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 we...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).展开更多
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 ...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.展开更多
文摘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).
基金the Natural Science Foundation of Anhui Province(No.1908085QH325).
文摘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.
