Time-Resolved Interactome Profiling Deconvolutes Secretory Protein Quality Control Dynamics

Protein-protein interactions play host to several cellular processes that depend heavily on both spatial and temporal regulation of these interactions. As such, understanding these parameters is necessary to fully comprehend and elucidate cellular processes and pathological disease states associated with them. Many advancements have allowed for more detailed characterization of these parameters, yet none are available to identify and quantify de novo protein-protein interactions with time-resolution at an organelle-wide scale. To address this, we have developed a quantitative mass-spectrometry method, time-resolved interactome profiling (TRIP). As a proof of concept, we utilize this methodology to elucidate alterations in interactions for the protein folding disease congenital hypothyroidism. We identify and deconvolute altered temporal engagement with pathways such as Hsp70/90 assisted folding, disulfide/redox processing, and N-glcosylation associated with hypothyroidism pathophysiology. We have coupled this methodology with functional siRNA screening and identified Vcp and Tex264 as key protein degradation components whose inhibition rescues mutant prohormone secretion. Ultimately, our results provide insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad application in investigating other protein folding diseases and cellular processes.