Cellular Protein Synthesis Research Articles

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Engineering Cyborg Bacteria Through Intracellular Hydrogelation.

Natural and artificial cells are two common chassis in synthetic biology. Natural cells can perform complex tasks through synthetic genetic constructs, but their autonomous replication often causes safety concerns for biomedical applications. In contrast, artificial cells based on nonreplicating materials, albeit possessing reduced biochemical complexity, provide more defined and controllable functions. Here, for the first time, the authors create hybrid material-cell entities termed Cyborg Cells. To create Cyborg Cells, a synthetic polymer network is assembled inside each bacterium, rendering them incapable of dividing. Cyborg Cells preserve essential functions, including cellular metabolism, motility, protein synthesis, and compatibility with genetic circuits. Cyborg Cells also acquire new abilities to resist stressors that otherwise kill natural cells. Finally, the authorsdemonstrate thetherapeutic potential by showing invasion into cancer cells. This work establishes a new paradigm in cellular bioengineering by exploiting a combination of intracellular man-made polymers and their interaction with the protein networks of living cells.

Pojavnost mikotoksina u hrani i piću

This paper describes the mycotoxins produced by fungi from the genera Fusarium, Penicillium, Aspergillus, Claviceps and other types of molds, their characteristics and importance. Mycotoxins are secondary fungi metabolites that serve as a defense mechanism in stressful conditions. Several hundred mycotoxins have been identified so far, and the most significant in terms of danger to human health and animals are aflatoxins, ochratoxin A, patulin, fumonisin, zearalenone and nivalenol/deoxynivalenol produced by toxigenic fungi from the genera Penicillium, Aspergillus, Claviceps, Stachybotris and Fusarium. Moisture and temperature are two factors that have a crucial influence on the development of the fungus and the synthesis of toxins. It is estimated that approximately 25% of agricultural crops are infected with fungi. Consequently, foods can also be contaminated with mycotoxins. By treating the stored grain with various chemicals, and in recent years, using environmentally friendly fungicides, the synthesis of mycotoxins is being prevented and the development of mycotoxicogenic fungi is being controlled. The mycotoxicosis can occur directly through the consumption of infected food, inhalation and skin contact, or indirectly through animals that eat contaminated feed. Mycotoxins have a pathological effect primarily on liver. Some mycotoxins also interfere with cellular protein synthesis, causing hypersensitivity and extreme immunodeficiency. Despite mycotoxins being resilient and remaining stable while undergoing chemical and thermal food processing, methods such as applying good hygiene and production practices from field to table can reduce their occurence.

Ontogenes and Their Role in Cellular Construction

The genes referred to as ontogenes are responsible for conditional mutations. Based on the results of the research of conditional mutations in D. melanogaster, we attempt to figure out the biological role of ontogenes. We conclude that ontogenes in the process of individual development control the construction of the living organisms of cells (cellular construction), which comprises the induction of cell division, determination of division plane, and the location of daughter cells after the division is completed. The process of morphogenesis consists of cellular construction and protein synthesis. Protein synthesis is controlled by protein-coding (Mendelian) genes. Mendelian genes are switched on by ontogenes. In terms of the two-component genome composed of Mendelian genes and ontogenes, we consider 1) the concept of biological character; 2) interspecific incompatibility; 3) ontogenesis; 4) phylogenesis; and 5) mutagenesis. Ontogenes, which control cellular construction, possess the specific features unusual for Mendelian genes, namely, 1) the activity in germ line tissue; 2) remote interaction; and 3) activity in a compacted state (heterochromatization). These specific features of ontogenes suggest that unlike the Mendelian genes with their chemical activity, ontogenes possess another type of activity (biophysical) involving induction of an electromagnetic field.

Metabotropic glutamate receptor 1 alpha: a unique receptor variant with variable implications for Alzheimer's disease pathogenesis.

Metabotropic glutamate receptor 1 alpha: a unique receptor variant with variable implications for Alzheimer's disease pathogenesis.

Improving physiological relevance of cell culture: the possibilities, considerations, and future directions of the ex vivo coculture model.

In vitro models provide an important platform for the investigation of cellular growth and atrophy to inform, or extend mechanistic insights from, logistically challenging in vivo trials. Although these models allow for the identification of candidate mechanistic pathways, many models involve supraphysiological dosages, nonphysiological conditions, or experimental changes relating to individual proteins or receptors, all of which limit translation to human trials. To overcome these drawbacks, the use of ex vivo human plasma and serum has been used in cellular models to investigate changes in myotube hypertrophy, cellular protein synthesis, anabolic and catabolic markers in response to differing age, disease states, and nutrient status. However, there are currently no concurrent guidelines outlining the optimal methodology for this model. This review discusses the key methodological considerations surrounding the use of ex vivo plasma and serum with a focus in application to skeletal muscle cell lines (i.e., C2C12, L6, and LHCN-M2) and human primary skeletal muscle cells (HSMCs) as a means to investigate molecular signaling in models of atrophy and hypertrophy, alongside future directions.

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression.

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24°C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24°C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

An unusual mode of baseline translation adjusts cellular protein synthesis capacity to metabolic needs.

In all domains of life, mechanisms exist that adjust translational capacity to nutrient restriction and other growth constraints. The mammalian target of rapamycin (mTOR) regulates the synthesis of ribosomal proteins and translation factors in mammalian cells via phosphorylation of the La-related protein 1 (LARP1). In the present model of starvation-induced translational silencing, LARP1 targets mRNAs carrying a 5' terminal oligopyrimidine (5'TOP) motif to shift these into subpolysomal ribonucleoprotein particles. However, how these mRNAs would be protected from degradation and rapidly made available to restore translation capacity when needed remained enigmatic. Here, to address this, we employ gradient profiling by sequencing (Grad-seq) and monosome footprinting. Challenging the above model, we find that 5'TOP mRNAs, instead of being translationally silenced during starvation, undergo low baseline translation with reduced initiation rates. This mode of regulation ensures a stable 5'TOP mRNA population under starvation and allows fast reversibility of the translational repression.

Profiling the Site of Protein CoAlation and Coenzyme A Stabilization Interactions.

Coenzyme A (CoA) is a key cellular metabolite known for its diverse functions in metabolism and regulation of gene expression. CoA was recently shown to play an important antioxidant role under various cellular stress conditions by forming a disulfide bond with proteins, termed CoAlation. Using anti-CoA antibodies and liquid chromatography tandem mass spectrometry (LC-MS/MS) methodologies, CoAlated proteins were identified from various organisms/tissues/cell-lines under stress conditions. In this study, we integrated currently known CoAlated proteins into mammalian and bacterial datasets (CoAlomes), resulting in a total of 2093 CoAlated proteins (2862 CoAlation sites). Functional classification of these proteins showed that CoAlation is widespread among proteins involved in cellular metabolism, stress response and protein synthesis. Using 35 published CoAlated protein structures, we studied the stabilization interactions of each CoA segment (adenosine diphosphate (ADP) moiety and pantetheine tail) within the microenvironment of the modified cysteines. Alternating polar-non-polar residues, positively charged residues and hydrophobic interactions mainly stabilize the pantetheine tail, phosphate groups and the ADP moiety, respectively. A flexible nature of CoA is observed in examined structures, allowing it to adapt its conformation through interactions with residues surrounding the CoAlation site. Based on these findings, we propose three modes of CoA binding to proteins. Overall, this study summarizes currently available knowledge on CoAlated proteins, their functional distribution and CoA–protein stabilization interactions.

Modeling and computational characterization of a Xanthomonas sp. Hypothetical protein identifies a remote ortholog of Burkholderia lethal factor 1

Burkholderia Lethal Factor 1 (BLF1) is a deamidase first characterized in Burkholderia pseudomallei. This enzyme inhibits cellular protein synthesis by deamidating a glutamine residue to a glutamic acid in its target protein, the eukaryotic translation initiation factor 4 A (eIF4A). In this work, we present the characterization of a hypothetical protein from Xanthomonas sp. Leaf131 as the first report of a BLF1 family ortholog outside of the Burkholderia genus. Although standard sequence similarity searches such as BLAST were not able to detect the homology between the Xanthomonas sp. Leaf131 hypothetical protein sequence and BLF1, our computed structure model for the Xanthomonas sp. hypothetical protein revealed structural similarities with an RMSD of 2.7 Å/164 Cα atoms and a TM-score of 0.72 when superposed. Structural comparisons of the Xanthomonas model structure against BLF1 and Escherichia coli cytotoxic necrotizing factor 1 (CNF1) revealed that the conserved signature LXGC motif and putative catalytic residues are structurally aligned thus signifying a level of functional or mechanistic similarity. Protein-protein docking analysis and molecular dynamics simulations also demonstrated that eIF4A could still be a possible target substrate for deamidation by XLF1 as it is for BLF1. We therefore propose that this Xanthomonas hypothetical protein be renamed as Xanthomonas Lethal Factor 1 (XLF1). Our work also provides further evidence of the utility of programs such as AlphaFold in bridging the computational function annotation transfer gap despite very low sequence identities of under 20%. Communicated by Ramaswamy H. Sarma

Endoplasmic Reticulum Stress, Oxidative Stress, and Rheumatic Diseases.

Background: The endoplasmic reticulum (ER) is a multi-functional organelle responsible for cellular homeostasis, protein synthesis, folding and secretion. It has been increasingly recognized that the loss of ER homeostasis plays a central role in the development of autoimmune inflammatory disorders, such as rheumatic diseases. Purpose/Main contents: Here, we review current knowledge of the contribution of ER stress to the pathogenesis of rheumatic diseases, with a focus on rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We also review the interplay between protein folding and formation of reactive oxygen species (ROS), where ER stress induces oxidative stress (OS), which further aggravates the accumulation of misfolded proteins and oxidation, in a vicious cycle. Intervention studies targeting ER stress and oxidative stress in the context of rheumatic diseases are also reviewed. Conclusions: Loss of ER homeostasis is a significant factor in the pathogeneses of RA and SLE. Targeting ER stress, unfolded protein response (UPR) pathways and oxidative stress in these diseases both in vitro and in animal models have shown promising results and deserve further investigation.

Attenuated Duck Hepatitis A Virus Infection Is Associated With High mRNA Maintenance in Duckling Liver via m6A Modification.

Host translation is generally modulated by viral infection, including duck hepatitis A virus (DHAV) infection. Previously, we reported that cellular protein synthesis in a cell model of duck embryo fibroblasts is significantly inhibited by DHAV infection but not viral proteins, suggesting that an important viral-host interaction occurs at the translational level. In this study, we aim to further understand the impact of DHAV virulence on cellular N6-methyladenosine (m6A) modification, which is essential to a wide variety of RNA biological processes, such as mRNA stabilization and translation. Using m6A antibody-based immunoprecipitation, m6A-seq, and LC–MS/MS, we observed that m6A-modified mRNA exists in both virulent and attenuated DHAV-infected duckling livers. Importantly, m6A levels in mRNA were much higher in attenuated DHAV-infected livers compared with virulent DHAV-infected livers, suggesting virulence-dependent regulation of m6A modification. Analysis of modification motifs indicated that GAAGAAG is the most enriched motif. Combined m6A-seq and RNA-seq data analysis indicated a generally positive correlation between m6A and mRNA expression levels in DHAV-infected duckling livers. GO analysis of genes with decreased or increased m6A levels showed that these genes were enriched in various terms, including oxidation–reduction processes and antiviral immune responses. Collectively, our work reveals DHAV virulence-dependent coordination between m6A modification and mRNA expression in duckling livers.

STAGE-DEPENDENT RESPONSES OF VASCULAR AND PARENCHYMAL CELLS IN THE HYPERTENSIVE RAT BRAIN

Objective: Long-term hypertension doubles all-cause dementia risk in aging by structural and functional alterations of the cerebral microvasculature and its surrounding tissue. However, therapeutical blood pressure lowering by itself is limited in its effectiveness depending on the progression of structural vessel remodeling, microvascular dysfunction and parenchymal tissue damage. Design and method: Therefore, the aim of this study was to investigate pathophysiological processes in different stages of hypertension. We explored acute, early- and late-chronic hypertension effects in the frontal brain of hypertensive rats, applying behavioral tests, histology, immunofluorescence, FACS and vascular/neural tissue RNA sequencing. Results: In response to initial increasing blood pressure and in absence of vascular pathology, microglia cells were found in close proximity to blood vessels and with morphological features of activation. Early chronic hypertensive animals showed frontal brain specific behavioral deficits, blood-brain-barrier leakage, leukocyte immigration, loss of glial reactivity and altered pathways related to cellular energy supply, protein synthesis and catabolism. In late chronic hypertension behavioral deficits and glial reactivity loss persisted. We also observed active angiogenesis, vascular fibroblast-related collagen aggregation and pathways related to decreased responsiveness to nutrients and increased metabolic demand. Additionally, we identified late-chronic vascular accumulation of insulin-like growth factor 1 (IGF1) binding proteins in brains of rats and humans with hypertensive cerebral small vessel disease, possibly attenuating protective IGF1 signaling in the cerebral microvasculature. Conclusions: Our results point towards stage-dependent reactions of vascular, glial, immune and neuronal cells in response to hypertension. Future studies have to verify, whether these mechanisms are valid in humans as well and whether they might be targeted to attenuate small vessel disease progression.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1’s shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1’s explicit role in blocking the IFN response.

Upregulation of tRNA-Ser-AGA-2-1 Promotes Malignant Behavior in Normal Bronchial Cells

Serine tRNAs (tRNASer) are frequently overexpressed in tumors and associated with poor prognosis and increased risk of recurrence in breast cancer. Impairment of tRNA biogenesis and abundance also impacts proteome homeostasis, and activates protein quality control systems. Herein, we aimed at testing whether increasing tRNASer abundance could foster tumor establishment through activation of the UPR. In order to do so, firstly we confirmed that the expression of tRNA-Ser-AGA-2-1 [hereafter tRNASer(AGA)] was upregulated by 1.79-fold in Stage I NSCLC tumors when compared to normal adjacent tissue. To study the impact of tRNASer(AGA) in early stage tumorigenesis, we induced its upregulation in a non-tumoral bronchial cell line, BEAS-2B. Upregulation of this tRNA increased cellular proliferation and protein synthesis rate, driven by eIF2α dephosphorylation and ATF4 activation downstream of PERK signaling. Futhermore, tRNASer(AGA) enhanced transformation potential in vitro, and promoted the establishment of slow growing tumors with aggressive features in nude mice. Our work highlights the importance of studying tRNA deregulation on early stage tumorigenesis, as they may be potential malignancy and aggressiveness biomarkers.

Rapid and quantitative evaluation of vaccinia virus-induced host shutoff using newly generated cell lines stably expressing secreted Gaussia luciferase.

Host shutoff, characterized by a global decline of cellular protein synthesis, is commonly observed in many viral infections, including vaccinia virus (VACV). Classic methods measuring host shutoff include the use of radioactive or nonradioactive probes to label newly synthesized proteins followed by radioautography or sodium dodecyl-sulfate polyacrylamide gel electrophoresisto resolve the proteins for follow-up detection. Although theseare highly reliable methods, they are time- and labor-consuming. Here, we generated two cell lines stably expressing secreted Gaussia luciferase. These reporter cells allow rapid, quantitative, and consecutive monitoring of host shutoff from a single infection sample. We evaluated host shutoff induced by wild-type and various mutant VACVs using the reporter cell lines. The results validated the utilities of the reporter cells and quantitatively characterized VACV-induced host shutoff at different stages of replication. Notably, the results also indicated additional major unidentified VACV shutoff factors. Our study provides new tools to study host shutoff. The reporter cells are also suitable for high throughput settings and rapid testing of clinically isolated viruses. In combination with classical methods, these tools will greatly facilitate understanding of virus-induced host shutoff, and protein synthesis shutoff caused by other physiologically relevant stresses.

Extracellular N6 -isopentenyladenosine (i6A) addition induces cotranscriptional i6A incorporation into ribosomal RNAs.

N6 -isopentenyladenosine (i6A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i6A has yet to be identified. Here we show that i6A addition to cell culture medium results in i6A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i6A was predominantly incorporated into 18S and 28S rRNAs, and i6A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i6A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i6A-modifying enzyme. These results indicate that upon cellular uptake of i6A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i6A concentrations, the cytotoxic effect of i6A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i6A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i6A in the context of intracellular nucleic acid anabolism and suggests investigation of i6A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.

The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network.

Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.

The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (−45%) and NAFLD (−35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.

Abstract TMP116: Pharmacological Inhibition Of Endoplasmic Reticulum Stress Prevents Intracranial Aneurysm Rupture

Background: The endoplasmic reticulum (ER) is responsible for cellular protein synthesis and folding. Cellular stimuli that perturb ER homeostasis create an imbalance between the protein-folding load and capacity of ER, causing unfolded or misfolded proteins to accumulate in the ER lumen, known as ER stress. Recent studies suggested that ER stress plays significant roles in the pathogenesis of inflammatory vascular diseases. Since inflammation is emerging as a vital component of the pathophysiology of intracranial aneurysms, we hypothesized that ER stress promotes the development of aneurysm rupture by inducing sustained vascular wall inflammation. We tested this hypothesis utilizing pharmacological approaches in mice. Methods: We used 10-week-old male C57BL/6J mice and induced intracranial aneurysms by combining an elastase injection and hypertension. We tested the effects of an ER stress activator (Tunicamycin) and inhibitor (4-phenylbutyric acid, 4-PBA) on the development of aneurysmal rupture. In addition, we assessed the roles of ER stress induced via deactivation of epidermal growth factor receptor (EGFR) by Erlotinib on aneurysm rupture. Results: The pharmacological inhibition of ER stress significantly decreased the rupture rate (P&lt;0.05, Fig. A). Meanwhile, there was a trend for the ER stress activator to increase the rupture rate (P=0.17, Fig.B). Moreover, the pharmacological inhibition of the EGFR pathway with Erlotinib significantly reduced the rupture rate (P&lt;0.05, Fig. C). In addition, Erlotinib treatment reduced the mRNA expression of ER stress markers GRP78 and CHOP (Fig.D-E). Conclusions: The pharmacological inhibition of ER stress decreased aneurysmal rupture in a mouse model of intracranial aneurysm. In addition, inhibition of EGFR decreased aneurysm rupture through the reduction of ER stress. Our findings suggest that ER stress and its upstream EGFR pathway may serve as novel therapeutic targets for preventing aneurysm rupture.