Cotranslational control of functional and pathological conformational switching of nascent polypeptides

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Project Summary/Abstract Many proteins have been identified to possess prion-like domains (PrLDs) capable of conformational switching between alternative three-dimensional structures. Conformational switching can be functional or pathological and the earliest point at which it could be regulated is during synthesis of the PrLD, especially for N-terminal PrLDs that emerge first from the ribosomal exit tunnel. However, the extent to which switching is regulated cotranslationally is largely unexplored, and our understanding of the physiological consequences of switching is in its infancy. Thus, elucidating ribosome-associated mechanisms and physiological impacts of conformational switching represents a critical barrier to advancing our understanding of how cells navigate the delicate balance between achieving proteostasis versus pathological protein misfolding. The applicant’s long-term goal is to decipher the mechanisms by which cells sense and respond to stress to maintain proteostasis, with a goal of better understanding the physiological significance of conformational switching in these processes. The overall objective of this application is to determine the contributions of cotranslational events in functional and pathological conformational switching of nascent chains, and the impact on gene expression of switching of a prion-forming protein. The central hypothesis is that conformational switching of nascent chains is governed by the interplay of translation kinetics and ribosome-associated factors in response to environmental cues and can result in both beneficial and pathogenic phenotypes. This hypothesis is based on the applicant’s published work and preliminary data, as well as published work from others. The rationale for the proposed research is that elucidating the contributions of ribosome-associated processes in conformational switching of nascent chains will revolutionize our understanding of proteostasis and pave the way for pharmacological manipulation to curtail pathogenic misfolding events. Using yeast prion-forming proteins and a human disease-associated protein sequence as models, this hypothesis will be tested by pursuing three specific aims: 1) Identify the roles of ribosome pausing and proteotoxic stress in modulating conformational switching of nascent chains; 2) Identify the roles of RAC and NAC in modulating conformational switching of nascent chains; and 3) Define physiological consequences of conformational switching of a prion-forming protein. The proposed work is innovative because it represents a substantial departure from the status quo by examining the earliest possible time-point in amyloidogenesis and by assessing the physiological consequences of functional conformational switching. The contribution of this work is expected to be detailed understanding of ribosome-associated mechanisms regulating cotranslational amyloid formation and the resulting physiological consequences. This contribution will be significant because cotranslational amyloid formation constitutes the earliest misfolding event against which pharmacological intervention could be targeted; thus, an understanding of the regulatory mechanisms governing the balance between functional and pathogenic conformational switching is urgently needed to advance the field.