Talk abstracts
Talk on Saturday 09:45-10:00am submitted by Michael Owens
The cellular morphologies of cancer-related DDX3X mutants are defined by their catalytic activities
Michael C. Owens (Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA), Hui Shen, Amber Yanas, Maria Saraí Mendoza-Figueroa, Ellen Lavorando (Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA), Xiaoyu Wei (Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA), Yale E. Goldman (Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA), Kathy Fange Liu (Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA)
Abstract:
DEAD-box RNA helicase 3, X-linked (DDX3X) is a key tumor suppressor. As such, heterozygous mutations of DDX3X are found in numerous cancers, such as medulloblastoma, breast cancer, and melanoma. Many of these mutations disrupt the enzymatic activity of DDX3X when expressed in vitro, although the exact mechanism for these disruptions remains unclear. Furthermore, when expressed in cells, several mutants of DDX3X phase separate into puncta without addition of an external stressor. To investigate the possible link between disrupted catalytic activity and aberrant puncta formation for DDX3X mutants, we selected twelve cancer-related mutants that span the conserved catalytic motifs of DDX3X and expressed them both in vitro and in cells. We found that five of these mutants formed unique “hollow” puncta in cells, where the mutants were enriched in a spherical shell surrounding a hollow core that lacked mutant DDX3X. When expressed recombinantly, “hollow” mutants had significantly decreased ATPase and strand separation activity relative to the wild-type protein. Using the wealth of structural information available for DDX3X at all stages of its catalytic cycle, we developed rescue mutations for two hollow mutants that both restored their catalytic activity in vitro and diminished the formation of hollow puncta in cells. Using both proximity ligation and immunofluorescence, we determined that DDX3X mutant hollow puncta trapped WT DDX3X, its Y chromosome-encoded homolog DDX3Y, and other proteins important for various signaling pathways. Collectively, these results point towards a model in which mutations that stall DDX3X’s catalytic cycle drive the formation of hollow cellular puncta that trap WT DDX3X or DDX3Y, preventing their normal functions and contributing to disease progression.
Keywords: DEAD-box helicases, liquid-liquid phase separation, sexually dimorphic protein homologs