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Cell Reports Jun 2021The formation of stress granules (SGs) is an essential aspect of the cellular response to many kinds of stress, but its adaptive role is far from clear. SG dysfunction...
The formation of stress granules (SGs) is an essential aspect of the cellular response to many kinds of stress, but its adaptive role is far from clear. SG dysfunction is implicated in aging-onset neurodegenerative diseases, prompting interest in their physiological function. Here, we report that during starvation stress, SGs interact with mitochondria and regulate metabolic remodeling. We show that SG formation leads to a downregulation of fatty acid β-oxidation (FAO) through the modulation of mitochondrial voltage-dependent anion channels (VDACs), which import fatty acids (FAs) into mitochondria. The subsequent decrease in FAO during long-term starvation reduces oxidative damage and rations FAs for longer use. Failure to form SGs, whether caused by the genetic deletion of SG components or an amyotrophic lateral sclerosis (ALS)-associated mutation, translates into an inability to downregulate FAO. Because metabolic dysfunction is a common pathological element of neurodegenerative diseases, including ALS, our findings provide a direction for studying the clinical relevance of SGs.
Topics: Amyotrophic Lateral Sclerosis; Cell Line, Tumor; Cell Lineage; Fatty Acids; HEK293 Cells; Humans; Induced Pluripotent Stem Cells; Lipid Droplets; Mitochondria; Neurons; Oxidation-Reduction; Permeability; Starvation; Stress Granules
PubMed: 34133922
DOI: 10.1016/j.celrep.2021.109237 -
Cell Mar 2017In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form...
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Topics: Amino Acid Sequence; Cytoplasmic Granules; Hot Temperature; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Intrinsically Disordered Proteins; Mutagenesis; Poly(A)-Binding Proteins; Proline; Protein Domains; Ribonucleases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Stress, Physiological
PubMed: 28283059
DOI: 10.1016/j.cell.2017.02.027 -
Molecular Cell Mar 2018Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are...
Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.
Topics: Adaptor Proteins, Signal Transducing; Autophagy-Related Proteins; Binding Sites; Cell Cycle Proteins; Cytoplasmic Granules; Female; HeLa Cells; Humans; Intrinsically Disordered Proteins; Models, Molecular; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Domains; Protein Folding; Stress, Physiological; Structure-Activity Relationship; Ubiquitins
PubMed: 29526694
DOI: 10.1016/j.molcel.2018.02.004 -
Proceedings of the National Academy of... Jul 2022Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine...
Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1β. We show that nsp1β is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1β residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.
Topics: Animals; Antiviral Restriction Factors; DNA Helicases; Immune Evasion; Poly-ADP-Ribose Binding Proteins; Porcine respiratory and reproductive syndrome virus; RNA Helicases; RNA Recognition Motif Proteins; Stress Granules; Swine; Viral Nonstructural Proteins; Virus Replication; eIF-2 Kinase
PubMed: 35858300
DOI: 10.1073/pnas.2201169119 -
Current Biology : CB May 2023Cytoplasmic stress granules (SGs) are phase-separated membrane-less organelles that form in response to various stress stimuli. SGs are mainly composed of non-canonical...
Cytoplasmic stress granules (SGs) are phase-separated membrane-less organelles that form in response to various stress stimuli. SGs are mainly composed of non-canonical stalled 48S preinitiation complexes. In addition, many other proteins also accumulate into SGs, but the list is still incomplete. SG assembly suppresses apoptosis and promotes cell survival under stress. Furthermore, hyperformation of SGs is frequently observed in various human cancers and accelerates tumor development and progression by reducing stress-induced damage of cancer cells. Therefore, they are of clinical importance. However, the precise mechanism underlying SG-mediated inhibition of apoptosis remains ill-defined. Here, using a proximity-labeling proteomic approach, we comprehensively analyzed SG-resident proteins and identified the executioner caspases, caspase-3 and -7, as SG components. We demonstrate that accumulation of caspase-3/7 into SGs is mediated by evolutionarily conserved amino acid residues within their large catalytic domains and inhibits caspase activities and consequent apoptosis induced by various stresses. Expression of an SG-localization-deficient caspase-3 mutant in cells largely counteracted the anti-apoptotic effect of SGs, whereas enforced relocalization of the caspase-3 mutant to SGs restored it. Thus, SG-mediated sequestration of executioner caspases is a mechanism underlying the broad cytoprotective function of SGs. Furthermore, using a mouse xenograft tumor model, we show that this mechanism prevents cancer cells from apoptosis in tumor tissues, thereby promoting cancer progression. Our results reveal the functional crosstalk between SG-mediated cell survival and caspase-mediated cell death signaling pathways and delineate a molecular mechanism that dictates cell-fate decisions under stress and promotes tumorigenesis.
Topics: Humans; Caspase 3; Caspases; Proteomics; Stress Granules; Cytoplasmic Granules; Apoptosis; Stress, Physiological
PubMed: 37119817
DOI: 10.1016/j.cub.2023.04.012 -
Cell Dec 2020Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins....
Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.
Topics: Activating Transcription Factor 4; Cytoplasmic Granules; Cytosol; HeLa Cells; Humans; Open Reading Frames; Protein Biosynthesis; RNA Transport; RNA, Messenger; Single Molecule Imaging; Stress, Physiological
PubMed: 33308477
DOI: 10.1016/j.cell.2020.11.010 -
Cell Death & Disease Jul 2023Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining...
Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining hypoxia-induced mitochondrial protein aggregation in C. elegans, we found that sublethal hypoxia, sodium azide, or heat shock-induced abundant ethidium bromide staining mitochondrial granules that preceded evidence of protein aggregation. Genetic manipulations that reduce cellular and organismal hypoxic death block the formation of these mitochondrial stress granules (mitoSG). Knockdown of mitochondrial nucleoid proteins also blocked the formation of mitoSG by a mechanism distinct from the mitochondrial unfolded protein response. Lack of the major mitochondrial matrix protease LONP-1 resulted in the constitutive formation of mitoSG without external stress. Ethidium bromide-staining RNA-containing mitochondrial granules were also observed in rat cardiomyocytes treated with sodium azide, a hypoxia mimetic. Mitochondrial stress granules are an early mitochondrial pathology controlled by LONP and the nucleoid, preceding hypoxia-induced protein aggregation.
Topics: Animals; Rats; Caenorhabditis elegans; Protein Aggregates; Ethidium; Sodium Azide; Stress Granules; Hypoxia; Mitochondrial Proteins
PubMed: 37468471
DOI: 10.1038/s41419-023-05988-6 -
Brain : a Journal of Neurology Jun 2023Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise...
Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders.
Topics: Humans; Mice; Animals; DNA Helicases; Heat-Shock Proteins; Protein Aggregates; Stress Granules; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; Spinocerebellar Ataxias; Ataxin-3; Mice, Transgenic; Machado-Joseph Disease
PubMed: 36511898
DOI: 10.1093/brain/awac473 -
Autophagy Jun 2023The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of...
The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage. Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame 3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1: TIA1 cytotoxic granule associated RNA binding protein.
Topics: Animals; Humans; DNA Helicases; Stress Granules; RNA Helicases; Poly-ADP-Ribose Binding Proteins; Proteomics; RNA Recognition Motif Proteins; Autophagy; COVID-19; SARS-CoV-2; TOR Serine-Threonine Kinases; Lysosomes; Cytoplasmic Granules; Mammals; Galectins
PubMed: 36394332
DOI: 10.1080/15548627.2022.2148900 -
Advanced Science (Weinheim,... Apr 2023Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that is suggested to be associated with hypoxia. This study is the first to identify...
Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that is suggested to be associated with hypoxia. This study is the first to identify a novel circular RNA (circRNA), circTBC1D14, whose expression is significantly upregulated in TNBC. The authors confirm that high circTBC1D14 expression is associated with a poor prognosis in patients with breast cancer. circTBC1D14-associated mass spectrometry and RNA-binding protein-related bioinformatics strategies indicate that FUS can interact with circTBC1D14, which can bind to the downstream flanking sequence of circTBC1D14 to induce cyclization. FUS is an essential biomarker associated with stress granules (SGs), and the authors find that hypoxic conditions can induce FUS-circTBC1D14-associated SG formation in the cytoplasm after modification by protein PRMT1. Subsequently, circTBC1D14 increases the stability of PRMT1 by inhibiting its K48-regulated polyubiquitination, leading to the upregulation of PRMT1 expression. In addition, FUS-circTBC1D14 SGs can initiate a cascade of SG-linked proteins to recognize and control the elimination of SGs by recruiting LAMP1 and enhancing lysosome-associated autophagy flux, thus contributing to the maintenance of cellular homeostasis and promoting tumor progression in TNBC. Overall, these findings reveal that circTBC1D14 is a potential prognostic indicator that can serve as a therapeutic target for TNBC treatment.
Topics: Humans; Amyotrophic Lateral Sclerosis; Triple Negative Breast Neoplasms; Stress Granules; Transcription Factors; Autophagy; Protein-Arginine N-Methyltransferases; Repressor Proteins; RNA-Binding Protein FUS
PubMed: 36806670
DOI: 10.1002/advs.202204988