Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

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Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

10.1101/721001 ◽

2019 ◽

Cited By ~ 8

Author(s):

Richard J. Wheeler ◽

Hyun O. Lee ◽

Ina Poser ◽

Arun Pal ◽

Thom Doeleman ◽

...

Keyword(s):

Phase Separation ◽

Neurodegenerative Disease ◽

Stress Granules ◽

Life Time ◽

Stress Granule ◽

Phase Behaviour ◽

Granule Proteins ◽

Lateral Sclerosis ◽

Liquid Liquid Phase Separation

AbstractAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with few avenues for treatment. Many proteins implicated in ALS associate with stress granules, which are examples of liquid-like compartments formed by phase separation. Aberrant phase transition of stress granules has been implicated in disease, suggesting that modulation of phase transitions could be a possible therapeutic route. Here, we combine cell-based and protein-based screens to show that lipoamide, and its related compound lipoic acid, reduce the propensity of stress granule proteins to aggregate in vitro. More significantly, they also prevented aggregation of proteins over the life time of Caenorhabditis elegans. Observations that they prevent dieback of ALS patient-derived (FUS mutant) motor neuron axons in culture and recover motor defects in Drosophila melanogaster expressing FUS mutants suggest plausibility as effective therapeutics. Our results suggest that altering phase behaviour of stress granule proteins in the cytoplasm could be a novel route to treat ALS.

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The Co-Chaperone HspBP1 Is a Novel Component of Stress Granules that Regulates Their Formation

Cells ◽

10.3390/cells9040825 ◽

2020 ◽

Vol 9 (4) ◽

pp. 825

Author(s):

Hicham Mahboubi ◽

Ossama Moujaber ◽

Mohamed Kodiha ◽

Ursula Stochaj

Keyword(s):

Stress Response ◽

Cellular Stress ◽

Stress Granules ◽

Biological Properties ◽

Cellular Stress Response ◽

Binding Domains ◽

Hsp70 Family ◽

Independent Functions

The co-chaperone HspBP1 interacts with members of the hsp70 family, but also provides chaperone-independent functions. We report here novel biological properties of HspBP1 that are relevant to the formation of cytoplasmic stress granules (SGs). SG assembly is a conserved reaction to environmental or pathological insults and part of the cellular stress response. Our study reveals that HspBP1 (1) is an integral SG constituent, and (2) a regulator of SG assembly. Oxidative stress relocates HspBP1 to SGs, where it co-localizes with granule marker proteins and polyA-RNA. Mass spectrometry and co-immunoprecipitation identified novel HspBP1-binding partners that are critical for SG biology. Specifically, HspBP1 associates with the SG proteins G3BP1, HuR and TIA-1/TIAR. HspBP1 also interacts with polyA-RNA in vivo and binds directly RNA homopolymers in vitro. Multiple lines of evidence and single-granule analyses demonstrate that HspBP1 is crucial for SG biogenesis. Thus, HspBP1 knockdown interferes with stress-induced SG assembly. By contrast, HspBP1 overexpression promotes SG formation in the absence of stress. Notably, the hsp70-binding domains of HspBP1 regulate SG production in unstressed cells. Taken together, we identified novel HspBP1 activities that control SG formation. These features expand HspBP1’s role in the cellular stress response and provide new mechanistic insights into SG biogenesis.

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Distinct stages in stress granule assembly and disassembly

eLife ◽

10.7554/elife.18413 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 224

Author(s):

Joshua R Wheeler ◽

Tyler Matheny ◽

Saumya Jain ◽

Robert Abrisch ◽

Roy Parker

Keyword(s):

Time Course ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Stress Granules ◽

Stress Granule ◽

Early Event ◽

Intrinsically Disordered

Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.

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Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

The Journal of Cell Biology ◽

10.1083/jcb.201709007 ◽

2018 ◽

Vol 217 (4) ◽

pp. 1303-1318 ◽

Cited By ~ 44

Author(s):

Benedikt Niewidok ◽

Maxim Igaev ◽

Abel Pereira da Graca ◽

Andre Strassner ◽

Christine Lenzen ◽

...

Keyword(s):

Liquid Phase ◽

Single Molecule ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Liquid Droplet ◽

Binding Protein ◽

Stress Granules

Stress granules (SGs) are cytosolic, nonmembranous RNA–protein complexes. In vitro experiments suggested that they are formed by liquid–liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain–binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs. Within SGs, single-molecule localization microscopy revealed distributed hotspots of immobilized G3BP1 and IMP1 that reflect the presence of relatively immobile nanometer-sized nanocores. We demonstrate alternating binding in nanocores and anomalous diffusion in the liquid phase with similar characteristics for both RBPs. Reduction of low-complexity regions in G3BP1 resulted in less detectable mobile molecules in the liquid phase without change in binding in nanocores. The data provide direct support for liquid droplet behavior of SGs in living cells and reveal transient binding of RBPs in nanocores. Our study uncovers a surprising disconnect between SG partitioning and internal diffusion and interactions of RBPs.

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Evaluating Stress Granules in Pancreatic Cancer In Vitro and In Vivo

Methods in Molecular Biology - Pancreatic Cancer ◽

10.1007/978-1-4939-8879-2_17 ◽

2018 ◽

pp. 183-195 ◽

Cited By ~ 1

Author(s):

Edward Sim ◽

Elena Irollo ◽

Elda Grabocka

Keyword(s):

Pancreatic Cancer ◽

Stress Granules

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0028 Sleep Duration Influences the Kinetics of Stress Granule Formation

SLEEP ◽

10.1093/sleep/zsaa056.027 ◽

2020 ◽

Vol 43 (Supplement_1) ◽

pp. A11-A12

Author(s):

M K Dougherty ◽

C Saul ◽

L Carman ◽

M D Nelson ◽

J C Tudor

Keyword(s):

Sleep Deprivation ◽

Sleep Duration ◽

Protein A ◽

Stress Granules ◽

Mtor Pathway ◽

Stress Granule ◽

Target Of Rapamycin ◽

Granule Formation ◽

One Way Anova

Abstract Introduction Stress granules are non-membrane bound aggregates of messenger ribonucleoproteins that are biomarkers of cellular stress. It has been shown in cells in vitro that suppression of the mammalian target of rapamycin (mTOR) pathway and its non-mammalian orthologue target of rapamycin (TOR) is associated with an increase in stress granule formation. It has also been shown that the mTOR pathway is suppressed in response to sleep deprivation in mice. Despite the possible connection via the TOR/mTOR pathway, there has not been any previous evidence linking sleep deprivation with stress granule formation. Methods Our present investigation uses the nematode Caenorhabditis elegans to model how stress granule formation and clearance are modified by sleep duration. We developed novel strains of C. elegans that model each type of sleep deprivation or enhancement and have RFP-labeled PAB-1 protein, a key component of stress granules. In addition to modifying sleep duration via genetic means, we also sleep deprived wildtype fluorescently labeled animals using mechanical disturbances. Results Animals with enhanced stress-induced sleep have stress granules that are smaller in size and cleared faster than wildtype, while sleep deprived animals have granules that are slower to clear (F11,473 = 7.752, ***p < 0.0001, one-way ANOVA). Animals that were manually deprived of stress-induced sleep were similarly slower to clear stress granules (F5,209 = 5.476 ***p < 0.0001, one-way ANOVA). Interestingly, animals genetically deprived of developmentally-timed sleep does not appear to have more stress granules in the middle of their sleep period than the sleeping wildtype stage (F2,42 = 2.659, p = 0.0729, one-way ANOVA). Conclusion This work demonstrates that the amount of sleep affects stress granule kinetics, which impacts the flow of genetic information inside cells. Support This work was supported by an R15GM122058 (NIH), John P. McNulty scholars program (SJU) and summer scholars program (SJU).

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Quantitative proteomics identifies the universally conserved ATPase Ola1p as a positive regulator of heat shock response in Saccharomyces cerevisiae

10.1101/2021.05.12.443772 ◽

2021 ◽

Author(s):

Stefan Dannenmaier ◽

Christine Desroches Altamirano ◽

Lisa Schueler ◽

Ying Zhang ◽

Johannes Hummel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Stress ◽

Heat Shock ◽

Heat Shock Response ◽

De Novo ◽

Stress Granules ◽

Cellular Stress Response ◽

Protein Ubiquitination ◽

Shock Response

The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in vitro studies and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When subsequently cells lacking Ola1p were relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared to wildtype cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved.

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High fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate

10.1101/2020.09.14.296673 ◽

2020 ◽

Author(s):

Brian D. Freibaum ◽

James Messing ◽

Peiguo Yang ◽

Hong Joo Kim ◽

J. Paul Taylor

Keyword(s):

Stress Granules ◽

Live Cells ◽

High Fidelity ◽

Limited Ability ◽

Intracellular Organization ◽

In Vitro Systems ◽

Protein Nucleic Acid ◽

Rnp Granules ◽

Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) is an important mechanism of intracellular organization that underlies the assembly of a variety of distinct RNP granules. Fundamental biophysical principles governing LLPS during RNP granule assembly have been revealed by simple in vitro systems consisting of several components, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in live cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells, and allows for a wide variety of studies of membraneless organelles.

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Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1

Molecular and Cellular Biology ◽

10.1128/mcb.00596-16 ◽

2016 ◽

Vol 37 (4) ◽

Cited By ~ 35

Author(s):

Lucas C. Reineke ◽

Wei-Chih Tsai ◽

Antrix Jain ◽

Jason T. Kaelber ◽

Sung Yun Jung ◽

...

Keyword(s):

Cell Fate ◽

Stress Granules ◽

Casein Kinase ◽

Casein Kinase 2 ◽

Stress Granule ◽

Translational Repression ◽

Transcriptional Responses ◽

Granule Formation ◽

Cell Fate Decisions

ABSTRACT Stress granules (SGs) are large macromolecular aggregates that contain translation initiation complexes and mRNAs. Stress granule formation coincides with translational repression, and stress granules actively signal to mediate cell fate decisions by signaling to the translation apparatus to (i) maintain translational repression, (ii) mount various transcriptional responses, including innate immunity, and (iii) repress apoptosis. Previous work showed that G3BP1 is phosphorylated at serine 149, which regulates G3BP1 oligomerization, stress granule assembly, and RNase activity intrinsic to G3BP1. However, the kinase that phosphorylates G3BP1 was not identified, leaving a key step in stress granule regulation uncharacterized. Here, using chemical inhibition, genetic depletion, and overexpression experiments, we show that casein kinase 2 (CK2) promotes stress granule dynamics. These results link CK2 activity with SG disassembly. We also show that casein kinase 2 phosphorylates G3BP1 at serine 149 in vitro and in cells. These data support a role for casein kinase 2 in regulation of protein synthesis by downregulating stress granule formation through G3BP1.

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