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Time-Resolved Interactome Profiling Deconvolutes Secretory Protein Quality Control Dynamics

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Madison Wright

LAB HEAD: Lars Plate

PROVIDER: PXD035681 | Pride | 2024-08-08

REPOSITORIES: Pride

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			Action	DRS
	Exp04-49-WT_TRIP_16plex_05May2021_0C_TechRep1.raw	Raw
	Exp04-49-WT_TRIP_16plex_05May2021_100C_TechRep1.raw	Raw
	Exp04-49-WT_TRIP_16plex_05May2021_10C_TechRep1.raw	Raw
	Exp04-49-WT_TRIP_16plex_05May2021_30C_TechRep1.raw	Raw
	Exp04-49-WT_TRIP_16plex_05May2021_60C_TechRep1.raw	Raw

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Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. We have analyzed the temporal changes in transcription following a short Notch activation treatment and related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However, neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus, feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were collected at 7 time points (0M-bM-^@M-^Y, 10M-bM-^@M-^Y, 20M-bM-^@M-^Y, 30M-bM-^@M-^Y, 40M-bM-^@M-^Y, 60M-bM-^@M-^Y and 100M-bM-^@M-^Y) after a 5 minute Notch stimulation as 3 independent replicates. Immunoprecipitation was performed with Su(H) antibody and compared to the total input DNA. Samples from replicates #1 and #2 were labelled with Cy5, and replicate #3 was labelled with Cy3 as a dye-swap. For time point 10M-bM-^@M-^Y, only 2 biological replicates were obtained.

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Time-Resolved Interactome Profiling Deconvolutes Secretory Protein Quality Control Dynamics

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