bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.01.29.830133

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

Hagai, M.-K.·Siany, A.·Kedersha, N.·Knafo, N.·Rivkin, N.·Danino, Y. M.·Cohen, N.·Olender, T.·Moens, T.·Higginbottom, A.·Cooper-Knock, J.·Eitan, C.·Toth Cohen, B.·Van Den Bocho, L.·Anderson, P.·Ivanov, P.·Geiger, T.·Hornstein, E.
View original source ↗Export metadata ↓

Abstract

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

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Hagai, M.-K., Siany, A., Kedersha, N., Knafo, N., Rivkin, N., Danino, Y. M., Cohen, N., Olender, T., Moens, T., Higginbottom, A., Cooper-Knock, J., Eitan, C., Toth Cohen, B., Van Den Bocho, L., Anderson, P., Ivanov, P., Geiger, T., Hornstein, E.. 2020-01-29. Spatio-temporal Proteomic Analysis of Stress Granule disassembly using APEX Reveals Regulation by SUMOylation and links to ALS pathogenesis. https://doi.org/10.1101/2020.01.29.830133

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Trans-branching of polyubiquitin chains orchestrates the DNA replication stress response

Polyubiquitin chain geometry dictates functional consequences of ubiquitylation. Although branched polyubiquitin chains are abundant in cells, little is known about their functions. Here we show that branching on the DNA replication factor PCNA, mediated by the ubiquitin-conjugating enzyme UBE2K and involving lysines 63 and 48 of ubiquitin, orchestrates the sequence of events in response to replication stress. By inducing VCP-dependent extraction of PCNA from chromatin, branching promotes re-priming of stalled forks and necessitates a BRCA1-dependent pathway of daughter-strand gap repair. Our study identifies hyper-accumulation of daughter-strand gaps as the mechanistic basis underlying the toxicity of inhibitors of the PCNA-specific isopeptidase, USP1, in BRCA1-deficient cells. Moreover, an unexpected preference of UBE2K to operate in trans suggests a general timing mechanism to organize hierarchies amongst ubiquitin signals.

molecular biology↗
bioRxiv2026

Impaired proteostasis is an early feature of the diabetic heart in humans and mice

Diabetes and obesity increase cardiac lipid levels leading to cardiomyopathy and heart failure. We hypothesized that intermittent fasting would reduce cardiac lipid levels. Surprisingly, intermittent fasting increased myocardial triglyceride content, but rescued mortality and attenuated cardiomyopathy in mice overexpressing cardiomyocyte acyl-CoA synthetase 1 (MHC-ACSL1). Lipid overload caused cardiomyocyte accumulation of polyubiquitinated protein aggregates containing desmin, a scaffolding intermediate filament protein, which intermittent fasting prevented. Furthermore, intermittent fasting reversed elevated myocardial C16:0 ceramide content, and knockdown of ceramide synthase CerS5 and CerS6 reduced palmitate-induced protein aggregation, highlighting a role for C16:0 ceramides in this pathology. Conversely, impairing aggrephagy with cardiomyocyte-specific p62 ablation induced heart failure in mice fed a high-fat diet, with paradoxically reduced cardiac lipid content. Crucially, non-failing diabetic human hearts also exhibited protein aggregate pathology. Taken together, these results demonstrate that impaired proteostasis characterizes cardiomyopathy from cardiac lipid overload and identify a promising new therapeutic target for this condition.

molecular biology↗
bioRxiv2026

Spatial profiling and neurovascular communication in the developing and adolescent cortex following prenatal alcohol exposure

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used the NanoString Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

molecular biology↗