Proximity labeling reveals an extensive steady-state stress granule interactome and insights to neurodegeneration

Abstract

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form in response to proteotoxic stress. Although SGs are distinct from aggregates observed in neurodegenerative disorders, they share protein components. We used APEX-mediated proximity labeling combined with quantitative mass spectrometry and high-throughput imaging to identify >100 previously unknown SG proteins in human cells, about 10% of which localize to SGs in a cell type-or stress type-dependent manner.Supporting a link between SG proteins and neurodegeneration, we demonstrate aberrant SG composition and subcellular distribution in iPSC-derived motor neurons from ALS patients, and identify several known and previously unidentified SG proteins that modify toxicity of mutant FUS and TDP-43 overexpression in Drosophila. We show that even in an unstressed steady-state, SG proteins form a densely-connected protein interaction network (PIN) and propose a model in which existing RNPs coalesce rapidly into microscopically visible granules that can act as gateways to pathological protein aggregation.

Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome

“…As members of the FET family of proteins, EWS and TAF-15 are known binding partners of FUS and were used as a positive control for interaction (Figure 4A, green bar) (dot plot for EWS and TAF15 in Supplementary Figure 3) (64). We were able to replicate that FUS WT and FUS P525L co-IP with UPF1, PABP1, G3BP1, and eIF2α as previously reported (Figure 4A, blue bar) (53,(65)(66)(67).…”

“…From this, we were able to visualize a multitude of proteins that overlap between ontology categories. We used this data along with the STRING interaction database to identify a subset of proteins from each ontology term that were either 1) previously identified binding partners for FUS WT (G3BP1, UPF1, PABP1, eIF2α) or 2) novel binding partners (VPS35, MOV10, CLTA) (14,41,53,65,67). As anticipated by the APEX2 datasets, we were able to confirm the interaction between all three FUS variants and the above targets utilizing two different methods: immunoprecipitation and immunofluorescence (Figure 4A/B).…”

RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

“…S1). Consistent with this observation, recent studies to comprehensively characterize the SG proteome failed to detect RBM45 within SGs [55,61].…”

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

“…An illustrative example is a cytoplasmic cluster (#17) formed by a group of RNA-binding proteins (including ATXN2L, NUFIP2 or FXR1, Fig. 4C) that separate into granules upon stress conditions (56)(57)(58)(59). Stress granules are not formed under the standard growth conditions used in our experiments, but the ability of our analysis to cluster these proteins together reveals an underlying specificity to their cytoplasmic localization (i.e., "texture") even in the absence of stress.…”