Chaperonin-containing TCP-1 Research Articles

Investigating Chaperonin-Containing TCP-1 subunit 2 as an essential component of the chaperonin complex for tumorigenesis

Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few definitive studies addressed the role of specific subunits in promoting the chaperonin’s function in cancer. To this end, we investigated the activity of CCT2 (CCTβ) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers.

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.

The TRiC/CCT Chaperonin and Its Role in Uncontrolled Proliferation.

The cell cycle is a sophisticated space-time regulated mechanism where a wide variety of protein modules and complexes associate functioning in a concerted manner to regulate and transfer the genetic material to daughter cells. CCT (chaperonin containing TCP-1, also known as TRiC) is a molecular machine that forms a high molecular weight complex (1000KDa). CCT is emerging as a key molecule during mitosis due to its essential role in the folding of many important proteins involved in cell division (Cdh1, Plk1, p27, Cdc20, PP2a regulatory subunits, tubulin or actin) suggesting its involvement in uncontrolled proliferation. The assembly is formed by eight different subunits called CCTα, β, γ, δ, ε, ζ, η and θ in mammals corresponding to CCT1-8 in yeast. CCT/TRiC is organized in a unique intra- and inter-ring arrangement. The chaperonin monomers share a common domain structure including an equatorial domain, which contains all the inter-ring contacts, most of the intra-ring contacts and the ATP binding site, whose binding and hydrolysis triggers the conformational changes that take place during the functional cycle. All chaperonins display an open substrate-receptive conformation, where the unfolded protein is recognized and trapped, and a closed conformation where the substrate is isolated from the bulk of the intracellular environment. In this chapter we discuss the complex set of intra- and inter-ring allosteric signals during chaperonin function.

Myelin Pathology: Involvement of Molecular Chaperones and the Promise of Chaperonotherapy.

The process of axon myelination involves various proteins including molecular chaperones. Myelin alteration is a common feature in neurological diseases due to structural and functional abnormalities of one or more myelin proteins. Genetic proteinopathies may occur either in the presence of a normal chaperoning system, which is unable to assist the defective myelin protein in its folding and migration, or due to mutations in chaperone genes, leading to functional defects in assisting myelin maturation/migration. The latter are a subgroup of genetic chaperonopathies causing demyelination. In this brief review, we describe some paradigmatic examples pertaining to the chaperonins Hsp60 (HSPD1, or HSP60, or Cpn60) and CCT (chaperonin-containing TCP-1). Our aim is to make scientists and physicians aware of the possibility and advantages of classifying patients depending on the presence or absence of a chaperonopathy. In turn, this subclassification will allow the development of novel therapeutic strategies (chaperonotherapy) by using molecular chaperones as agents or targets for treatment.

Bioinformatics analysis of the prognostic value of CCT6A and associated signalling pathways in breast cancer.

Breast cancer (BC) is the most frequently diagnosed type of cancer and the leading cause of cancer-associated mortality among women worldwide. However, the molecular basis for the pathogenesis of BC requires further exploration. Recent studies have demonstrated that chaperonin-containing TCP1 subunit 6A (CCT6A) efficiently suppresses transforming growth factor-β-mediated metastasis by inhibiting the function of SMAD family member 2 in lung cancer. However, the functional significance of CCT6A in other types of cancer, including BC, remains to be investigated. Therefore, this study evaluated CCT6A expression in BC samples, and further analysed its association with survival, clinicopathological parameters and related signalling pathways using online datasets. The present study indicated that CCT6A expression was significantly higher in BC tissues compared with in surrounding noncancerous tissues at both mRNA and protein levels. Furthermore, increased CCT6A expression was significantly associated with poor survival, including overall survival, relapse-free survival, distant metastasis-free survival and post progression survival, in patients with BC. Pathway finder analysis indicated that CCT6A was significantly associated with the cell cycle, and its expression was significantly positively correlated with cyclin (CCN)B2 and CCNA2 expression. Taken together, to the best of our knowledge, the present study is the first to indicate that CCT6A may serve a significant role in BC tumour progression.

TRiC/CCT chaperonins are essential for organ growth by interacting with insulin/TOR signaling in Drosophila

Organ size is regulated by intercellular signaling for cell growth and proliferation. The TOR pathway mediates a key signaling mechanism for controlling cell size and number in organ growth. Chaperonin containing TCP-1 (CCT) is a complex that assists protein folding and function, but its role in animal development is largely unknown. Here we show that the CCT complex is required for organ growth by interacting with the TOR pathway in Drosophila. Reduction of CCT4 results in growth defects by affecting both cell size and proliferation. Loss of CCT4 causes preferential cell death anterior to the morphogenetic furrow in the eye disc and within wing pouch in the wing disc. Depletion of any CCT subunit in the eye disc results in headless phenotype. Overgrowth by active TOR signaling is suppressed by CCT RNAi. The CCT complex physically interacts with TOR signaling components including TOR, Rheb, and S6K. Loss of CCT leads to decreased phosphorylation of S6K and S6 while increasing phosphorylation of Akt. Insulin/TOR signaling is also necessary and sufficient for promoting CCT complex transcription. Our data provide evidence that the CCT complex regulates organ growth by directly interacting with the TOR signaling pathway.

HNRNP Q suppresses polyglutamine huntingtin aggregation by post‐transcriptional regulation of vaccinia‐related kinase 2

Misfolded proteins with abnormal polyglutamine (polyQ) expansion cause neurodegenerative disorders, including Huntington's disease. Recently, it was found that polyQ aggregates accumulate as a result of vaccinia-related kinase 2 (VRK2)-mediated degradation of TCP-1 ring complex (TRiC)/chaperonin-containing TCP-1 (CCT), which has an essential role in the prevention of polyQ protein aggregation and cytotoxicity. The levels of VRK2 are known to be much higher in actively proliferating cells but are maintained at a low level in the brain via an unknown mechanism. Here, we found that basal levels of neuronal cell-specific VRK2 mRNA are maintained by post-transcriptional, rather than transcriptional, regulation. Moreover, heterogeneous nuclear ribonucleoprotein Q (HNRNP Q) specifically binds to the 3'untranslated region of VRK2 mRNA in neuronal cells to reduce the mRNA stability. As a result, we found a dramatic decrease in CCT4 protein levels in response to a reduction in HNRNP Q levels, which was followed by an increase in polyQ aggregation in human neuroblastoma cells and mouse cortical neurons. Taken together, these results provide new insights into how neuronal HNRNP Q decreases VRK2 mRNA stability and contributes to the prevention of Huntington's disease, while also identifying new prognostic markers of HD.

The structure and evolution of eukaryotic chaperonin-containing TCP-1 and its mechanism that folds actin into a protein spring.

Actin is folded to its native state in eukaryotic cytosol by the sequential allosteric mechanism of the chaperonin-containing TCP-1 (CCT). The CCT machine is a double-ring ATPase built from eight related subunits, CCT1-CCT8. Non-native actin interacts with specific subunits and is annealed slowly through sequential binding and hydrolysis of ATP around and across the ring system. CCT releases a folded but soft ATP-G-actin monomer which is trapped 80 kJ/mol uphill on the folding energy surface by its ATP-Mg2+/Ca2+ clasp. The energy landscape can be re-explored in the actin filament, F-actin, because ATP hydrolysis produces dehydrated and more compact ADP-actin monomers which, upon application of force and strain, are opened and closed like the elements of a spring. Actin-based myosin motor systems underpin a multitude of force generation processes in cells and muscles. We propose that the water surface of F-actin acts as a low-binding energy, directional waveguide which is recognized specifically by the myosin lever-arm domain before the system engages to form the tight-binding actomyosin complex. Such a water-mediated recognition process between actin and myosin would enable symmetry breaking through fast, low energy initial binding events. The origin of chaperonins and the subsequent emergence of the CCT-actin system in LECA (last eukaryotic common ancestor) point to the critical role of CCT in facilitating phagocytosis during early eukaryotic evolution and the transition from the bacterial world. The coupling of CCT-folding fluxes to the cell cycle, cell size control networks and cancer are discussed together with directions for further research.

Elucidation of Therapeutic Peptide Binding Partners from Isolated Mitochondria.

CT20p is a protein derived from the C-terminus of Bax. It has selective cytotoxicity for cancer cells, such as the sensitive triple-negative MDA-MB-231 breast adenocarcinoma cells, but not normal cells like the resistant MCF-10A epithelial breast cells. To understand the reason for the peptide’s selective toxicity, a "pull-down" experiment with biotinylated CT20p (biotin-CT20p) and whole-cell protein lysates from breast cancer and normal cells were performed. These studies revealed that CT20p binds to a cytosolic protein called chaperonin-containing TCP-1 (CCT), a molecular chaperone that folds actin and tubulin. However, this method could not detect possible rare interactions made by CT20p with mitochondrial proteins. To determine whether CT20p is associated with mitochondrial proteins as part of the mechanism by which it induces cell death, mitochondrial protein lysates from MDA-MB-231 and MCF-10A cells were isolated and a streptavidin-agarose pulldown procedure using biotin-CT20p was performed. Protein interactions were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The results of the experimental procedure showed that biotin-CT20p did not "pull down" any observable mitochondrial proteins from the sensitive MDA-MB-231 cells, indicating that the peptide may not interact with mitochondrial proteins in breast cancer cells. Rather, the interactions observed with biotin-CT20p were with mitochondrial proteins derived from resistant MCF-10A cells, indicating that these interactions were not driving the cancer-selective cell death process. The absence of CT20p-associated proteins from the mitochondrial lysates of MDA-MB-231 breast cancer cells supports the hypothesis that CT20p, unlike the parent protein, Bax, exerts its cytotoxic effects via a cytosolic protein.

The substrate specificity of eukaryotic cytosolic chaperonin CCT.

The cytosolic chaperonin CCT (chaperonin containing TCP-1) is an ATP-dependent double-ring protein machine mediating the folding of members of the eukaryotic cytoskeletal protein families. The actins and tubulins are obligate substrates of CCT because they are completely dependent on CCT activity to reach their native states. Genetic and proteomic analysis of the CCT interactome in the yeast Saccharomyces cerevisiae revealed a CCT network of approximately 300 genes and proteins involved in many fundamental biological processes. We classified network members into sets such as substrates, CCT cofactors and CCT-mediated assembly processes. Many members of the 7-bladed propeller family of proteins are commonly found tightly bound to CCT isolated from human and plant cells and yeasts. The anaphase promoting complex (APC/C) cofactor propellers, Cdh1p and Cdc20p, are also obligate substrates since they both require CCT for folding and functional activation. In vitro translation analysis in prokaryotic and eukaryotic cell extracts of a set of yeast propellers demonstrates their highly differential interactions with CCT and GroEL (another chaperonin). Individual propeller proteins have idiosyncratic interaction modes with CCT because they emerged independently with neo-functions many times throughout eukaryotic evolution. We present a toy model in which cytoskeletal protein biogenesis and folding flux through CCT couples cell growth and size control to time dependent cell cycle mechanisms.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Suppression of CCT3 inhibits malignant proliferation of human papillary thyroid carcinoma cell.

Papillary thyroid carcinoma (PTC) is the most frequently occurring subtype of thyroid cancer. A certain portion of PTCs can progress to recurrent metastatic cancer. Currently, there remains no effective molecular target therapy for PTCs. As a subunit of the chaperonin containing TCP1 (CCT) complex, CCT3 is involved in various biological processes. CCT3 has been reported to drive the proliferation of hepatocellular carcinoma cells. Nevertheless, it remains unknown whether CCT3 regulates the development of PTC. The present study examined CCT3 protein expression in 30 PTC samples from patients undergoing thyroidectomy. A significant increase was observed in CCT3 expression in the PTC samples compared with the matched adjacent normal thyroid tissues. Lentiviral-mediated small interfering RNAs were used to knock down CCT3 in K1 cells. It was observed that the expression of CCT3 was significantly suppressed in K1 cells infected with lentivirus containing a CCT3-targeting short hairpin RNA. Our results showed that CCT3 knockdown markedly decreased the proliferation and cell cycle progression of K1 cells. In addition, the knockdown of CCT3 induced apoptosis of K1 cell. Taken together, the findings of the present study indicated that CCT3 presents as a potential molecular marker of PTC and regulates the development of PTC in humans.

Mutation in the Zebrafish cct2 Gene Leads to Abnormalities of Cell Cycle and Cell Death in the Retina: A Model of CCT2-Related Leber Congenital Amaurosis.

PurposeThe compound heterozygous mutations in the β subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates.MethodsA cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays. Phenotypic rescue of mutant phenotype was investigated by injection of CCT2 RNA into zebrafish embryos.ResultsThe cct2 mutation (L394H-7del) led to the synthesis of a mutated cctβ protein with the L394H replacement and deletion of 7 amino acid residues (positions 395–401). The homozygous cct2-L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2-L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was affected, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCTβ rescued the small eye phenotype, reduced retinal cell death, and restored the levels of CCTβ protein and the major client protein Gβ1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle.ConclusionsThe retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.

Targeting chaperonin containing TCP1 (CCT) as a molecular therapeutic for small cell lung cancer.

Identifying new druggable targets is desired to meet the needs for effective cancer treatments. To this end, we previously reported the efficacy of a therapeutic peptide called CT20p that displays selective cytotoxicity through inhibition of a multi-subunit, protein-folding complex called Chaperonin-Containing TCP-1 (CCT). To investigate the role of CCT in cancer progression, we examined protein levels of CCT subunits in liver, prostate, and lung cancer using human tissue microarrays. We found that these cancers expressed higher levels of CCT2 as compared to normal tissues. Small cell lung cancer (SCLC) stood out as having statistically significant difference in CCT2. Higher levels of CCT2 in tumors from lung cancer patients were also associated with decreased survival. Using SCLC cell lines, we observed detectable amounts of CCT subunits and cells were susceptible to killing by CT20p. Treatment with CT20p, delivered to cells using polymeric nanoparticles, was cytotoxic to all SCLC cell lines, decreasing the levels of CCT client proteins like STAT3. In contrast, treatment with a STAT3 inhibitor was effective in one of the SCLC cell lines. While we found that CCT levels could vary in cell lines, normal tissues had low levels of CCT and minimal toxicity to liver or kidney function was observed in mice treated with CT20p. These results indicate that in SCLC, changes in CCT levels could be used as a biomarker for diagnosis and that targeting CCT for inhibition with CT20p is a promising treatment approach for those cancers such as SCLC that currently lack targeted therapeutics.

CT20p as a therapeutic for lung cancer with elevated chaperonin containing TCP1 (CCT) expression levels.

e23163 Background: We previously reported, in breast cancer, the effectiveness of a novel therapeutic peptide called CT20p that displays cancer-selective cytotoxicity. CT20p targets for inhibition a macromolecular complex called Chaperonin-Containing TCP-1 (CCT) that is responsible for folding actin and tubulin and other proteins, like STAT3, into their active forms. CCT is composed of 8 subunits (CCT1-8) that are highly conserved among species. In breast cancer cases, we found that the genes for CCT were frequently amplified (CCT2), upregulating the chaperonin’s activity, which correlated with decreased patient survival. Methods: To determine whether CCT is active in other cancers and thereby a broad therapeutic target, we examined the protein levels of CCT2 in colon, liver, prostate and lung cancer by immunohistochemistry (IHC), using human tissue microarrays (TMAs). Genomic data from The Cancer Genome Atlas (TCGA) was used to support IHC findings. Immunoblot probing of three CCT subunits (CCT2, CCT4, CCT5) was performed to determine their relative expression level in five small cell lung cancer (SCLC) cell lines compared to normal lung cells and these cells tested for susceptibility to killing by CT20p. Results: Results of IHC staining for CCT2 were interpreted on a scale of 1 to 4 (with 4 being the strongest staining). We found that with the exception of colon cancer, all cancers expressed high levels of CCT2 with advancing stage. We identified a significant correlation with cancer progression in both small cell lung carcinoma (SCLC) and squamous cell lung carcinoma (SqCLC). While individual levels of the three probed CCT subunits varied amongst the SCLC cell lines, overall CCT expression was higher in the SCLC cells when compared to normal lung cells and correlated with susceptibility to killing by CT20p. Using the TCGA database, CCT gene alterations were detected in clinical lung cancer cases, with amplification of CCT4 gene in SqCLC cases linked to decreased survival. Conclusions: These results indicate that targeted inhibition of CCT through CT20p treatment is a promising treatment approach for those cancers like SCLC that currently lack effective therapeutics to sustain progression-free survival.

A novel therapeutic to enhance the immunogenicity of breast cancer cells.

119 Background: Breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. A possible solution is to generate dying breast cancer cells that operate like a vaccine to stimulate tumor-specific immunity. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes and sensitize tumor cells to killing by natural killer (NK) cells. However, the current doses of drugs used to induce ICD may be too high to translate into clinically relevant regimens. Methods: Our group discovered a novel peptide, CT20p, that is effective at nanomolar concentrations and can be delivered to breast tumors using targeted nanoparticles. CT20p works by inhibiting a unique protein folding complex, called chaperonin-containing TCP-1 (CCT), which is highly expressed in cancer cells but not normal cells. Using triple negative breast cancer (TNBC) and normal cell lines as well as patient-derived tumor cells, we examined the biological consequences of CCT inhibition by CT20p in terms of the induction of endoplasmic reticulum (ER) stress, through the accumulation of unfolded proteins, and the release of the danger signals associate with ICD that could stimulate innate immunity. Results: Dying TNBC cells treated with CT20p displayed alterations in PERK, a mediator of the unfolded protein response (UPR), which resulted in the membrane translocation of calreticulin (Crt), an “eat me”’ signal and a key marker of ICD. CT20p-killed TNBC cells were more readily phagocytosed, and NK cells more effectively killed remaining cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p. Conclusions: Our study suggests that inhibition of CCT by CT20p generates dying cancer cells that stimulate innate immune responses, through the release of signals like Crt, to attract phagocytes and promote NK cell cytotoxicity. By enhancing the immunogenicity of breast cancer cells, CT20p could increase responsiveness to to checkpoint inhibitors or cellular immunotherapy, hence providing more complete protection from breast cancer recurrence and metastasis.

The chaperonin CCT inhibits assembly of \u03b1-synuclein amyloid fibrils by a specific, conformation-dependent interaction

The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington’s disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson’s disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism.

PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer.

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin β1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin β1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

CCT complex restricts neuropathogenic protein aggregation via autophagy.

Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.

CCT2 Mutations Evoke Leber Congenital Amaurosis due to Chaperone Complex Instability.

Leber congenital amaurosis (LCA) is a hereditary early-onset retinal dystrophy that is accompanied by severe macular degeneration. In this study, novel compound heterozygous mutations were identified as LCA-causative in chaperonin-containing TCP-1, subunit 2 (CCT2), a gene that encodes the molecular chaperone protein, CCTβ. The zebrafish mutants of CCTβ are known to exhibit the eye phenotype while its mutation and association with human disease have been unknown. The CCT proteins (CCT α-θ) forms ring complex for its chaperon function. The LCA mutants of CCTβ, T400P and R516H, are biochemically instable and the affinity for the adjacent subunit, CCTγ, was affected distinctly in both mutants. The patient-derived induced pluripotent stem cells (iPSCs), carrying these CCTβ mutants, were less proliferative than the control iPSCs. Decreased proliferation under Cct2 knockdown in 661W cells was significantly rescued by wild-type CCTβ expression. However, the expression of T400P and R516H didn’t exhibit the significant effect. In mouse retina, both CCTβ and CCTγ are expressed in the retinal ganglion cells and connecting cilium of photoreceptor cells. The Cct2 knockdown decreased its major client protein, transducing β1 (Gβ1). Here we report the novel LCA mutations in CCTβ and the impact of chaperon disability by these mutations in cellular biology.

The interactome of CCT complex – A computational analysis

The eukaryotic chaperonin, CCT (Chaperonin Containing TCP1 or TriC-TCP-1 Ring Complex) has been subjected to physical and genetic analyses in S. cerevisiae which can be extrapolated to human CCT (hCCT), owing to its structural and functional similarities with yeast CCT (yCCT). Studies on hCCT and its interactome acquire an additional dimension, as it has been implicated in several disease conditions like neurodegeneration and cancer. We attempt to study its stress response role in general, which will be reflected in the aspects of human diseases and yeast physiology, through computational analysis of the interactome. Towards consolidating and analysing the interactome data, we prepared and compared the unique CCT-interacting protein lists for S. cerevisiae and H. sapiens, performed GO term classification and enrichment studies which provide information on the diversity in CCT interactome, in terms of protein classes in the data set. Enrichment with disease-associated proteins and pathways highlight the medical importance of CCT. Different analyses converge, suggesting the significance of WD-repeat proteins, protein kinases and cytoskeletal proteins in the interactome. The prevalence of proteasomal subunits and ribosomal proteins suggest a possible cross-talk between protein-synthesis, folding and degradation machinery. A network of chaperones and chaperonins that function in combination can also be envisaged from the CCT interactome-Hsp70 interactome analysis.