腾冲嗜酸热两面菌伴侣素ATcpnβ底物结合特性研究

嗜酸嗜热古菌腾冲嗜酸热两面菌(Acidianus tengchongensis)来源的Ⅱ型伴侣素ATcpnβ已获得晶体结构解析,其顶端结构域突触下端相应于Ⅰ型伴侣素GroEL的重要底物结合位点处的氨基酸多为极性氨基酸,将其突变为疏水性氨基酸时,突变体对变性底物的捕获能力显著增强.表面等离子共振研究表明,ATcpnβ对于化学变性底物的再折叠中间体的其捕获作用不依赖于Mg2+/ATP.前期对该伴侣素冷冻电镜观察和结构解析表明,ATP的存在并不能驱动ATcpnβ从开放构型向闭合构型转变,但是表面等离子共振研究表明,ATcpnβ对热变性过程中已经聚集的底物的捕获作用依赖于Mg2+/ATP,说明Mg2+/ATP可以介导ATcpnβ顶端结构域一定的构象变化,引起顶端结构域疏水残基的进一步暴露,从而能够与大分子聚集体紧密结合.两个方面的研究均表明,伴侣素蛋白与变性底物的结合仍然以疏水相互作用为主,并且伴侣素蛋白与变性底物的结合受Mg2+/ATP的结合调控,与伴侣素蛋白疏水面的暴露程度相关.

Overexpression of chaperonin containing TCP1, subunit 3 predicts poor prognosis in hepatocellular carcinoma

AIM:To investigate the value of chaperonin containing TCP1,subunit 3(CCT3) to predict the prognosis of patients with hepatocellular carcinoma(HCC) and determine its function in HCC progression.METHODS:CCT3 expression levels were examined in human non-cancerous liver tissues and a variety of HCC cell lines by quantitative real-time PCR and immunoblotting.CCT3 expression was suppressed by small interfering RNA.The effects of reducing CCT3 expression in HCC cells were tested.The3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide(MTT) assay,cell counting experiment,cell cycle assay,apoptosis assay and invasion assay were employed to evaluate cell functions in vitro.Immunohistochemistry was performed on HCC specimens.In addition,CCT3 expression in HCC specimens was also assessed at the protein and mRNA level.Associations between clinicopathological characteristics and prognosis were analyzed,along with the possible mechanisms involved in CCT3's function in HCC progression.RESULTS:The expression levels of CCT3 mRNA and protein were upregulated in HCC cell lines in contrast to adjacent non-cancerous tissues.Reducing CCT3 expression not only suppressed cell proliferation in cell counts,MTT assay,cell cycle assay and induced cell apoptosis(P < 0.05 vs negative control),but also inhibited the tumor cell invasion capacity in vitro {P< 0.01 vs negative control).Overexpression of CCT3 in the nuclei of cancer cells in HCC specimens(58of 104 patients,55.8%) was associated with poor prognosis in HCC patients(3-year survival rate,55.5%vs 84.2%,P = 0.020) after hepatectomy.Mechanistic analyses showed that signal transducer and activator of transcription 3(STAT3) activation was decreased even when stimulated by interleukin-6 after knocking down CCT3 in the HepG2 cell line.CONCLUSION:Overexpression of CCT3 in the nuclei of cancerous cells is associated with HCC progression.CCT3 may be a target that affects the activation of STAT3 in HCC.

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).

Fusion Proteins Cpn10-E^(rns) with Properties of Generating CSFV-Neutralized Antibodies

When pigs are infected with classical swine fever virus （ CSFV）, the antibody primarily targets the structural giycoprotein E^rns of the virus. Previous investigations have demonstrated that ETM has low or no virus neutralizing capacity. In this study, candidate subunit marker vaccine, chaperonin 10 （Cpnl0）-E^rns, which possess the property of generating neutralized antibodies against lethal challenge of virulent CSFV was developed. The gene of ETM was isolated from Hog cholera lapinized virus （HCLV） -infected spleen cells of rabbits via RT-PCR method and fused to the downstream region of the cpn10 gene; the products of recombinant fusion protein （ cpn10-E^rns ） induced expression in Escherichia coli, and the products were purified by affinity chromatography. During the course of vaccination, the candidate vaccines cpn10-E^rns were used for the immunization of guinea pigs, and they induced a strong antibody response against cpn10-E^rns. The antibodies can be immobilized by coating inactivated CSFV particles, indicating that these antibodies can recognize CSFV. Neutralization assay was carried out on rabbits according to National Regulations on Veterinary Drug. The results clearly indicate that the typical fever of rabbits induced by the live attenuated HCLV could be inhibited by preincubation with the antisera （dilution 1： 4） induced by cpn10-E^rns, but not inhibited by preincubation with the antisera induced only by E^rns. Analogous results were observed for the group of the rabbits immunized with cpn10-E^rns, which were protected against the typical fever induced by the challenge with HCLV. The findings of this study formed the basis of a new means for developing subunit marker vaccine against CSFV.

Cytosolic chaperonin CCT possesses GTPase activity

Cytosolic chaperonin CCT (also known as TRiC) is a hetero-oligomeric cage-like molecular chaperone that assists in protein folding by ATPase cycle-dependent conformational changes. However, role of the nucleo-tide binding and hydrolysis in CCT-assisted protein folding is still poorly understood. We purified CCT by using ATP-Sepharose and other columns, and found that CCT possesses ability to hydrolyze GTP, with an activity level very similar to the ATPase activity. CCT was more resistant to proteinase K treatment in the presence of GTP or ATP. These results suggest that the GTPase activity of CCT may play a role in chaperone-assisted protein folding.

Suppression of Sup35 amyloid fibril formation by group II chaperonin from <i>Thermoplasma acidophilum</i>

The Group II chaperonin from Thermoplasma acidophilum was added to the in vitro amyloid fibrillation reaction of yeast Sup35NM protein to assess its effects. By measuring the formation of Sup35NM fibrils in real time using the fluorescent dye Thioflavin T, we found that the addition of T. acidophilum-cpn α16, α1, and β1 proteins suppressed fibril formation. Addition of a 0.1 molar-equivalent T. acidophilum-cpn α16 relative to Sup35NM prolonged the initial lag-time of fibril formation and decreased the rate of fibril extension. Addition of 1 or 3 molar-equivalents of T. acidophilum-cpn monomers also produced a similar effect. Delayed addition of these chaperonins after the initial lag phase did not suppress fibril formation. Interestingly, these effects were also observed upon adding only the apical domain segments of α and β-subunits, and we also found that deletion of the helical protrusion in the apical domain of these segments led to an abolishment of the suppression effects. A synthetic peptide whose sequence corresponded to the helical protrusion also displayed a suppression effect, which indicated that archaeal group II chaperonin binds to Sup35NM through the helical protrusion of the apical domain. These findings suggest that group II chaperonin might be actively involved in suppressing amyloid fibril formation, in addition to acting as a protein folding assistant.

Heat shock proteins: Molecules with assorted functions

Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in allstudied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several folds.Hsps undergo ATP-driven conformational changes to stabilize unfolded proteins or unfold them for translocationacross membranes or mark them for degradation. They are broadly classified in several families according to theirmolecular weights and functional properties. Extensive studies during the past few decades suggest that Hsps play avital role in both normal cellular homeostasis and stress response. Hsps have been reported to interact with numeroussubstrates and are involved in many biological functions such as cellular communication, immune response, proteintransport, apoptosis, cell cycle regulation, gametogenesis and aging. The present review attempts to provide a briefoverview of various Hsps and summarizes their involvement in diverse biological activities.

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.

Protomer Roles in Chloroplast Chaperonin Assembly and Function

The individual roles of three chloroplast CPN60 protomers （CPN60α, CPN60β1, and CPN60β2） and whether and how they are assembled into functional chaperonin complexes are investigated in Chlamydomonas reinhardtii. Protein complexes containing all three potential subunits were identified in Chlamydomonas, and their co-expression in Escherichia coil yielded a homogeneous population of oligomers containing all three subunits （CPN60α1β11β2）, with a molecular weight consistent with a tetradecameric structure. While homo-oligomers of CPN60β could form, they were dramatically reduced when CPN60α was present and homo-oligomers of CPN60β2 were readily changed into hetero-oligomers in the presence of ATP and other protomers. ATP hydrolysis caused CPN60 oligomers to disassemble and drove the purified protomers to reconstitute oligomers in vitro, suggesting that the dynamic nature of CPN60 oligomers is dependent on ATP. Only hetero-oligomeric CPN60α1β1β2, containing CPN60α, CPN60β1, and CPN60β2 subunits in a 5：6：3 ratio, cooperated functionally with GroES. The combination of CPN60α and CPN60β subunits, but not the individual subunits alone, complemented GroEL function in E. coil with subunit recognition specificity. Down-regulation of the CPN60α subunit in Chlamydomonas resulted in a slow growth defect and an inability to grow autotrophically, indicating the essential role of CPN60α in vivo.

Structural variation of molecular chaperone group Ⅱ by crystallography and cryo-electron microscopy

Chaperonins, a class of molecular chaperones, are oligomeric complexes acting as a protein-folding chamber in an ATP-dependent manner. Chaperonins have been classifed

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.

Explicit solvent molecular dynamics simulations of chaperonin-assisted rhodanese folding

Chaperonins are known to facilitate the productive folding of numerous misfolded proteins. Despite their established importance, the mechanism of chaperonin-assisted protein folding remains unknown. In the present article, all-atom explicit solvent molecular dynamics （MD） simulations have been performed for the first time on rhodanese folding in a series of cavity-size and cavity-charge chaperonin mutants. A compromise between stability and flexibility of chaperonin structure during the substrate folding has been observed and the key factors affecting this dynamic process are discussed.

Flexible interwoven termini determine the thermal stability of thermosomes

Group II chaperonins,which assemble as double-ring complexes,assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner.The mo-lecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated.Here,we selected the group II chaperonins(cpn-αand cpn-β),also called thermosomes,from Acidianus tengchongensis and in-vestigated their assembly and thermal stability.We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities.Cpn-βis more thermally stable than cpn-α,while the thermal stability of the hetero thermo-some cpn-αβis intermediate.Cryo-electron microscopy reconstructions of cpn-αand cpn-βrevealed the interwo-ven densities of their non-conserved fl exible N/C-termini around the equatorial planes.The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interac-tions in the assembly and thermal stability of the ther-mosomes.