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The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction.

ABSTRACT: Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.

SUBMITTER: Dun MD

PROVIDER: S-EPMC3196097 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction.

Dun Matthew D MD Smith Nathan D ND Baker Mark A MA Lin Minjie M Aitken R John RJ Nixon Brett B

The Journal of biological chemistry 20110831 42

Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface ...[more]

PMID: 21880732

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Project description:Uncontrolled proliferation as a result of dysregulated cell cycling is one of the hallmarks of cancer. Therapeutically targeting pathways that control the cell cycle would improve patient outcomes. However, the development of drug resistance and a limited number of inhibitors that target multiple cell cycle modulators are challenges that impede stopping the deregulated growth that leads to malignancy. To advance the discovery of new druggable targets for cell cycle inhibition, we investigated the role of Chaperonin-Containing TCP1 (CCT or TRiC) in breast cancer cells. CCT, a type II chaperonin, is a multi-subunit protein-folding complex that interacts with many oncoproteins and mutant tumor suppressors. CCT subunits are highly expressed in a number of cancers, including breast cancer. We found that expression of one of the CCT subunits, CCT2, inversely correlates with breast cancer patient survival and is subject to copy number alterations through genomic amplification. To investigate a role for CCT2 in the regulation of the cell cycle, we expressed an exogenous CCT2-FLAG construct in T47D and MCF7 luminal A breast cancer cells and examined cell proliferation under conditions of two-dimensional (2D) monolayer and three-dimensional (3D) spheroid cultures. Exogenous CCT2 increased the proliferation of cancer cells, resulting in larger and multiple spheroids as compared to control cells. CCT2-expressing cells were also able to undergo spheroid growth reversal, re-attaching, and resuming growth in 2D cultures. Such cells gained anchorage-independent growth. CCT2 expression in cells correlated with increased expression of MYC, especially in spheroid cultures, and other cell cycle regulators like CCND1 and CDK2, indicative of a novel activity that could contribute to the increase in cell growth. Statistically significant correlations between CCT2, MYC, and CCND1 were shown. Since CCT2 is located on chromosome 12q15, an amplicon frequently found in soft tissue cancers as well as breast cancer, CCT2 may have the basic characteristics of an oncogene. Our findings suggest that CCT2 could be an essential driver of cell division that may be a node through which pathways involving MYC, cyclin D1 and other proliferative factors could converge. Hence the therapeutic inhibition of CCT2 may have the potential to achieve multi-target inhibition, overcoming the limitations associated with single agent inhibitors.

Project description:Chaperonin containing TCP1 (CCT/TRiC) is a multi-subunit protein folding complex that enables the cancer phenotype to emerge from the mutational landscape that drives oncogenesis. We and others linked increased expression of CCT subunits to advanced tumor stage and invasiveness that inversely correlates with cancer patient outcomes. In this study, we examined the expression of the second CCT subunit, CCT2, using genomic databases of adult and pediatric tumors and normal tissues, and found that it was highly expressed in pediatric cancers, showing a significant difference compared to normal tissues. Histologic staining confirmed that CCT subunits are highly expressed in tumor tissues, which was exemplified in neuroblastoma. Using two neuroblastoma cells, MYCN-amplified, IMR-32 cells, and non-amplified, SK-N-AS cells, we assessed baseline levels for CCT subunits and found expressions comparable to the highly invasive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. Exogenous expression of CCT2 in both SK-N-AS and IMR-32 cells resulted in morphological changes, such as larger cell size and increased adherence, with significant increases in the CCT substrates, actin, and tubulin, as well as increased migration. Depletion of CCT2 reversed these effects and reduced cell viability. We evaluated CCT as a therapeutic target in IMR-32 cells by testing a novel peptide CCT inhibitor, CT20p. Treatment with CT20p induced cell death in these neuroblastoma cells. The use of CCT2 as a biological indicator for detection of neuroblastoma cells shed in blood was examined by spiking IMR-32 cells into human blood and using an anti-CCT2 antibody for the identification of spiked cancer cells with the CellSearch system. Results showed that using CCT2 for the detection of neuroblastoma cells in blood was more effective than the conventional approach of using epithelial markers like cytokeratins. CCT2 plays an essential role in promoting the invasive capacity of neuroblastoma cells and thus offers the potential to act as a molecular target in the development of novel therapeutics and diagnostics for pediatric cancers.

Dataset Information

The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction.

Publications

The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction.

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