RESUMEN
Ribosome biogenesis is among the most resource-intensive cellular processes, with ribosomal proteins accounting for up to half of all newly synthesized proteins in eukaryotic cells. During stress, cells shut down ribosome biogenesis in part by halting rRNA synthesis, potentially leading to massive accumulation of aggregation-prone 'orphan' ribosomal proteins (oRPs). Here we show that, during heat shock in yeast and human cells, oRPs accumulate as reversible peri-nucleolar condensates recognized by the Hsp70 co-chaperone Sis1/DnaJB6. oRP condensates are liquid-like in cell-free lysate but solidify upon depletion of Sis1 or inhibition of Hsp70. When cells recover from heat shock, oRP condensates disperse in a Sis1- and Hsp70-dependent manner, and the oRP constituents are incorporated into functional ribosomes in the cytosol, enabling cells to efficiently resume growth. Preserving biomolecules in reversible condensates-like mRNAs in cytosolic stress granules and oRPs at the nucleolar periphery-may be a primary function of the Hsp70 chaperone system.
Asunto(s)
Proteínas Ribosómicas , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Since a majority of ovarian tumors recur in a drug-resistant form leaving patients few treatment options, the goal of this study was to explore phenotypic and molecular characteristics of a cisplatin-resistant ovarian cancer cell line (OVCAR8R) as compared to its cisplatin-sensitive syngeneic counterpart (OVCAR8) and to explore the effectiveness of a novel chemotherapeutic, Withaferin A (WA). In addition to unique morphological characteristics, the small heat shock proteins (Hsps) αB-Crystallin (HspB5) and Hsp27 are constitutively expressed along with increased expression of vimentin in OVCAR8R cells, while OVCAR8 cells do not endogenously express these Hsps, supporting that Hsp overexpression may confer resistance to chemotherapy and promote more aggressive tumor types. WA increases apoptosis in a dose-dependent manner in OVCAR8 cells, while OVCAR8R cells remain more viable at comparable doses of WA coincident with the upregulation of αB-Crystallin. To determine the significance of αB-Crystallin in conferring a more aggressive phenotype, αB-Crystallin was silenced by CRISPR-Cas9 in OVCAR8R cells. The morphology of the OVCAR8R clones in which αB-Crystallin was silenced reverted to the morphology of the original cisplatin-sensitive OVCAR8 cells. Further, cisplatin-resistant OVCAR8R cells constitutively express higher levels of vimentin and migrate more readily than cisplatin-sensitive OVCAR8 and OVCAR8R cells in which αB-Crystallin was silenced. Transient overexpression of wildtype αB-Crystallin, but not a chaperone-defective-mutant, alters the morphology of these cells to closely resemble the cisplatin-resistant OVCAR8R cells and protects versus WA-induced apoptosis. Together, this research supports the potential effectiveness of WA as a therapy for ovarian cancer cells that have not yet acquired resistance to platinum-based therapies, and importantly, underscores that αB-Crystallin contributes to a more aggressive cellular phenotype and as such, may be a promising molecular target for a better clinical outcome.
