Cytoplasmic Stress Response Research Articles

Nucleo-cytoplasmic distribution of SAP18 reveals its dual function in splicing regulation and heat stress response in Arabidopsis

The dynamic shuttling of proteins between the nucleus and cytoplasm orchestrates vital functions in eukaryotes. Here, we unveil multifaceted functions of Arabidopsis Sin3-associated protein 18 kDa (SAP18) in regulating development and heat stress tolerance. Proteomic analysis demonstrated that SAP18 is a core component of the nuclear Apoptosis- and Splicing-Associated Protein (ASAP) complex in Arabidopsis, contributing to the precise splicing of genes associated with leaf development. Genetic analysis further confirmed SAP18's critical role in different developmental processes as part of the ASAP complex, including leaf morphogenesis and flowering time. Interestingly, upon heat shock SAP18 translocates from the nucleus to cytoplasmic stress granules and processing bodies. The heat-sensitive phenotype of SAP18 loss-of-function mutant revealed its novel role in plant thermoprotection. Our findings significantly expand our understanding of SAP18 relevance for plant growth, linking nuclear splicing with cytoplasmic stress responses, and providing new perspectives for future exploration of plant thermotolerance mechanisms.

DEAD box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress

The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1’s RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.

Plasticity of nuclear and cytoplasmic stress responses of RNA-binding proteins.

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.

Integrating mitochondrial translation into the cellular context.

Mitochondrial-encoded subunits of the oxidative phosphorylation system assemble with nuclear-encoded subunits into enzymatic complexes. Recent findings showed that mitochondrial translation is linked to other mitochondrial functions, as well as to cellular processes. The supply of mitochondrial-encoded proteins is coordinated by the coupling of mitochondrial protein synthesis with assembly of respiratory chain complexes. MicroRNAs imported from the cytoplasm into mitochondria were, surprisingly, found to act as regulators of mitochondrial translation. In turn, translation in mitochondria controls cellular proliferation, and mitochondrial ribosomal subunits contribute to the cytoplasmic stress response. Thus, translation in mitochondria is apparently integrated into cellular processes.

PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1α-mediated unfolded protein response

SummaryPoly(ADP-ribose) polymerases (PARPs) (also known as ADP-ribosyl transferase D proteins) modify acceptor proteins with ADP-ribose modifications of varying length (reviewed in refs 1–3). PARPs regulate key stress response pathways, including DNA damage repair and the cytoplasmic stress response2,3,4,5,6. Here, we show that PARPs also regulate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). Human PARP16/ARTD15 is a tail-anchored ER transmembrane protein required for activation of the functionally related ER stress sensors PERK and IRE1α during the UPR. The third identified ER stress sensor, ATF6, is not regulated by PARP16. Similar to other PARPs that function during stress, PARP16 enzymatic activity is up-regulated during ER stress when it (ADP-ribosyl)ates itself, PERK and IRE1α. (ADP-ribosyl)ation by PARP16 is sufficient for activating PERK and IRE1α in the absence of ER stress, and is required for PERK and IRE1 α activation during the UPR. Modification of PERK and IRE1α by PARP16 increases their kinase activities and the endonuclease activity of IRE1α. Interestingly, the C-terminal luminal tail of PARP16 is required for PARP16 function during ER stress, suggesting that it transduces stress signals to the cytoplasmic PARP catalytic domain.

Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondii

A key problem in the treatment of numerous pathogenic eukaryotes centers on their development into latent forms during stress. For example, the opportunistic protist Toxoplasma gondii converts to latent cysts (bradyzoites) responsible for recrudescence of disease. We report that Toxoplasma eukaryotic initiation factor-2alpha (TgIF2alpha) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2alpha remains phosphorylated in bradyzoites, explaining how these cells maintain their quiescent state. Furthermore, we have characterized novel eIF2 kinases; one in the endoplasmic reticulum and a likely regulator of the unfolded protein response (TgIF2K-A) and another that is a probable responder to cytoplasmic stresses (TgIF2K-B). Significantly, our data suggest that 1) the regulation of protein translation through eIF2 kinases is associated with development, 2) eIF2alpha phosphorylation is employed by cells to maintain a latent state, and 3) endoplasmic reticulum and cytoplasmic stress responses evolved in eukaryotic cells before the early diverging Apicomplexa. Given its importance to pathogenesis, eIF2 kinase-mediated stress responses may provide opportunities for novel therapeutics.

The Extracytoplasmic Stress Factor, σE, Is Required to Maintain Cell Envelope Integrity in Escherichia coli

Extracytoplasmic function or ECF sigma factors are the most abundant class of alternative sigma factors in bacteria. Members of the rpoE subclass of ECF sigma factors are implicated in sensing stress in the cell envelope of Gram-negative bacteria and are required for virulence in many pathogens. The best-studied member of this family is rpoE from Escherichia coli, encoding the σE protein. σE has been well studied for its role in combating extracytoplasmic stress, and the members of its regulon have been largely defined. σE is required for viability of E. coli, yet none of the studies to date explain why σE is essential in seemingly unstressed cells. In this work we investigate the essential role of σE in E. coli by analyzing the phenotypes associated with loss of σE activity and isolating suppressors that allow cells to live in the absence of σE. We demonstrate that when σE is inhibited, cell envelope stress increases and envelope integrity is lost. Many cells lyse and some develop blebs containing cytoplasmic material along their sides. To better understand the connection between transcription by σE and cell envelope integrity, we identified two multicopy suppressors of the essentiality of σE, ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the σE regulon. Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response. Our results are consistent with a model in which overexpression of ptsN reduces stress in the cell envelope, thereby promoting survival in the absence of σE.

Hfq Modulates the σ E -Mediated Envelope Stress Response and the σ 32 -Mediated Cytoplasmic Stress Response in Escherichia coli

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the sigma(S)-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the sigma(E)- and sigma(32)-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the sigma(E)-mediated envelope stress response and are defective in sigma(E)-mediated repression of outer membrane proteins. We also show that the sigma(32)-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the "long-term adaptation" of sigma(32) to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli.

Expression and Assembly of a Functional Type IV Secretion System Elicit Extracytoplasmic and Cytoplasmic Stress Responses inEscherichia coli

Conditions perturbing protein homeostasis are known to induce cellular stress responses in prokaryotes and eukaryotes. Here we show for the first time that expression and assembly of a functional type IV secretion (T4S) machinery elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. After induction of T4S genes by a nutritional upshift and assembly of functional DNA transporters encoded by plasmid R1-16, host cells activated the CpxAR envelope stress signaling system, as revealed by induction or repression of downstream targets of the CpxR response regulator. Furthermore, we observed elevated transcript levels of cytoplasmic stress genes, such as groESL, with a concomitant increase of sigma(32) protein levels in cells expressing T4S genes. A traA null mutant of plasmid R1-16, which lacks the functional gene encoding the major pilus protein pilin, showed distinctly reduced stress responses. These results corroborated our conclusion that the activation of bacterial stress networks was dependent on the presence of functional T4S machinery. Additionally, we detected increased transcription from the rpoHp(1) promoter in the presence of an active T4S system. Stimulation of rpoHp(1) was dependent on the presence of CpxR, suggesting a hitherto undocumented link between CpxAR and sigma(32)-regulated stress networks.

Redox Regulatory Mechanisms in Cellular Stress Responses

Reactive oxygen species are produced in a highly localized and specific pattern in biological stress responses. The present review examines the redox regulatory aspects of a number of molecular stress response mechanisms in both prokaryotes and eukaryotes. The present review provides examples representing both the cytoplasmic stress response, often studied as the heat shock response, as well as the stress response of the endoplasmic reticulum, known as the unfolded protein response. The examples have been selected to illustrate the variety of ways that redox signals mediate and affect stress responses. Redox regulatory mechanisms are intricately embedded in both the cytoplasmic and endoplasmic reticulum stress responses at multiple levels. Many different stimuli, both internal and external, activate endogenous production of reactive oxygen species as a necessary part of the intracellular communication system that activates stress responses.

Stimulation of N-Linked Glycosylation and Lipid-Linked Oligosaccharide Synthesis by Stress Responses in Metazoan Cells

ABSTRACTEndoplasmic reticulum (ER) stress responses comprising the unfolded protein response (UPR) are activated by conditions that disrupt folding and assembly of proteins inside the ER lumenal compartment. Conditions known to be proximal triggers of the UPR include saturation of chaperones with misfolded protein, redox imbalance, disruption of Ca2+ levels, interference with N-linked glycosylation, and failure to dispose of terminally misfolded proteins. Potentially, ER stress responses can reprogram cells to correct all of these problems and thereby restore ER function to normal. This article will review literature on stimulation of N-linked glycosylation by ER stress responses, focusing on metazoan systems. The mechanisms involved will be contrasted with those mediating stimulation of N-linked glycosylation by cytoplasmic stress responses. This information will interest readers who study the biological roles of stress responses, the functions of N-linked glycans, and potential strategies for treatment of genetic disorders of N-linked glycosylation.

Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli.

We report here the transcriptome analyses of highly expressed genes that are subject to catabolite repression or activation mediated by the cyclic AMP receptor protein (Crp). The results reveal that many operons encoding enzymes of central carbon metabolic pathways (e.g., Krebs cycle enzymes), as well as transporters and enzymes that initiate carbon metabolism, are subject to direct Crp-mediated catabolite repression. By contrast, few enzyme-encoding genes (direct regulation) but many ribosomal protein- and tRNA-encoding genes (indirect regulation) are subject to Crp-dependent glucose activation. Additionally, Crp mediates strong indirect catabolite repression of many cytoplasmic stress response proteins, including the major chaperone proteins, five ATP-dependent protease complexes, and several cold and heat shock proteins. These results were confirmed by (i) phenotypic analyses, (ii) real-time PCR studies, (iii) reporter gene fusion assays, and (iv) previously published reports about representative genes. The results serve to define and extend our appreciation of the Crp regulon.

Evolutionary changes in heat-inducible gene expression in lines of Escherichia coli adapted to high temperature.

The involvement of heat-inducible genes, including the heat-shock genes, in the acute response to temperature stress is well established. However, their importance in genetic adaptation to long-term temperature stress is less clear. Here we use high-density arrays to examine changes in expression for 35 heat-inducible genes in three independent lines of Escherichia coli that evolved at high temperature (41.5 degrees C) for 2,000 generations. These lines exhibited significant changes in heat-inducible gene expression relative to their ancestor, including parallel changes in fkpA, gapA, and hslT. As a group, the heat-inducible genes were significantly more likely than noncandidate genes to have evolved changes in expression. Genes encoding molecular chaperones and ATP-dependent proteases, key components of the cytoplasmic stress response, exhibit relatively little expression change; whereas genes with periplasmic functions exhibit significant expression changes suggesting a key role for the extracytoplasmic stress response in the adaptation to high temperature. Following acclimation at 41.5 degrees C, two of the three lines exhibited significantly improved survival at 50 degrees C, indicating changes in inducible thermotolerance. Thus evolution at high temperature led to significant changes at the molecular level in heat-inducible gene expression and at the organismal level in inducible thermotolerance and fitness.

Regulation of the dolichol pathway in human fibroblasts by the endoplasmic reticulum unfolded protein response.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells triggers the unfolded protein response (UPR), which activates transcription of several genes encoding ER chaperones and folding enzymes. This study reports that conversion of dolichol-linked Man(2-5)GlcNAc(2) intermediates into mature Glc(3)Man(9)GlcNAc(2) oligosaccharides in primary human adult dermal fibroblasts is also stimulated by the UPR. This stimulation was not evident in several immortal cell lines and did not require a cytoplasmic stress response. Inhibition of dolichol-linked Glc(3)Man(9)GlcNAc(2) synthesis by glucose deprivation could be counteracted by the UPR, improving the transfer of Glc(3)Man(9)GlcNAc(2) to asparagine residues on nascent polypeptides. Glycosidic processing of asparagine-linked Glc(3)Man(9)GlcNAc(2) in the ER leads to the production of monoglucosylated oligosaccharides that promote interaction with the lectin chaperones calreticulin and calnexin. Thus, control of the dolichol-linked Glc(3)Man(9)GlcNAc(2) supply gives the UPR the potential to maintain efficient protein folding in the ER without new synthesis of chaperones or folding enzymes.

Regulation of Translational Initiation during Cellular Responses to Stress

Chemicals and conditions that damage proteins, promote protein misfolding, or inhibit protein processing trigger the onset of protective homeostatic mechanisms resulting in "stress responses" in mammalian cells. Included in these responses are an acute inhibition of mRNA translation at the initiation step, a subsequent induction of various protein chaperones, and the recovery of mRNA translation. Separate, but closely related, stress response systems exist for the endoplasmic reticulum (ER), relating to the induction of specific "glucose-regulated proteins" (GRPs), and for the cytoplasm, pertaining to the induction of the "heat shock proteins" (HSPs). Activators of the ER stress response system, including Ca(2+)-mobilizing and thiol-reducing agents, are discussed and compared to activators of the cytoplasmic stress system, such as arsenite, heavy metal cations, and oxidants. An emerging integrative literature is reviewed that relates protein chaperones associated with cellular stress response systems to the coordinate regulation of translational initiation and protein processing. Background information is presented describing the roles of protein chaperones in the ER and cytoplasmic stress response systems and the relationships of chaperones and protein processing to the regulation of mRNA translation. The role of chaperones in regulating eIF-2 alpha kinase activities, eIF-2 cycling, and ribosomal loading on mRNA is emphasized. The putative role of GRP78 in coupling rates of translation to processing is modeled, and functional relationships between the HSP and GRP chaperone systems are discussed.