Cotranslational control of functional and pathological conformational switching of nascent polypeptides

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.

Grant Number: 2R15GM119081-03
NIH Institute/Center: NIH
Principal Investigator: Dale Cameron