的直接底物,在小鼠体内过表达USP14可维持FASN的稳定表达并导致脂肪肝[23]。最近研究表明,蛋白酶体系统是调控初级纤毛形成的核心系统之一,并且纤毛相关蛋白与蛋白酶体亚基具有相互作用[31-34]。本研究中OSBPL2的生物学功能与脂质代谢关系密切,而USP14可以通过抑制K48链调控OSBPL2的泛素水平,进而稳定OSBPL2的表达。上述结果提示,USP14可能通过调控OSBPL2的表达进而参与脂质代谢和听觉细胞纤毛的调控。

  • 本研究证实了去泛素化酶USP14对OSBPL2的去泛素化修饰的调控作用,从泛素化和去泛素化修饰角度研究OSBPL2的生物学功能将为探索OSBPL2突变致聋的分子发病机制提供新思路。

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  • 参考文献

    • [1] KENTALA H,WEBER ⁃BOYVAT M,OLKKONEN V M.OSBP ⁃ related protein family:mediators of lipid transport and signaling at membrane contact sites[J].Int Rev Cell Mol Biol,2016,321:299-340

    • [2] PIETRANGELO A,RIDGWAY N D.Bridging the molecu⁃ lar and biological functions of the oxysterol ⁃ binding pro⁃ tein family[J].Cell Mol Life Sci,2018,75(17):3079-3098

    • [3] 王天明,曾华沙,王盈,等.OSBPL2基因缺陷型巴马小型猪肥胖相关特征的分析[J].南京医科大学学报(自然科学版),2020,40(9):1281-1288

    • [4] LAITINEN S,LEHTO M,LEHTONEN S,et al.ORP2,a homolog of oxysterol binding protein,regulates cellular cholesterol metabolism[J].J Lipid Res,2002,43(2):245-255

    • [5] ZHANG C,ZHANG H,ZHANG M,et al.OSBPL2 defi⁃ ciency upregulate SQLE expression increasing intracellu⁃ lar cholesterol and cholesteryl ester by AMPK/SP1 and SREBF2 signalling pathway[J].Exp Cell Res,2019,383(2):111512

    • [6] YAO J,ZENG H,ZHANG M,et al.OSBPL2 ⁃ disrupted pigs recapitulate dual features of human hearing loss and hypercholesterolaemia[J].J Genet Genomics,2019,46(8):379-387

    • [7] WANG T,WEI Q,LIANG L,et al.OSBPL2 is required for the binding of COPB1 to ATGL and the regulation of lipid droplet lipolysis[J].iScience,2020,23(7):101252

    • [8] WANG H,LIN C,YAO J,et al.Deletion of OSBPL2 in au⁃ ditory cells increases cholesterol biosynthesis and drives reactive oxygen species production by inhibiting AMPK activity[J].Cell Death Dis,2019,10(9):627

    • [9] WANG T,ZHANG T,TANG Y,et al.Oxysterol ⁃ binding protein ⁃ like 2 contributes to the developmental progres⁃ sion of preadipocytes by binding to β ⁃ catenin[J].Cell Death Discov,2021,7(1):109

    • [10] GLICKMAN M H,CIECHANOVER A.The ubiquitin⁃pro⁃ teasome proteolytic pathway:destruction for the sake of construction[J].Physiol Rev,2002,82(2):373-428

    • [11] LIU B,CHEN J,ZHANG S.Emerging role of ubiquitin ⁃ specific protease 14 in oncogenesis and development of tumor:therapeutic implication[J].Life Sci,2019,239:116875

    • [12] DE POOT S A H,TIAN G,FINLEY D.Meddling with fate:the proteasomal deubiquitinating enzymes[J].J Mol Biol,2017,429(22):3525-3545

    • [13] LEE B H,LEE M J,PARK S,et al.Enhancement of prote⁃ asome activity by a small ⁃ molecule inhibitor of USP14 [J].Nature,2010,467(7312):179-184

    • [14] LEE B H,LU Y,PRADO M A,et al.USP14 deubiqui⁃ tinates proteasome⁃bound substrates that are ubiquitinated at multiple sites[J].Nature,2016,532(7599):398-401

    • [15] FAESEN A C,LUNA ⁃VARGAS M P,SIXMA T K.The role of UBL domains in ubiquitin ⁃ specific proteases[J].Biochem Soc Trans,2012,40(3):539-545

    • [16] CHAKRABORTY J,VON STOCKUM S,MARCHESAN E,et al.USP14 inhibition corrects an in vivo model of im⁃ paired mitophagy[J].EMBO Mol Med,2018,10(11):e9014

    • [17] SRINIVASAN V,BRUELLE C,SCIFO E,et al.Dynamic interaction of USP14 with the chaperone HSC70 mediates crosstalk between the proteasome,ER signaling,and au⁃ tophagy[J].iScience,2020,23(1):100790

    • [18] SHARMA A,ALSWILLAH T,SINGH K,et al.USP14 regu⁃ lates DNA damage repair by targeting RNF168 ⁃ depen⁃ dent ubiquitination[J].Autophagy,2018,14(11):1976-1990

    • [19] LI H,QUAN J,ZHAO X,et al.USP14 negatively regu⁃ lates RIG⁃I⁃mediated IL⁃6 and TNF⁃α production by in⁃ hibiting NF ⁃κB activation[J].Mol Immunol,2021,130:69-76

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    • [23] LIU B,JIANG S,LI M,et al.Proteome ⁃wide analysis of USP14 substrates revealed its role in hepatosteatosis via stabilization of FASN[J].Nat Commun,2018,9(1):4770

    • [24] LI H,ZHAO Z,LING J,et al.USP14 promotes K63 ⁃ linked RIG ⁃ I deubiquitination and suppresses antiviral immune responses[J].Eur J Immunol,2019,49(1):42-53

    • [25] CHEN M,MENG Q,QIN Y,et al.TRIM14 inhibits cGAS degradation mediated by selective autophagy receptor p62 to promote innate immune responses[J].Mol Cell,2016,64(1):105-119

    • [26] WERTZ I E,MURRAY J M.Structurally⁃defined deubiq⁃ uitinase inhibitors provide opportunities to investigate dis⁃ ease mechanisms[J].Drug Discov Today Technol,2019,31:109-123

    • [27] KIM H T,GOLDBERG A L.UBL domain of Usp14 and other proteins stimulates proteasome activities and pro⁃ tein degradation in cells[J].PNAS,2018,115(50):E11642-E11650

    • [28] KIM H T,GOLDBERG A L.The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activi⁃ ties and ubiquitin ⁃ independent proteolysis[J].J Biol Chem,2017,292(23):9830-9839

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