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

Chaperonin Containing TCP1 (CCT) as a Target for Cancer Therapy

Treatments for aggressive cancers like triple negative breast cancer (TNBC) and small-cell lung cancer (SCLC) have not improved and remain associated with debilitating side effects. There is an unmet medical need for better, druggable targets and improved therapeutics. To this end, we investigated the role of Chaperonin-Containing TCP1 (CCT), an evolutionarily conserved protein-folding complex composed of eight subunits (CCT1-8), in oncogenesis. Our laboratory was the first to report that the CCT2 subunit is highly expressed in breast cancer and could be therapeutically targeted. To determine whether CCT is a marker of disease progression in other cancers, we analyzed CCT2 gene expression in liver, prostate and lung cancer, using publicly available genetic databases, and confirmed findings by assessing CCT2 and client proteins, like STAT3, in tumor tissues by immunohistochemistry. We found that CCT2 was high in all cancers, especially SCLC, and correlated with decreased patient survival. We tested CT20p, the peptide therapeutic developed by our laboratory to inhibit CCT, on SCLC and primary lung cells, finding that CT20p was only cytotoxic to SCLC cells. Since SCLC currently lacks targeted therapeutics, our work yielded a new targeted agent that could improve lung cancer mortality. To establish a mechanism of action for CT20p, we partially knocked out CCT2 in TNBC cells, which decreased tumorigenicity in mice and reduced levels of essential proteins like STAT3. To confirm, we overexpressed CCT2 in non-tumorigenic cells and conferred tumor-like characteristics such as increased migration and elevated STAT3. These studies positioned us to develop and validate a strategy for discovery of new small molecule inhibitors of CCT. We thus advanced the field of cancer research by demonstrating that CCT could have diagnostic potential for cancers, such as SCLC and TNBC, that are a significant cause of human death and showed that targeting CCT is a promising therapeutic approach