Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation.

The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.

Cells are sensitive to changes in their environment. For example, maintaining normal salt levels in the blood, also called tonicity, is essential for the health of individual cells and the organism as a whole. Tonicity controls the movement of water in and out of the cell: high levels of salt inside the cell draw water in, while high levels of salt outside the cell draw water out. If salt levels in the environment surrounding the cells become too high, too much water will be drawn out, causing the cells to shrink. Changes in tonicity can cause the cell to become stressed. Initially, cells adapt to this stress by switching on sets of genes that help restore fluid balance and allow the cell to regain its normal shape and size. If the increase in tonicity exceeds tolerable stress levels and harms the cell, this initiates an inflammatory response which ultimately leads to cell death. However, it remained unclear how cells switch from adapting to responding with inflammation. Now, Farabaugh et al. have used an experimental system which mimics high salt to identify the mechanism that allows cells to switch between these two responses. The experiments showed that when salt levels are too high, cells switch on a stress sensing protein called PACT, which activates another protein called PKR. When PACT was deleted from mouse cells, this led to a decrease in the activity of inflammatory genes, and prevented the cells from self-destructing. Other proteins that are involved in the adaptive and inflammatory response are the NF-κB family of proteins and TonEBP. Farabaugh et al. found that under low intensity stress, when salt levels outside the cell are slightly too high, a family member of NF-κB works with TonEBP to switch on adaptive genes. But, if salt levels continue to rise, PACT activates and turns on PKR. This blocks the interaction between NF-κB and TonEBP, allowing another family member of NF-κB to interact with TonEBP instead. This switches the adaptive response off and the inflammatory response on. There are many diseases that involve changes in tonicity, including diabetes, cancer, inflammatory bowel disease, and dry eye syndrome. Understanding the proteins involved in the adaptive and inflammatory response could lead to the development of drugs that help to protect cells from stress-induced damage.

Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1ß. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer.

Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to α-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110α upregulates GPT2 gene expression through an AKT-independent, PDK1-RSK2-ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not with WT PIK3CA. Together, these data establish oncogenic PIK3CA mutations as a cause of glutamine dependency in CRCs and suggest that targeting glutamine metabolism may be an effective approach to treat CRC patients harbouring PIK3CA mutations.

Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation.

c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic ß cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic ß cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.

BACKGROUND & AIMS: Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS: Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS: CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, the products of which heterodimerize to form an amino acid transporter in HT-29 cells after bacterial infection; levels of SLC7A5-SLC3A2 were increased in tissues from patients with UC compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5-SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3-deficient cells. CONCLUSIONS: CAP-D3 down-regulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2α.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.

ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.

Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.

Structural characterization of the ribosomal P1A-P2B protein dimer by small-angle X-ray scattering and NMR spectroscopy.

The five ribosomal P-proteins, denoted P0-(P1-P2)2, constitute the stalk structure of the large subunit of eukaryotic ribosomes. In the yeast Saccharomyces cerevisiae, the group of P1 and P2 proteins is differentiated into subgroups that form two separate P1A-P2B and P1B-P2A heterodimers on the stalk. So far, structural studies on the P-proteins have not yielded any satisfactory information using either X-ray crystallography or NMR spectroscopy, and the structures of the ribosomal stalk and its individual constituents remain obscure. Here we outline a first, coarse-grained view of the P1A-P2B solution structure obtained by a combination of small-angle X-ray scattering and heteronuclear NMR spectroscopy. The complex has an elongated shape with a length of 10 nm and a cross section of approximately 2.5 nm. 15N NMR relaxation measurements establish that roughly 30% of the residues are present in highly flexible segments, which belong primarily to the linker region and the C-terminal part of the polypeptide chain. Secondary structure predictions and NMR chemical shift analysis, together with previous results from CD spectroscopy, indicate that the structured regions involve alpha-helices. NMR relaxation data further suggest that several helices are arranged in a nearly parallel or antiparallel topology. These results provide the first structural comparison between eukaryotic P1 and P2 proteins and the prokaryotic L12 counterpart, revealing considerable differences in their overall shapes, despite similar functional roles and similar oligomeric arrangements. These results present for the first time a view of the structure of the eukaryotic stalk constituents, which is the only domain of the eukaryotic ribosome that has escaped successful structural characterization.