The role of the Chaperonin containing t-complex polypeptide 1, subunit 8 (CCT8) in B-cell non-Hodgkin's lymphoma.

The chaperonin containing t-complex polypeptide 1 (CCT) is known to mediate folding of proteins. CCT, subunit 8 (CCT8), is the θ subunit of CCT complex chaperonin. CCT8 has been reported to be dysregulated in several tumor tissues. In this study, we investigated the role of CCT8 in B-cell non-Hodgkin's lymphoma (NHL). Clinically, the expression levels of CCT8 in reactive lymphoid hyperplasia (RLH) and B-cell NHL specimens were investigated using immunohistochemical analysis. We found that CCT8 was highly expressed in proliferating germinal center cells compared with the quiescent cells of the follicular mantle zone. Furthermore, CCT8 was highly expressed in progressive lymphomas than in indolent lymphomas. Kaplan-Meier curve showed that high expression of CCT8 was significantly associated with shorter overall survival in patients with diffuse large B-cell lymphoma. Moreover, we demonstrated that CCT8 could promote the proliferation of B-cell NHL cells. In addition, we found that CCT8 could accelerate the G1/S transition in B-cell NHL. Finally, we demonstrated that overexpression of CCT8 could reverse cell adhesion-mediated drug resistance (CAM-DR) phenotype. Our study may shed new insights into the important role of CCT8 in cancer development.

TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells.

Phosphorothioate (PS) antisense oligonucleotides (ASOs) have been successfully developed as drugs to reduce the expression of disease-causing genes. PS-ASOs can be designed to induce degradation of complementary RNAs via the RNase H pathway and much is understood about that process. However, interactions of PS-ASOs with other cellular proteins are not well characterized. Here we report that in cells transfected with PS-ASOs, the chaperonin T-complex 1 (TCP1) proteins interact with PS-ASOs and enhance antisense activity. The TCP1-ß subunit co-localizes with PS-ASOs in distinct nuclear structures, termed phosphorothioate bodies or PS-bodies. Upon Ras-related nuclear protein (RAN) depletion, cytoplasmic PS-body-like structures were observed and nuclear concentrations of PS-ASOs were reduced, suggesting that TCP1-ß can interact with PS-ASOs in the cytoplasm and that the nuclear import of PS-ASOs is at least partially through the RAN-mediated pathway. Upon free uptake, PS-ASOs co-localize with TCP1 proteins in cytoplasmic foci related to endosomes/lysosomes. Together, our results indicate that the TCP1 complex binds oligonucleotides with TCP1-ß subunit being a nuclear PS-body component and suggest that the TCP1 complex may facilitate PS-ASO uptake and/or release from the endocytosis pathway.

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.

New insights into the functions and localization of nuclear CCT protein complex in K562 leukemia cells.

PURPOSE: The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) plays an important role in maintaining cellular homeostasis by assisting the folding of many proteins and is also well known for the critical roles in disease. However, the functions of CCT complex have not been established globally, especially when translocating into nuclear. The purpose of this study is to explore the function of CCT in nuclear and present a strategy in clinical proteomics studies. EXPERIMENTAL DESIGN: Blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with mass spectrometry was applied to separate and identify CCT protein complexes. RESULTS: We isolated the CCT complex in K562 nucleus and identified a novel CCT complex containing 40 protein components involved in protein folding, RNA processing, apoptosis, and cell metabolism. The interactions between four candidate proteins and CCT were confirmed by immunoblotting. Computational biological analyses and independent biochemical assays validated the overall quality of interactions. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support clues that CCT might play an unexpected role in various biological processes including RNA processing. And we envision future applications for this system searching for new clues of CCT in disease and readily be applied to the clinic.

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.