High expression of chaperonin-containing TCP1 subunit 3 may induce dismal prognosis in multiple myeloma.

The prognosis role of CCT3 in MM and the possible pathways it involved were studied in our research. By analyzing ten independent datasets (including 48 healthy donors, 2220 MM, 73 MGUS, and 6 PCL), CCT3 was found to express higher in MM than healthy donors, and the expression level was gradually increased from MGUS, SMM, MM to PCL (all P < 0.01). By analyzing three independent datasets (GSE24080, GSE2658, and GSE4204), we found that CCT3 was a significant indicator of poor prognosis (all P < 0.01). KEGG and GSEA analysis showed that CCT3 expression was associated with JAK-STAT3 pathway, Hippo signaling pathway, and WNT signaling pathway. In addition, different expressed genes analysis revealed MYC, which was one of the downstream genes regulated by JAK-STAT3 pathway, was upregulated in MM. This confirms that JAK-STAT3 signaling pathway may promote the progress of disease which was regulated by CCT3 expression. Our study revealed that CCT3 may play a supporting role at the diagnosis of myeloid, and high expression of CCT3 suggested poor prognosis in MM. CCT3 expression may promote the progression of MM mainly by regulating MYC through JAK-STAT3 signaling pathway.

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

BACKGROUND: Joint 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. METHODS: In 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. RESULTS: Results 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). CONCLUSION: The 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.

Characterization of CCTα and evaluating its expression in the mud crab Scylla paramamosain when challenged by low temperatures alone and in combination with high and low salinity.

Chaperonin containing the T-complex polypeptide-1 (CCT), which is known to be involved in intracellular assembly and folding of proteins, is a class of chaperonin omnipresent in all forms of life. Previous studies showed that CCT played a vital role in cold hardiness of various animals. In order to understand the response of the polypeptide complex to low temperature challenge and other environmental stresses, a subunit of CCT (CCTα) was cloned from the mud crab Scylla paramamosain by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA SpCCTα was of 1972 bp and contained a 1668 bp open reading frame (ORF) encoding a polypeptide of 555 amino acids with four conserved motifs. The messenger ribonucleic acid (mRNA) levels of SpCCTα in ten tissues of adult S. paramamosain was subsequently examined and the highest expression was found in muscle, followed by gill, hepatopancreas, thoracic ganglion, hemocyte, heart, cerebral ganglion, stomach, eyestalk ganglion, and epidermis. The expressions of SpCCTα in the muscle of sub-adult crabs (pre-acclimated to 28 °C) subjected to the challenges of both lower temperatures (25, 20, 15, and 10 °C) alone and low temperatures (15 and 10 °C) in combination with salinity of 35 and 10 were further investigated by fluorescent quantitative real-time PCR (qPCR). It was revealed that when exposed to lower temperatures alone, the mRNA transcripts of the SpCCTα gene in the muscle were generally induced for significant higher expression at 10 °C treatment than the 25, 20, and 15 °C treatments; meanwhile, exposure to 15 °C also frequently led to significantly higher expression than those at 20 and 25 °C. This finding indicated that the up-regulation of SpCCTα was closely related to the cold hardiness of S. paramamosain. The results of an additional experiment challenging the sub-adult crabs with various combinations of low temperatures with different salinity conditions generally demonstrated that at both 10 and 15 °C, the expression of SpCCTα under the high salinity of 35 was significantly lower than that at low salinity of 10, implying that the damages caused by low temperatures with high salinity were less than that under low salinity.

Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta.

Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.

Essential role of the chaperonin CCT in rod outer segment biogenesis.

PURPOSE: While some evidence suggests an essential role for the chaperonin containing t-complex protein 1 (CCT) in ciliogenesis, this function remains poorly understood mechanistically. We used transgenic mice, previously generated in our lab, and characterized by a genetically-induced suppression of CCT in rod photoreceptors as well as a malformation of the rod sensory cilia, the outer segments, to gain new insights into this underlying molecular mechanism. METHODS: The CCT activity in rod photoreceptors of mice was suppressed by overexpressing the chaperonin inhibitor, phosducin-like protein short, and the ensuing changes of cellular morphology were analyzed by light and electron microscopy. Protein expression levels were studied by fluorescent microscopy and Western blotting. RESULTS: Suppressing the chaperonin made the photoreceptors incompetent to build their outer segments. Specifically, the CCT-deficient rods appeared unable to expand the outer segment plasma membrane, and accommodate growth of this compartment. Seeking the molecular mechanisms underlying such a shortcoming, we found that the affected rods could not express normal levels of Bardet-Biedl Syndrome (BBS) proteins 2, 5, and 7 and, owing to that deficiency, were unable to assemble the BBSome, a multisubunit complex responsible for ciliary trafficking. A similar effect in response to the chaperonin suppression was also observed in cultured ciliated cells. CONCLUSIONS: Our data provide new evidence indicating the essential role of the chaperonin CCT in the biogenesis of vertebrate photoreceptor sensory cilia, and suggest that it may be due to the direct participation of the chaperonin in the posttranslational processing of selected BBS proteins and assembly of the BBSome.

Human CCT4 and CCT5 chaperonin subunits expressed in Escherichia coli form biologically active homo-oligomers.

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.

Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is a specific regulator of fibroblast motility and contractility.

Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult) wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta) is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF) stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA) reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (alpha-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less alpha-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased alpha-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of alpha-SMA with siRNA reduced both basal and growth factor-induced fibroblast motility. These results indicate that CCT-eta is a specific regulator of fibroblast motility and contractility and may be a key determinant of the scarless wound healing phenotype by means of its specific regulation of alpha-SMA expression.