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.

A Unique ISR Program Determines Cellular Responses to Chronic Stress.

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

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.

The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress.

The ribosomal uL10 protein, formerly known as P0, is an essential element of the ribosomal GTPase-associated center responsible for the interplay with translational factors during various stages of protein synthesis. In eukaryotic cells, uL10 binds two P1/P2 protein heterodimers to form a pentameric P-stalk, described as uL10-(P1-P2)2, which represents the functional form of these proteins on translating ribosomes. Unlike most ribosomal proteins, which are incorporated into pre-ribosomal particles during early steps of ribosome biogenesis in the nucleus, P-stalk proteins are attached to the 60S subunit in the cytoplasm. Although the primary role of the P-stalk is related to the process of translation, other extraribosomal functions of its constituents have been proposed, especially for the uL10 protein; however, the list of its activities beyond the ribosome is still an open question. Here, by the combination of biochemical and advanced fluorescence microscopy techniques, we demonstrate that upon nucleolar stress induction the uL10 protein accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein. Importantly, using a novel approach, FRAP-AC (FRAP after photoConversion), we have shown that the ribosome-free pool of uL10 represents a population of proteins released from pre-existing ribosomes. Taken together, our data indicate that the presence of uL10 on the ribosomes is affected in stressed cells, thus it might be considered as a regulatory element responding to environmental fluctuations.

Coordinated Regulation of the Neutral Amino Acid Transporter SNAT2 and the Protein Phosphatase Subunit GADD34 Promotes Adaptation to Increased Extracellular Osmolarity.

Cells respond to shrinkage induced by increased extracellular osmolarity via programmed changes in gene transcription and mRNA translation. The immediate response to this stress includes the induction of expression of the neutral amino acid transporter SNAT2. Increased SNAT2-mediated uptake of neutral amino acids is an essential adaptive mechanism for restoring cell volume. In contrast, stress-induced phosphorylation of the α subunit of the translation initiation factor eIF2 (eIF2α) can promote apoptosis. Here we show that the response to mild hyperosmotic stress involves regulation of the phosphorylation of eIF2α by increased levels of GADD34, a regulatory subunit of protein phosphatase 1 (PP1). The induction of GADD34 was dependent on transcriptional control by the c-Jun-binding cAMP response element in the GADD34 gene promoter and posttranscriptional stabilization of its mRNA. This mechanism differs from the regulation of GADD34 expression by other stresses that involve activating transcription factor 4 (ATF4). ATF4 was not translated during hyperosmotic stress despite an increase in eIF2α phosphorylation. The SNAT2-mediated increase in amino acid uptake was enhanced by increased GADD34 levels in a manner involving decreased eIF2α phosphorylation. It is proposed that the induction of the SNAT2/GADD34 axis enhances cell survival by promoting the immediate adaptive response to stress.

In vivo formation of Plasmodium falciparum ribosomal stalk - a unique mode of assembly without stable heterodimeric intermediates.

BACKGROUND: The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins. METHODS: We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods. RESULTS: All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)2form represents the most favorable architecture for parasite P-proteins. CONCLUSION: The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1-P2 heterodimeric intermediate. GENERAL SIGNIFICANCE: On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.

Metabolic adaptation of skeletal muscle to hyperammonemia drives the beneficial effects of l-leucine in cirrhosis.

BACKGROUND & AIMS: Increased skeletal muscle ammonia uptake with loss of muscle mass adversely affects clinical outcomes in cirrhosis. Hyperammonemia causes reduced protein synthesis and sarcopenia but the cellular responses to impaired proteostasis and molecular mechanism of l-leucine induced adaptation to ammonia induced stress were determined. METHODS: Response to activation of amino acid deficiency sensor, GCN2, in the skeletal muscle from cirrhotic patients and the portacaval anastomosis (PCA) rat were quantified. During hyperammonemia and l-leucine supplementation, protein synthesis, phosphorylation of eIF2α, mTORC1 signaling, l-leucine transport and response to l-leucine supplementation were quantified. Adaptation to cellular stress via ATF4 and its target GADD34 were also determined. RESULTS: Activation of the eIF2α kinase GCN2 and impaired mTORC1 signaling were observed in skeletal muscle from cirrhotic patients and PCA rats. Ammonia activated GCN2 mediated eIF2α phosphorylation (eIF2α-P) and impaired mTORC1 signaling that inhibit protein synthesis in myotubes and MEFs. Adaptation to ammonia induced stress did not involve translational reprogramming by activation transcription factor 4 (ATF4) dependent induction of the eIF2α-P phosphatase subunit GADD34. Instead, ammonia increased expression of the leucine/glutamine exchanger SLC7A5, l-leucine uptake and intracellular l-leucine levels, the latter not being sufficient to rescue the inhibition of protein synthesis, due to potentially enhanced mitochondrial sequestration of l-leucine. l-leucine supplementation rescued protein synthesis inhibition caused by hyperammonemia. CONCLUSIONS: Response to hyperammonemia is reminiscent of the cellular response to amino acid starvation, but lacks the adaptive ATF4 dependent integrated stress response (ISR). Instead, hyperammonemia-induced l-leucine uptake was an adaptive response to the GCN2-mediated decreased protein synthesis. LAY SUMMARY: Sarcopenia or skeletal muscle loss is the most frequent complication in cirrhosis but there are no treatments because the cause(s) of muscle loss in liver disease are not known. Results from laboratory experiments in animals and muscle cells were validated in human patients with cirrhosis to show that ammonia plays a key role in causing muscle loss in patients with cirrhosis. We identified a novel stress response to ammonia in the muscle that decreases muscle protein content that can be reversed by supplementation with the amino acid l-leucine.

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.

A self-defeating anabolic program leads to ß-cell apoptosis in endoplasmic reticulum stress-induced diabetes via regulation of amino acid flux.

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing ß-cells in type 2 diabetes mellitus. ß-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in ß-cells in vivo. Paradoxically, chronic ER stress in ß-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in ß-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Genome-wide identification and quantitative analysis of cleaved tRNA fragments induced by cellular stress.

Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation-deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNA(Met). We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin-mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders.

Solution structure of the natively assembled yeast ribosomal stalk determined by small-angle X-ray scattering.

The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.

Identification of a novel alternatively spliced isoform of the ribosomal uL10 protein.

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10ß. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases - trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10ß protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10ß is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10ß undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10ß, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.

Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR.

The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

Pentameric organization of the ribosomal stalk accelerates recruitment of ricin a chain to the ribosome for depurination.

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.

Biophysical properties of the eukaryotic ribosomal stalk.

The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.

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.

Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2 of Plasmodium falciparum in a Mouse Model.

Malaria remains one the most infectious and destructive protozoan diseases worldwide. Plasmodium falciparum, a protozoan parasite with a complex life cycle and high genetic variability responsible for the difficulties in vaccine development, is implicated in most malaria-related deaths. In the course of study, we prepared a set of antigens based on P-proteins from P. falciparum and determined their immunogenicity in an in vivo assay on a mouse model. The pentameric complex P0-(P1-P2)2 was prepared along with individual P1, P2, and P0 antigens. We determined the level of cellular- and humoral-type immunological response followed by development of specific immunological memory. We have shown that the number of Tc cells increased significantly after the first immunization with P2 and after the second immunization with P1 and P0-(P1-P2)2, which highly correlated with the number of Th1 cells. P0 appeared as a poor inducer of cellular response. After the third boost with P1, P2, or P0-(P1-P2)2, the initially high cellular response dropped to the control level accompanied by elevation of the number of activated Treg cells and a high level of suppressive TGF-ß. Subsequently, the humoral response against the examined antigens was activated. Although the titers of specific IgG were increasing during the course of immunization for all antigens used, P2 and P0-(P1-P2)2 were found to be significantly stronger than P1 and P0. A positive correlation between the Th2 cell abundance and the level of IL-10 was observed exclusively after immunization with P0-(P1-P2)2. An in vitro exposure of spleen lymphocytes from the immunized mice especially to the P1, P2, and P0-(P1-P2)2 protein caused 2-3-fold higher cell proliferation than that in the case of lymphocytes from the nonimmunized animals, suggesting development of immune memory. Our results demonstrate for the first time that the native-like P-protein pentameric complex represents much stronger immune potential than individual P-antigens.

GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells.

GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome.

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.