Chaperonin-containing T-complex Polypeptide Research Articles

Complementary DNA Cloning and Tissue Expression Profile of Chaperonin-Containing T-Complex Polypeptide 1-Beta in Pinctada maxima Exposed to Cold Stress

Complementary DNA Cloning and Tissue Expression Profile of Chaperonin-Containing T-Complex Polypeptide 1-Beta in Pinctada maxima Exposed to Cold Stress

Chaperonin CCT5 binding with porcine parvovirus NS1 promotes the interaction of NS1 and COPƐ to facilitate viral replication

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in first pregnant sows. The non-structure protein NS1 of PPV is a multifunctional protein playing a key role in viral replication. Chaperonin-containing T-complex polypeptide complex (CCT), containing CCT1-CCT8 subunits, belongs to the type II chaperones that interact with proteins to help in folding and maintaining. In this study, CCT5, for the first time, was found to be one of the host interacting proteins of PPV NS1, and CCT5 was directly bound with NS1. Interference of CCT5 expression by specific siRNA and knockout of CCT5 expression by CRISPR/Cas9 suppressed PPV replication, while overexpression of CCT5 promoted PPV replication in PK-15 cells. The interaction of CCT5 and PPV NS1 was dependent on the 36–42 aa motif at the N-terminal end of NS1. More importantly, CCT5 was also found interacting with COPƐ, which has previously been demonstrated to promote PPV replication by regulating type I interferon. Interference and knockout of CCT5 expression significantly reduced the interaction of PPV NS1 and host protein COPƐ, and promoted the IFN-β expression. These results show that CCT5 mediates the interaction of PPV NS1 and COPƐ to regulate viral replication, providing new insight into the mechanism of PPV replication.

Chaperonin Mechanisms: Multiple and (Mis)Understood?

The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact.

Cdna Cloning and Expression Analysis of Chaperonin Containing T-Complex Polypeptide 1-Alpha in the Pearl Oyster Pinctada maxima Exposed to Cold Stress

The complete cDNA sequence of chaperonin-containing T-complex polypeptide 1-alpha (CCT-alpha) gene of the pearl oyster Pinctada maxima was determined using rapid amplification of cDNA ends technique and characterized with bioinformatic analysis. Its expression profile in different organs and under cold stress was characterized using real-time quantitative polymerase chain reaction. Results revealed that the complete cDNA sequence of P. maxima CCT-alpha has 1,803 base pairs (bp), including a 1,608-bp open reading framework that codes 535 amino acids. The protein has three conservative functional domains: equatorial, apical, and intermediate domains. The equatorial domain is 20 amino acids shorter than that of other species, and contains the three signature sequences of CCT family protein. The CCT-alpha of P. maxima has nine conservative ATP-binding sites and six protein-binding sites, and has high homology with Aplysia californica (80.7%), Delia antiqua (77.4%), Danio rerio (77.1%), Xenopus laevis (76.7%), Gallus gallus (76.4%), and Homo sapiens (75.4%). The CCT-alpha shows tissue-specific expression profile in P. maxima, with expression levels higher in gill, mantle, and adductor muscle than in hepatopancreas and heart. Cold stress markedly increased mRNA contents of CCT-alpha in the organs of P. maxima, but different organs showed distinct cold stress response pattern. Chaperonin-containing T-complex polypeptide 1-alpha transcription in adductor muscle, hepatopancreas, and heart increased at 2 h, and reached peak values at 6 h, maintained high levels at 12 h, and significantly decreased at 24 h. The product in gill and mantle decreased at 2 h, began to increase at 6 h, and showed a large increase at 12 h, but reduced to below the initial level at 24 h.

Resistance of Acta2R149C/+ mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex

Pathogenic variants of the gene for smooth muscle α-actin (ACTA2), which encodes smooth muscle (SM) α-actin, predispose to heritable thoracic aortic disease. The ACTA2 variant p.Arg149Cys (R149C) is the most common alteration; however, only 60% of carriers have a dissection or undergo repair of an aneurysm by 70 years of age. A mouse model of ACTA2 p.Arg149Cys was generated using CRISPR/Cas9 technology to determine the etiology of reduced penetrance. Acta2R149C/+ mice had significantly decreased aortic contraction compared with WT mice but did not form aortic aneurysms or dissections when followed to 24 months, even when hypertension was induced. In vitro motility assays found decreased interaction of mutant SM α-actin filaments with SM myosin. Polymerization studies using total internal reflection fluorescence microscopy showed enhanced nucleation of mutant SM α-actin by formin, which correlated with disorganized and reduced SM α-actin filaments in Acta2R149C/+ smooth muscle cells (SMCs). However, the most prominent molecular defect was the increased retention of mutant SM α-actin in the chaperonin-containing t-complex polypeptide folding complex, which was associated with reduced levels of mutant compared with WT SM α-actin in Acta2R149C/+ SMCs. These data indicate that Acta2R149C/+ mice do not develop thoracic aortic disease despite decreased contraction of aortic segments and disrupted SM α-actin filament formation and function in Acta2R149C/+ SMCs. Enhanced binding of mutant SM α-actin to chaperonin-containing t-complex polypeptide decreases the mutant actin versus WT monomer levels in Acta2R149C/+ SMCs, thus minimizing the effect of the mutation on SMC function and potentially preventing aortic disease in the Acta2R149C/+ mice.

CCTδ colocalizes with actin and β-tubulin: Insight into its involvement in the cytoskeleton formation of the intracellular parasite Nosema bombycis

The chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone protein that is widely present in eukaryotic cytoplasm and can assist in the folding of newly synthesized proteins. The CCT complex consists of eight completely different subunits, among which the δ subunit plays an extremely important role in the folding and assembly of cytoskeleton proteins as an individual or complex with other subunits. In this study, we identified the CCTδ in the microsporidian Nosema bombycis (NbCCTδ) for the first time. The NbCCTδ gene contains a complete ORF of 1497 bp in length that encodes a 498 amino acid polypeptide. NbCCTδ is expressed throughout the entire lifecycle of N. bombycis and rather higher in early stage of proliferation. Indirect immunofluorescence results showed that NbCCTδ was colocalized with actin and β-tubulin during the proliferative and sporogonic phases of N. bombycis. RNA interference down-regulated the expression of the NbCCTδ gene. These results imply that NbCCTδ may participate in cytoskeleton formation and proliferation of N. bombycis.

Human ribosomal protein and proteasomal subunit suppress cct mutations and reduce alpha-synuclein toxicity in Saccharomyces cerevisiae

CCT (Chaperonin containing T-complex polypeptide), the chaperonin complex in S. cerevisiae, bears striking similarities to human CCT and serves as a model system to study properties of the human chaperonin complex. In this study, we employ temperature-sensitive (ts) mutants of CCT complex in S. cerevisiae to screen a human glioma cDNA library for identifying human proteins, which on overexpression, can rescue mutations in the CCT complex. Among other proteins, we identify a human ribosomal protein, RPS6p and a proteasomal subunit, PSMA6p as suppressors of cct mutations. As compromised protein folding may result in protein aggregation in the cell, we also studied the effect of the suppressors on protein aggregation. Both the genes reduced toxicity due to alpha-synuclein aggregation in a cog6Δ mutant. RPS6 was more potent in suppressing the ts mutations and in reducing alpha-synuclein toxicity. Yeast homologs of RPS6 and PSMA6 showed similar functions, thus confirming cross-species conservation of the identified functions.

Overexpression of chaperonin containing T-complex polypeptide subunit zeta 2 (CCT6b) suppresses the functions of active fibroblasts in a rat model of joint contracture

BackgroundJoint contracture is a fibrous disease characterized as joint capsule fibrosis that results in joint dysfunction and disability. The purpose of this study was to analyze the biological activities of chaperonin containing T-complex polypeptide (CCT) subunits and to determine the role of CCT chaperone in joint contracture in a rat model.MethodsIn this study, the rat model of joint contracture was established by immobilizing the rat knee for 8 weeks. Then, fibroblasts were isolated from the posterior joint capsule and were cultured for functional analysis such as qRT-PCR, Western blot, transwell assay, and collagen assay. The effect of CCT subunit was determined by employing a lentivirus containing target gene and transfecting it into fibroblasts.ResultsResults of qRT-PCR and Western blot showed that among all CCT subunits, CCT6b significantly decreased in the fibroblasts from contractive joints compared to cells from normal joints (p < 0.05). Overexpression of CCT6b by transfection of lentivirus containing CCT6b gene to active fibroblasts significantly inhibited fibrous marker (α-SMA, COL-1) expressions, fibroblast migration, and collagen synthesis (all p < 0.05). Moreover, fibrosis-related chaperone CCT7 expression was decreased with CCT6b overexpression (p < 0.05).ConclusionThe biological activities of CCT subunits in fibroblasts from the joint contracture rat model were analyzed in this study. CCT6b significantly decreased in the active fibroblasts, and overexpression of CCT6b significantly inhibited fibroblast functions. These findings indicate that CCT6b appears to be a potential molecular biomarker and therapeutic target for the novel therapies of joint contracture.

Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC.

The eukaryotic group II chaperonin, the chaperonin-containing t-complex polypeptide 1 (CCT), plays an important role in cytosolic proteostasis. It has been estimated that as much as 10% of cytosolic proteins interact with CCT during their folding process. CCT is composed of 8 different paralogous subunits. Due to its complicated structure, molecular and biochemical investigations of CCT have been difficult. In this study, we constructed an expression system for CCT from a thermophilic fungus, Chaetomium thermophilum (CtCCT), by using E. coli as a host. As expected, we obtained recombinant CtCCT with a relatively high yield, and it exhibited fairly high thermal stability. We showed the advantages of the overproduction system by characterizing CtCCT variants containing ATPase-deficient subunits. For diffracted X-ray tracking experiment, we removed all surface exposed cysteine residues, and added cysteine residues at the tip of helical protrusions of selected two subunits. Gold nanocrystals were attached onto CtCCTs via gold-thiol bonds and applied for the analysis by diffracted X-ray tracking. Irrespective of the locations of cysteines, it was shown that ATP binding induces tilting motion followed by rotational motion in the CtCCT molecule, like the archaeal group II chaperonins. When gold nanocrystals were attached onto two subunits in the high ATPase activity hemisphere, the CtCCT complex exhibited a fairly rapid response to the motion. In contrast, the response of CtCCT, which had gold nanocrystals attached to the low-activity hemisphere, was slow. These results clearly support the possibility that ATP-dependent conformational change starts with the high-affinity hemisphere and progresses to the low-affinity hemisphere.

Transient Kinetic Analysis of ATP Hydrolysis by the CCT/TRiC Chaperonin

The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis. The phases were assigned by (i) determining their dependence on ATP and K+ concentrations and (ii) by measuring their sensitivity to the mutation Gly345→Asp in subunit CCT4, which decreases cooperativity in ATP binding. The values of the observed rate constants corresponding to the burst phases are found to decrease with increasing ATP and K+ concentrations, thereby indicating that the apo state of CCT/TRiC is in equilibrium between several conformations and that “conformational selection” by ATP takes place before hydrolysis. The amplitude of the lag phase, which follows, decreases with increasing ATP concentrations, thus indicating that it reflects a transition between states with low affinity for ATP and a state with high affinity for ATP that is predominant under steady-state conditions. A kinetic model based on the data is suggested, in which CCT/TRiC is in equilibrium between a relatively large number of states that are distinguished kinetically, in agreement with its proposed sequential allosteric mechanism.

Chaperonin-containing t-complex protein-1 subunit β as a possible biomarker for the phase of glomerular hyperfiltration of diabetic nephropathy.

In cell model, we discovered the association between chaperonin-containing t-complex polypeptide 1 subunit β (TCP-1β) and early diabetic nephropathy (DN). In this study, we further explored the relationships between TCP-1β and type 2 diabetic mellitus (DM). To mimic the clinical hyperfiltration state, a type 2 DM mice model was established by feeding a high-fat diet in combination with treatment of streptozotocin and nicotinamide. Blood and urine were collected to determine creatinine clearance (C cr), and kidney tissues were harvested for evaluation of TCP-1β expression by immunohistochemistry and Western blot. Meanwhile, clinical subjects of healthy controls and type 2 DM were recruited to strengthen the evidence with urine TCP-1β. Results showed that C cr and the expression of TCP-1β in kidney were significantly higher one week after hyperglycemia development, suggesting that the hyperfiltration state was successfully established in the mice model. TCP-1β was expressed predominantly on renal tubules. By using the estimated glomerular filtration rate to index progression in clinical investigation, urine TCP-1β level was associated with the hyperfiltration phase in type 2 DM patients. Conclusively, we confirmed that TCP-1β is a possible biomarker for early nephropathy of type 2 DM, but further mechanistic study to elucidate its cause and pathway is needed.

Monoclonal Antibody against α-Actinin 4 from Human Umbilical Vein Endothelial Cells Inhibits Endothelium-Dependent Vasorelaxation

Background: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. Methods: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. Results: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. Conclusions: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Molecular Characterization and Phylogenetic Analysis ofBabesiasp. NV-1 Detected from Wild American Mink (Neovison vison) in Hokkaido, Japan

Babesiosis is a tick-borne protozoan disease affecting many mammalian species worldwide, caused by the intraerythrocytic multiplication of Babesia spp. The present study aimed to detect the presence of Babesia sp. in 13 American mink from Hokkaido, Japan. One of 13 animals was positive, as indicated by nested PCR targeting the 18S ribosomal RNA (SSU rDNA) and subunit 7 (eta) of the chaperonin-containing t-complex polypeptide 1 (CCT7) genes from species of Babesia and Theileria. Sequencing of the PCR product of SSU rDNA revealed 99% homology to the isolates of Babesia sp. SAP#131 found in raccoons in Hokkaido, whereas that of the CCT7 gene showed 80% homology to the isolates of Babesia gibsoni in dogs as determined by BLAST analysis. We refer to the cognate sequence as Babesia sp. NV-1. Phylogenetic analyses of SSU rDNA and CCT7 genes from Babesia sp. NV-1 revealed them to be most closely related to the Babesia sp. SAP#131 from a raccoon in Hokkaido and to canine B. gibsoni, respectively. Here, we provide the first molecular evidence of the Babesia sp. NV-1 parasite in feral American mink ( Neovison vison ) in Hokkaido, Japan.

Anti-Retinal Antibodies in Serum of Laser-Treated Rabbits

Retinal injuries that affect the photoreceptors and/or the retinal pigment epithelium (RPE) may result in the leakage of retinal proteins into the systemic circulation. This study was designed to determine whether an immune response is elicited after an acute retinal injury resulting in circulating anti-retinal antibodies in the serum. Fifty laser burns of different grades (minimally visible lesion [MVL], grade II [GII], or grade III [GIII] lesions) were created in the retinas of Dutch Belted rabbits. The degree of laser burns was confirmed by fundus imaging and histology. Serum samples were collected from the animals 3 months after the retinal injury. Candidate autoantigens were identified by two-dimensional (2-D) Western blots of rabbit retinal lysate probed with sera from either control or laser-treated animals. Candidate autoantigens were further characterized by immunostaining to confirm their retinal localization. Seven and 11 protein spots were selected from the MVL and GII laser-treated samples, respectively, for autoantigen identification. No protein spots were detected in the GIII laser-treated samples. Four candidate autoantigens were common to both MVL and GII lesions: dihydropyrimidinase-related protein 2, fructose-bisphosphate aldolase C, chaperonin-containing T-complex polypeptide 1 subunit zeta, and pyruvate kinase isozyme. Laser-induced retinal injuries resulted in circulating anti-retinal antibodies that were detectable 3 months after the injury. The response appeared to vary with the severity of the laser retinal damage. The identification of the candidate antigens in this study suggest that this approach may permit future development of new diagnostic methods for retinal injuries.

Experimental Models of Type-2 Diabetic Nephropathy

Experimental Models of Type-2 Diabetic Nephropathy

Identification and Phylogenetic Analysis of Japanese Macaque Babesia-1 (JM-1) detected from a Japanese Macaque (Macaca fuscata fuscata)

We demonstrate here the identification and phylogenetic characterization of Babesia microti (B. microti)-like parasite detected from a splenectomized Japanese macaque (Macaca fuscata fuscata) at a facility for laboratory animal science. On Day 133 after splenectomy, intra-erythrocytic parasites were found on light microscopic examination, and the level of parasitemia reached 0.3% on blood smear. Molecular characterization of the parasite using nested-polymerization chain reactions targeting the 18S rRNA, β-tubulin, and subunit 7 (eta) of the chaperonin-containing t-complex polypeptide 1 (CCT7) genes were identified as a B. microti-like parasite, designated the Japanese Macaque Babesia-1 (JM-1).

Chaperonin containing T-complex polypeptide (CCT) subunit expression in oral mucosal wounds and fibroblasts

Mucosal wound healing in adults has been reported to feature diminished scar formation compared to healing skin wounds. We sought to determine if the expression pattern of chaperonin containing T-complex polypeptide (CCT) subunits in mucosal wounds and fibroblasts is different from that observed in skin wounds and fibroblasts. We found that CCT-beta is the only subunit message to be reduced in wounded mucosa versus unwounded control, and this reduction was confirmed at the protein level. In contrast, mRNA levels of CCT-zeta, -delta, -eta, and -epsilon were significantly increased in mucosal wounds. The increase in CCT-eta was also confirmed at the protein level. Expression levels of CCT-alpha, -beta, -delta; -epsilon, and -theta mRNAs were significantly increased in adult mucosal fibroblasts in culture compared to skin-derived fibroblasts. Western blot analyses confirmed a modest increase in CCT-beta in adult mucosal fibroblasts relative to skin fibroblasts, but CCT-eta protein was unaffected. These differences may contribute to the reported difference in healing outcomes between these two tissue types.

Involvement of F-Actin in Chaperonin-Containing t-Complex 1 Beta Regulating Mouse Mesangial Cell Functions in a Glucose-Induction Cell Model

The aim of this study is to investigate the role of chaperonin-containing t-complex polypeptide 1 beta (CCT2) in the regulation of mouse mesangial cell (mMC) contraction, proliferation, and migration with filamentous/globular-(F/G-) actin ratio under high glucose induction. A low CCT2 mMC model induced by treatment of small interference RNA was established. Groups with and without low CCT2 induction examined in normal and high (H) glucose conditions revealed the following major results: (1) low CCT2 or H glucose showed the ability to attenuate F/G-actin ratio; (2) groups with low F/G-actin ratio all showed less cell contraction; (3) suppression of CCT2 may reduce the proliferation and migration which were originally induced by H glucose. In conclusion, CCT2 can be used as a specific regulator for mMC contraction, proliferation, and migration affected by glucose, which mechanism may involve the alteration of F-actin, particularly for cell contraction.

Babesia microti-Group Parasites Compared Phylogenetically by Complete Sequencing of the CCT.ETA. Gene in 36 Isolates

Babesia microti, the erythroparasitic cause of human babesiosis, has long been taken to be a single species because classification by parasite morphology and host spectrum blurred distinctions between the parasites. Phylogenetic analyses of the 18S ribosomal RNA gene (18S rDNA) and, more recently, the beta-tubulin gene have suggested inter-group heterogeneity. Intra-group relationships, however, remain unknown. This study was conducted to clarify the intra- and inter-group phylogenetic features of the B. microti-group parasites with the eta subunit of the chaperonin-containing t-complex polypeptide l (CCTeta) gene as a candidate genetic marker for defining the B. microti group. We prepared complete sequences of the CCTeta gene from 36 piroplasms and compared the phylogenetic trees. The B. microti-group parasites clustered in a monophyletic assemblage separate from the Babesia sensu stricto and Theileria genera and subdivided predominantly into 4 clades (U.S., Kobe, Hobetsu, Munich) with highly significant evolutionary distances between the clades. B. rodhaini branched at the base of the B. microti-group parasites. In addition, a unique intron presence/absence matrix not observable in 18S rDNA or beta-tubulin set the B. microti group entirely apart from either Babesia sensu stricto or Theileria. These results have strong implications for public health, suggesting that the B. microti-group parasites are a full-fledged genus comprising, for now, four core species, i.e., U.S., Kobe, Hobetsu, and Munich species nova. Furthermore, the CCTeta gene is an instructive and definitive genetic marker for analyzing B. microti and related parasites.

Differential expression of chaperonin containing T-complex polypeptide (CCT) subunits during fetal and adult skin wound healing

Integumentary wound healing in early fetal life is regenerative and proceeds without scar formation. Expressomic analysis of this phenomenon by differential display has previously determined that the eta subunit of the cytosolic chaperonin containing T-complex polypeptide (CCT) is downregulated in the healing fetal wound milieu. We now report that no other CCT subunit shares this distinct pattern of gene regulation as determined by limiting dilution reverse transcriptase polymerase chain reaction (RT-PCR); all seven of the remaining CCT subunits demonstrate no change in messenger RNA (mRNA) expression in healing fetal wounds compared to unwounded control tissue. The alpha subunit, however, did evidence reduced message levels in healing adult wound tissue. We herein report on the cloning and sequence of the complementary DNA (cDNA) for rabbit CCT-alpha and confirm its wound specific decrease in adult tissues through quantitative real-time RT-PCR assay. We also confirm that quantitative evaluation of CCT-alpha and CCT-zeta mRNA expression shows no change in healing fetal wounds.