TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

大鼠肝再生相关基因Tax1bp1与绿色荧光蛋白融合表达的研究

目的构建大鼠肝再生相关基因Tax1bp1的真核表达载体,融合绿色荧光蛋白并在再生肝中高效表达,观察目的蛋白的表达情况。方法利用反转录聚合酶链式反应(RT-PCR)技术以大鼠再生肝为材料克隆Tax1bp1基因的开放阅读框(ORF),然后亚克隆到表达载体上构建重组表达载体pEGFP-N1-Tax1bp1,再用尾静脉液压法转入大鼠再生肝中进行绿色荧光蛋白融合表达。结果 Tax1bp1ORF克隆正确,融合绿色荧光蛋白重组表达载体pEGFP-N1-Tax1bp1构建成功;部分肝切除转基因6、12、24、48 h后均有荧光表达,Tax1bp1融合蛋白在上述时间点绿色荧光融合蛋白细胞转染率分别为8.7%、11.2%、14.5%和1.2%,转染率趋势同空载体绿色荧光蛋白表达情况一致。结论重组表达载体pEGFP-N1-Tax1bp1构建成功,并实现了绿色荧光融合蛋白的高效表达,为进一步研究肝再生相关Tax1bp1基因功能奠定了基础。

TAX1BP1调控B细胞自噬和凋亡的研究

目的探究TAX1BP1在B细胞自噬与凋亡中的作用。方法慢病毒感染B淋巴瘤细胞(Raji细胞),敲低TAX1BP1表达。利用雷帕霉素处理Raji细胞,通过Western blot、细胞免疫荧光染色和流式细胞术,观察自噬流强弱以及细胞凋亡情况。通过检测自噬相关通路P53蛋白表达变化,初步探索调控机制。结果荧光显微镜观察慢病毒感染阳性率在90%以上。与阴性组比较,shTAX1BP1组细胞中TAX1BP1的mRNA表达水平降低80%、蛋白表达水平降低90%。经10 nmol/L雷帕霉素处理后,与阴性组相比较,shTAX1BP1组LC3II/I比值显著下降(P<0.01),凋亡相关蛋白Bcl-2表达显著升高,Bax、Caspase-3表达显著下降。敲低TAX1BP1降低Raji细胞自噬水平,减少Raji细胞凋亡。敲低TAX1BP1增加P53蛋白表达,推测TAX1BP1可能通过P53相关通路调控Raji细胞的自噬与凋亡。结论敲低TAX1BP1可抑制Raji细胞自噬与凋亡,初步预测这一生物学作用可能与P53信号通路有关。本研究为进一步探讨SLE的发病机制奠定了一定的实验基础。

选择性自噬受体TAX1BP1的研究进展及意义

自噬是真核细胞内主要的降解系统之一,在清除细胞内受损物质方面发挥着重要作用。近年来,自噬与疾病的关系成为研究的热门话题。自噬功能的异常往往影响着疾病的发生、发展及预后。细胞通过自噬途径选择性地清除某些细胞质成分的过程称为选择性自噬。选择性自噬的发生通常需要自噬受体的参与,不同的自噬受体发挥的具体功能也不尽相同。其中,Tax1结合蛋白1(Tax1-binding protein 1,TAX1BP1)作为选择性自噬接头蛋白的一员,主要由一个SKIP羧基同源性域(SKIP carboxyl homology,SKICH)、一个微管相关蛋白I轻链3结合结构域(LC3-interacting region,LIR)、三个卷曲螺旋和一个羧基末端泛素锌指结合(ubiquitin-binding zinc finger,UBZ)结构域构成。这些结构域介导了TAX1BP1与其他蛋白质的相互作用,并在一定程度上对TAX1BP1在细胞中的功能产生影响。TAX1BP1同时调节NF-κB、JNK等信号通路;它广泛地参与到线粒体自噬、异体自噬以及溶酶体自噬等自噬进程中去;TAX1BP1的异常表达与炎症反应、恶性肿瘤及循环系统疾病的发生密切相关。该文主要对TAX1BP1的结构功能、调节的信号通路、与自噬的关系,以及其生理意义进行系统的总结,为今后TAX1BP1的研究提供新思路。

TAX1BP1 protects against myocardial infarction-associated cardiac anomalies through inhibition of inflammasomes in a RNF34/MAVS/NLRP3-dependent manner

Acute myocardial infarction(MI),one of the most common cardiovascular emergencies,is a leading cause of morbidity and mortality.Ample evidence has revealed an essential role for inflammasome activation and autophagy in the pathogenesis of acute MI.Tax1-binding protein 1(TAX1BP1),an adaptor molecule involved in termination of proinflammatory signaling,serves as an important selective autophagy adaptor,but its role in cardiac ischemia remains elusive.This study examined the role of TAX1BP1 in myocardial ischemic stress and the underlying mechanisms involved.Levels of TAX1BP1 were significantly downregulated in heart tissues of patients with ischemic heart disease and in a left anterior descending(LAD)ligation-induced model of acute MI.Adenovirus carrying TAX1BP1 was delivered into the myocardium.The acute MI induced procedure elicited an infarct and cardiac dysfunction,the effect of which was mitigated by TAX1BP1 overexpression with little effect from viral vector alone.TAX1BP1 nullified acute MI-induced activation of the NLRP3 inflammasome and associated mitochondrial dysfunction.TAX1BP1 overexpression suppressed NLRP3 mitochondrial localization by inhibiting the interaction of NLRP3 with mitochondrial antiviral signaling protein(MAVS).Further investigation revealed that ring finger protein 34(RNF34)was recruited to interact with TAX1BP1 thereby facilitating autophagic degradation of MAVS through K27-linked polyubiquitination of MAVS.Knockdown of RNF34 using siRNA nullified TAX1BP1 yielded protection against hypoxia-induced MAVS mitochondrial accumulation,NLRP3 inflammasome activation and associated loss of mitochondrial membrane potential.Taken together,our results favor a cardioprotective role for TAX1BP1 in acute MI through repression of inflammasome activation in a RNF34/MAVS-dependent manner.

Tax1结合蛋白1对B细胞功能的抑制作用

目的探讨Tax1结合蛋白1(TAX1BP1)对B细胞增殖、活化、免疫球蛋白分泌等功能的影响。方法利用免疫磁珠法分选小鼠脾脏CD19^+B细胞,转染siRNA下调TAX1BP1的表达;并利用TAX1BP1慢病毒感染B细胞株Raji细胞系建立稳定下调TAX1BP细胞系。体外用LPS刺激B细胞后通过CCK8、流式细胞术和ELISA法分别评价TAX1BP1对B细胞增殖、B细胞表面共刺激分子表达、细胞周期以及免疫球蛋白分泌的影响。结果与对照组相比,敲低TAX1BP1促进了B细胞的增殖(P=0.008)及Raji细胞的增殖(P<0.0001),上调了B细胞表面活化分子CD69和CD80的表达(P<0.001),促进了免疫球蛋白lgG及lgM的生成(P<0.05)。结论TAX1BP1可以通过抑制B细胞增殖、活化、抗体产生等多方面调节B细胞功能。