Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules

Abstract

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.

Protein and metabolite composition of Arabidopsis stress granules

“…b). These six proteins have SG‐localised homologs in either human or yeast (Jain et al ., ; Markmiller et al ., ; Youn et al ., ) , cells, and this was also confirmed for 27 additional proteins identified in our study (Table S1). This total number of 33 conserved SG proteins was designated as the conserved SG proteome.…”

“…Proximity‐labeling experiments have previously revealed a preexisting interaction network between core SG proteins present in unstressed human cells (Markmiller et al ., ; Youn et al ., ). We investigated whether the same might be true in Arabidopsis by investigating Rbp47b protein interactors in unstressed cells using a TAP protocol.…”

“…In line with recent results based on human cell lines (Markmiller et al ., ; Youn et al ., ), our data also point to the presence of a preexisting interaction network between core SG proteins that normally occurs in unstressed cells. The TAP experiments using Rbp47b as bait and in the unstressed cells revealed a list of interactors with well known associations with Arabidopsis SGs.…”

Expanding APEX2 Substrates for Proximity‐Dependent Labeling of Nucleic Acids and Proteins in Living Cells

“…Eukaryotic cells feature highly compartmentalized cellular structures.Inthe past decade,anumber of spatially restricted chemical labeling techniques have been developed to facili-tate profiling of subcellular proteomes.T hese techniques often capitalize on genetically targetable enzymes that catalyze the formation of highly reactive intermediates,s uch as biotin-conjugated phenoxyl radicals [1,2] or biotinyl 5'adenylate [3,4] in the cellular environment, which then react with neighboring biomolecules to achieve proximity-dependent labeling.A mong these,e nhanced ascorbate peroxidase APEX2 offers both high reactivity and high spatiotemporal resolution, and has been instrumental in revealing the proteomes of an array of subcellular compartments,including mitochondria, [1,[5][6][7] primary cilia, [8] neuronal synapses, [9] stress granules, [10] and G-protein-coupled receptor signaling complexes. [11,12] More recently,APEX2-mediated proximity labeling has been applied to map the subcellular transcriptome, either through crosslinking RNAt ob iotinylated proteins (APEX-RIP [13] and Proximity-CLIP [14] )o rb yd irectly biotinylating RNA( APEX-seq [15] ).…”

Mild heat stress induces transcription of the β‐casein gene via unfolded protein response‐activated XBP1 signaling in undifferentiated mammary epithelial cells

“…In a study using AD-293 cells derived from human fetal kidney, the expression levels of UPR factors, including GRP78, CHOP, and XBP1s increased, and the phosphorylation of elF2 was induced with a mild heat stimulus at 39°C -40°C. The cellular response to stress accordingly differs depending on cell type, stress type, and stress intensity (Markmiller et al, 2018). Most of the research on the relationship between heat stress and the UPR has been under conditions of strong heat stress at 42°C -43°C for 1-2 hr (Hou, Lin, Hou, & Liu, 2014;Kim et al, 2013), whereas only few reports have examined the effect of mild heat stress at 39°C over an extended time period.…”