Biology subjects

Kedersha, N.

Publications and source records attributed to Kedersha, N..

4 recordsLinked to original sources

bioRxiv2021

Caprin-1 binding to the critical stress granule protein G3BP1 is regulated by pH

G3BP is the central hub within the protein-RNA interaction network of stress-induced bio-molecular condensates known as stress granules (SG). The SG-associated proteins Caprin-1 and USP10 exhibit mutually exclusive binding to the structured NTF2-domain of G3BP1, thereby regulating G3BP1-mediated condensation, but with opposite effects: Caprin-1 promotes but USP10 inhibits SG formation. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1 derived short linear motif (SLiM), which provides a molecular understanding for the mutually exclusive binding of USP10 and Caprin-1 to G3BP1. Caprin-1 but not USP10 contacts two G3BP1-NTF2 histidine residues, which was confirmed using biochemical, biophysical and cellular biological binding assays. G3BP1/Caprin-1 interactions disrupted via point mutations resulted in fewer and smaller SG condensates. In addition, biochemical binding assays demonstrated reduced binding of Caprin-1 to G3BP1 at lower pH values. Finally, ratiometric pH sensitive measurements of SGs revealed a substantial drop in pH compared to the adjacent cytosol, suggesting that reduced pH can fine-tune and regulate the G3BP1-mediated interaction network via a NTF2-mediated pH-sensitive SLiM-selection mechanism.

biochemistry↗

bioRxiv2020

Spatio-temporal Proteomic Analysis of Stress Granule disassembly using APEX Reveals Regulation by SUMOylation and links to ALS pathogenesis

Cytoplasmic stress granules (SG) form in response to a variety of cellular stresses by phase-separation of proteins associated with non-translating mRNAs. SG provide insight into the biology of neurodegeneration, including amyotrophic lateral sclerosis (ALS) because they approximate some of the molecular conditions for nucleation of insoluble aggregates in neuropathological inclusions. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SG undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of SG temporal disassembly sequence, using multi-bait APEX proximity-proteomics. We discover 109 novel SG-proteins and characterize at proteomic resolution two biophysically distinct SG substructures. We further demonstrate that dozens of additional proteins are recruited to SG specifically during disassembly, indicating that it is a highly regulated process. The involved proteins link SG disassembly, to mitochondrial biology and the cytoskeleton. Parallel analysis with C9ORF72-associated dipeptides, which are found in patients with ALS and frontotemporal dementia, demonstrated compositional changes in SG during the course of disassembly and focused our attention on the roles SUMOylation in SG disassembly. We demonstrate that broad SUMOylation of SG-proteins is required for SG disassembly and is impaired by C9ORF72-associated dipeptides, representing an unexplored potential molecular mechanism of neurodegeneration. Altogether, out study fundamentally increases the knowledge about SG composition in human cells by dissecting the SG spatio-temporal proteomic landscape, provides an in-depth resource for future work on SG function and reveals basic and disease-relevant mechanisms of SG disassembly. HighlightsO_LIMulti bait APEX proximity labelling reveals 109 novel SG proteins and two distinct SG substructures. C_LIO_LIProteomic dissection of SG temporal disassembly under basal conditions and with a model of neurodegeneration. C_LIO_LIDisassembly-engaged proteins (DEPs) include SUMO ligases that are recruited during normal disassembly and dysregulated in ALS-like conditions. C_LIO_LIPervasive SG protein SUMOylation during SG disassembly is impaired by ALS-like conditions. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/830133v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@15899daorg.highwire.dtl.DTLVardef@1dcd399org.highwire.dtl.DTLVardef@c7ef12org.highwire.dtl.DTLVardef@20c096_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

Bisphenol A promotes stress granule assembly and modulates the integrated stress response

Bisphenol-A (BPA) is a ubiquitous precursor of polycarbonate plastics that is found in the blood and serum of >92% of Americans. While BPA has been well documented to act as a weak estrogen receptor (ER) agonist, its effects on cellular stress are unclear. Here, we demonstrate that high-dose BPA causes stress granules (SGs) in human cells. A common estrogen derivative, {beta}-estradiol, does not trigger SGs, indicating the mechanism of SG induction is not via the ER pathway. We also tested other structurally related environmental contaminants including the common BPA substitutes BPS and BPF, the industrial chemical 4-nonylphenol (4-NP) and structurally related compounds 4-EP and 4-VP, and the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The variable results from these related compounds suggest that structural homology is not a reliable predictor of the capacity of a compound to cause SGs. Also, we demonstrate that BPA acts primarily through the PERK pathway to generate canonical SGs. Finally, we show that chronic exposure to a low physiologically relevant dose of BPA disrupts SG assembly by inhibiting SGs upon additional acute stress. Our work identifies additional effects of BPA beyond endocrine disruption that may have consequences for human health.

cell biology↗

bioRxiv2019

Revisiting G3BP-S149 phosphorylation and its impact on stress granule assembly

Stress granules (SGs) are cytoplasmic, non-membranous RNA/protein structures that assemble in response to environmental stress. G3BP is a critical SG-nucleating protein, and its ability to regulate SGs has been reported to be regulated by serine 149 phosphorylation. We now report that the constructs engineered to contain non-phosphorylatable and phosphomimetic (G3BP1-S149A and G3BP1-S149E, respectively) mutations used in many studies include additional unintended mutations (A54T/S149A and S99P/S149E) one of which (S99P) is responsible for the effects on SG assembly attributed to S149E. Specifically, the S99P mutation alone reduces SG nucleation and impairs the ability to rescue SG assembly in {Delta}{Delta}G3BP1/2 U2OS KO cells, challenging the widely-stated conclusion that de-phosphorylation of serine 149 in G3BP1 promotes SG assembly. We used comparative mass spectrometry analysis of both (1) ectopically expressed GFP-G3BP1 in {Delta}{Delta}G3BP1/2 U2OS KO and (2) endogenous G3BP1 in wild-type U2OS, with and without sodium arsenite treatment, in an attempt to reproduce earlier findings, but found no significant changes in S149 phosphorylation that correlate with arsenite-induced SG formation.

cell biology↗