Involvement of T-complex protein 1-ring complex/chaperonin containing T-complex protein 1(TRiC/CCT) in retrograde axonal transport through tau phosphorylation

The cytosolic chaperonin T-complex protein 1-ring complex(TRiC)or chaperonin containing T-complex protein 1(CCT)is essential in de novo folding of approximately 10%of the eukaryotic,newly translated polypeptides as well as misfolded proteins.There is a close link between the TRiC/CCT cytosolic chaperonin and neurodegenerative diseases(Lopez et al.,2015).A lot of research suggests that CCT plays neuroprotective roles in neurodegenerative diseases including Huntington’s disease(Lopez et al.,2015).Either overexpression of a single or all eight subunits(CCT1-8)or treatment of the substrate-binding apical domain of yeast CCT1(ApiCCT1)prevented mutant Huntingtin aggregation and improved cellular and neuronal functions(Zhao et al.,2016).Importantly,our recent study has demonstrated that both CCT and ApiCCT could reduce mutant Huntingtin level and enhance both anterograde and retrograde axonal transport of brain-derived neurotrophic factor.These results led to restoration of the trophic status of striatal neurons from a bacterial artificial chromosome transgenic mouse model of Huntington’s disease(Zhao et al.,2016).Axonal transport is regulated by many factors including microtubule-associated protein tau,which promotes tubulin polymerization and stabilizes microtubules.Impaired interaction between tau and microtubules plays a vital role in the pathogenesis of multiple neurodegenerative diseases(Wang and Mandelkow,2016).Interestingly,tau phosphorylation is also observed in brains of several Huntington’s disease mouse models and Huntington’s disease patients(Gratuze et al.,2016).In a recent study,we explored if CCT subunit has any effect on axonal transport in a tau-dependent pathway(Chen et al.,2018b).We focused on the retrograde axonal transport of brain-derived neurotrophic factor,as neurotrophic factor-mediated signaling in the form of signaling endosome is essential in both the developing and the mature nervous system and dysregulation of trafficking of neurotrophic factors is tightly linked to disorders of the nervous system(Chen et al.,2018a).We found that the expression of a single CCT subunit(CCT5)significantly promoted retrograde axonal transport of brain-derived neurotrophic factor in primary cortical neurons.Mechanically,CCT regulated the level of cyclin-dependent kinase 5(CDK5)/p35/p25 and,subsequently contributed to CCT-induced tau phosphorylation,which induced detachment of tau from microtubules(Chen et al.,2018b)(Figure 1).

皱纹盘鲍伴侣蛋白CCTζ的分子克隆及其在不同锌含量日粮处理下的表达分析(英文)

伴侣蛋白CCT在蛋白折叠、抗胁迫以及调节细胞生长等生理过程中担负重要作用。研究利用同源克隆和RACE技术克隆了皱纹盘鲍伴侣蛋白CCTζ(HdhCCTζ)cDNA全长序列,并对CCTζ基因的序列特征进行了分析。HdhCCTζcDNA序列长1960 bp,开放阅读框为1599 bp,编码532个氨基酸,生物学软件推测其编码蛋白相对分子量为58.46 kD,等电点为6.53。BLAST分析结果表明HdhCCTζ与哺乳动物、鸟和鱼类等物种的CCT具有较高的同源性。生物信息学分析结果显示,HdhCCTζ蛋白序列中包含3个典型的CCT信号基序(35RTNLGPKGTLKML47,56LTKDGNVLLHEMQIQHP72和84QDDITGDGT92)以及1个腺苷三磷酸结合基序(89GDGTTS94)。此外,在HdhCCTζ蛋白序列中还包含3个糖基化位点(23NISA26,128NKSL131and234NVSL237)。实时荧光定量PCR检测结果显示,HdhCCTζ在皱纹盘鲍组织中为组成型表达,但主要集中在性腺、血淋巴和肝胰腺中表达。使用不同锌含量(6.69 mg/kg、33.85 mg/kg、710.63 mg/kg和3462.5 mg/kg)的日粮来饲喂幼鲍20周后,取其肝脏和血细胞总RNA,利用实时荧光定量PCR技术进行HdhCCTζmRNA的表达水平分析。分析结果显示,随着日粮中锌含量的增加,HdhCCTζmRNA在血淋巴和肝胰腺中的表达水平呈现上调的趋势。相比于锌适量组(33.85 mg/kg),过量的锌(3462.5 mg/kg)却能够下调HdhCCTζmRNA的表达水平。上述结果表明,HdhCCTζ的表达受到日粮中锌添加量的影响,而高表达量的HdhCCTζ可能在提高皱纹盘鲍机体抗胁迫过程中发挥重要作用。

CCT4 suppression inhibits tumor growth in hepatocellular carcinoma by interacting with Cdc20

Background:The chaperonin containing t-complex(CCT)proteins play an important role in cell cycle-related protein degradation in yeast and mammals.The role of the chaperonin containing t-complex 4(CCT4),one subtype of CCT proteins,in the progress of hepatocellular carcinoma(HCC)was not fully elucidated.Here,we aimed to explore the mechanisms of CCT4 in HCC.Methods:In this study,we used the UALCAN platform to analyze the relationship between CCT4 and HCC,and the association of CCT4 with the overall survival(OS)of HCC patients was also analyzed.CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot(WB)assay.Lentivirus vector was used to knock down the CCT4 expression,and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines.Cell counting kit-8(CCK-8)and 5-ethynyl-20-deoxyuridine(EdU)assays were used to detect the cell proliferation,and flow cytometry(FCM)was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells.Co-immunoprecipitation(co-IP)assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC.Data were calculated from at least three replicate experiments and expressed as mean±standard deviation.Student’s t test,paired t test,and Kaplan–Meier analysis were used to compare across different groups.Results:We found CCT4 was upregulated in HCC tissues compared with normal tissues,and its high expression was associated with poor prognosis(P<0.001).CCT4 was significantly increased in HCC tumor tissues compared with normal tissues(0.98±0.12 vs.0.23±0.05,t=7.73,P<0.001).After being transfected with CCT4 short-hairpin RNA(shRNA),CCT4 was decreased in mRNA level and protein level in both Huh7(mRNA level:0.41±0.07 vs.1.01±0.11,t=8.09,P=0.001;protein level:0.61±0.03 vs.0.93±0.07,t=7.19,P=0.002)and Hep3b cells(mRNA level:0.55±0.11 vs.1.04±0.15,t=4.51,P=0.011;protein level:0.64±0.10 vs.0.95±0.08,t=4.32,P=0.012).CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7(OD value of 3 days:0.60±0.14 vs.0.97±0.16,t=3.13,P=0.036;OD value of 4 days:1.03±0.07 vs.1.50±0.12,t=5.97,P=0.004)and Hep3b(OD value of 3 days:0.69±0.14 vs.1.10±0.11,t=3.91,P=0.017;OD value of 4 days:1.12±0.12 vs.1.48±0.13,t=3.55,P=0.024)cells.EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7(EdU positive rate:[31.25±3.41]%vs.[58.72±3.78]%,t=9.34,P=0.001)and Hep3b cells(EdU positive rate:[44.13±7.02]%vs.[61.79±3.96]%,t=3.79,P=0.019).FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells(apoptosis rate of Huh7:[9.10±0.80]%vs.[3.66±0.64]%,t=-9.18,P=0.001;apoptosis rate of Hep3b:[6.69±0.72]%vs.[4.20±0.86]%,t=-3.84,P=0.018).We also found that CCT4 could regulate anaphase-promoting complex(APC)Cdc20 activity via interacting with Cdc20.Furthermore,CCT4 knockdown induced securin(0.65±0.06 vs.0.44±0.05,t=-4.69,P=0.009)and B-cell lymphoma-2(Bcl-2)interacting mediator of cell death(Bim;0.96±0.06 vs.0.61±0.09,t=securin inhibited cell growth by downregulating cyclin D1(0.65±0.05 vs.1.04-±5.65,0.07,Pt==0.005)accumulation.The upregulation of 8.12,P=0.001),and the accumulation of Bim inhibited Bcl-2(0.77±0.04 vs.0.87±0.04,t=3.00,P=0.040)and activated caspase 9(caspase 9:0.77±0.04 vs.0.84±0.05,t=1.81,P=0.145;cleaved caspase 9:0.64±0.06 vs.0.16±0.07,t=1.81,P=0.001),which led to elevated apoptosis.Conclusions:Overall,these results showed that CCT4 played an important role in HCC pathogenesis through,at least partly,interacting with Cdc20.