Monday, December 30, 2024
12/30/2024
Abstract. Most biological processes require the production and degradation of cellular proteins. Maintaining a balanced protein homeostasis (proteostasis) is therefore essential to cellular fitness. Furthermore, lapses in proteostasis have been linked to the molecular basis of a wide variety of genetic diseases. Nevertheless, our understanding of how the cell buffers adaptive swings in proteostasis and how this is perturbed by mutations remains incomplete. This is especially true for integral membrane proteins (MPs), which account for a quarter of the proteome and include most drug targets. The production of folded, functional, and properly localized MPs is both inefficient and sensitive to the effects of mutations that promote misfolding. The net efficiency of this process is established by the interactions that nascent MPs form with chaperones that mediate quality control. Most MPs form interactions with an array of QC proteins in the endoplasmic reticulum (ER) that establish the balance between the degradation of immature protein and the export of mature protein from the secretory pathway. We recently demonstrated that this balance is highly sensitive to the propensity of nascent MPs to adopt alternative topologies with respect to the membrane. We found that mutations that promote these defects impact MP expression and ligand binding in a manner that shapes both MP evolution and the molecular basis of disease. Furthermore, we also found that the mechanical forces generated by formation of alternative topologies can alter the outcomes of ribosomal protein synthesis. However, it remains unclear how certain topologies are selectively recognized by molecular chaperones and how these interactions ultimately shape MP biosynthesis and degradation. In the following, we outline innovative approaches to identify specific conformational defects that promote the interaction of nascent MPs with various molecular chaperones including the ER membrane protein complex and calnexin- two mechanistically distinct intramembrane chaperones. By combining CRISPR with deep mutational scanning (DMS), we will determine which classes of mutations and their associated conformational defects promote interactions with these chaperones and how this ultimately shapes mutational tolerance. Additionally, we will build on our recent discoveries to gain insights into how these cotranslational processes impact the fidelity membrane protein biosynthesis itself. We describe the discovery of a novel ribosomal frameshift site within the CFTR transcript and provide evidence suggesting this motif selectively terminates translation of a common misfolded variant responsible for most cases of cystic fibrosis (ΔF508). These findings point to a novel role of ribosomal frameshifting in the regulation of membrane protein homeostasis. We outline ongoing efforts to identify factors that modulate this regulation and to discover and characterize comparable motifs in other disease linked MPs including the voltage-gated potassium channel KCNQ1. Together, our findings will provide mechanistic insights into the regulation of membrane protein biosynthesis and quality control as well as how these constraints factor into the molecular basis of proteostatic diseases of MP misfolding.
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