Cellular Protein Synthesis Research Articles

Review of Ribosome Interactions with SARS-CoV-2 and COVID-19 mRNA Vaccine.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causing pathogen of the unprecedented global Coronavirus Disease 19 (COVID-19) pandemic. Upon infection, the virus manipulates host cellular machinery and ribosomes to synthesize its own proteins for successful replication and to facilitate further infection. SARS-CoV-2 executes a multi-faceted hijacking of the host mRNA translation and cellular protein synthesis. Viral nonstructural proteins (NSPs) interact with a range of different ribosomal states and interfere with mRNA translation. Concurrent mutations on NSPs and spike proteins contribute to the epidemiological success of variants of concern (VOCs). The interactions between ribosomes and SARS-CoV-2 represent attractive targets for the development of antiviral therapeutics and vaccines. Recently approved COVID-19 mRNA vaccines also utilize the cellular machinery, to produce antigens and trigger immune responses. The design features of the mRNA vaccines are critical to efficient mRNA translation in ribosomes, and are directly related to the vaccine’s efficacy, safety, and immunogenicity. This review describes recent knowledge of how the SARS-CoV-2 virus’ genomic characteristics interfere with ribosomal function and mRNA translation. In addition, we discuss the current learning of the design features of mRNA vaccines and their impacts on translational activity in ribosomes. The understanding of ribosomal interactions with the virus and mRNA vaccines offers the foundation for antiviral therapeutic discovery and continuous mRNA vaccine optimization to lower the dose, to increase durability and/or to reduce adverse effects.

がん細胞におけるLAT1のアミノ酸の取込みへの寄与とLAT1阻害薬によるタンパク質合成抑制効果

がん細胞におけるLAT1のアミノ酸の取込みへの寄与とLAT1阻害薬によるタンパク質合成抑制効果

Robust T cell activation requires an eIF3-driven burst in T cell receptor translation.

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here, we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3'-untranslated regions (3'-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3'-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

Xenobiotic metabolism and its physiological consequences in high-Antarctic Notothenioid fishes

The Antarctic ecosystem is progressively exposed to anthropogenic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). So far, it is largely unknown if PAHs leave a mark in the physiology of high-Antarctic fish. We approached this issue via two avenues: first, we examined the functional response of the aryl hydrocarbon receptor (Ahr), which is a molecular initiating event of many toxic effects of PAHs in biota. Chionodraco hamatus and Trematomus loennbergii served as representatives for high-Antarctic Notothenioids, and Atlantic cod, Gadus morhua as non-polar reference species. We sequenced and cloned the Ahr ligand binding domain (LBD) of the Notothenioids and deployed a GAL4-based luciferase reporter gene assay expressing the Ahr LBD. Benzo[a]pyrene (BaP), beta-naphthoflavone and chrysene were used as ligands for the reporter gene assay. Second, we investigated the energetic costs of Ahr activation in isolated liver cells of the Notothenioids during acute, non-cytotoxic BaP exposure. In the reporter assay, the Ahr LBD of Atlantic cod and the Antarctic Notothenioids were activated by the ligands tested herein. In the in vitro assays with isolated liver cells of high-Antarctic Notothenioids, BaP exposure had no effect on overall respiration, but caused shifts in the respiration dedicated to protein synthesis. Thus, our study demonstrated that high-Antarctic fish possess a functional Ahr that can be ligand-activated in a concentration-dependent manner by environmental contaminants. This is associated with altered cost for cellular protein synthesis. Future studies have to show if the toxicant-induced activation of the Ahr pathway may lead to altered organism performance of Antarctic fish.

Ribosome dysfunction in osteoarthritis.

Purpose of reviewTranslation of genetic information encoded within mRNA molecules by ribosomes into proteins is a key part of the central dogma of molecular biology. Despite the central position of the ribosome in the translation of proteins, and considering the major proteomic changes that occur in the joint during osteoarthritis development and progression, the ribosome has received very limited attention as driver of osteoarthritis pathogenesis.Recent findingsWe provide an overview of the limited literature regarding this developing topic for the osteoarthritis field. Recent key findings that connect ribosome biogenesis and activity with osteoarthritis include: ribosomal RNA transcription, processing and maturation, ribosomal protein expression, protein translation capacity and preferential translation.SummaryThe ribosome as the central cellular protein synthesis hub is largely neglected in osteoarthritis research. Findings included in this review reveal that in osteoarthritis, ribosome aberrations have been found from early-stage ribosome biogenesis, through ribosome build-up and maturation, up to preferential translation. Classically, osteoarthritis has been explained as an imbalance between joint tissue anabolism and catabolism. We postulate that osteoarthritis can be interpreted as an acquired ribosomopathy. This hypothesis fine-tunes the dogmatic anabolism/katabolism point-of-view, and may provide novel molecular opportunities for the development of osteoarthritis disease-modifying treatments.

Role of vacuum assisted closure in the management of infected wound in orthopaedics

Background and objective: Wounds are a major source of morbidity, lead to considerable disability, and are associated with increased mortality; therefore, they have a significant impact on public health and the expenditure of healthcare resources [1]. Vacuum-assisted closure (VAC) uses negative pressure to assist wound healing. Negative pressure drains fluid from the wound, thus removing the substrate for growth of microorganisms. Negative pressure may also accelerate granulation tissue formation and promote angiogenesis. The mechanical stimulation of cells by tensile forces may also play a role by increasing cellular proliferation and protein synthesis. Negative pressure wound therapy (NPWT) involves the use of a negative pressure therapy or suction device to aspirate and remove fluids, debris, and infectious materials from the wound bed to promote the formation of granulation tissue. Materials and Methods: A total of 30 post-operative infected wound cases presented in between December 2019 to August 2021 were taken for study. Each case was examined clinically in systematic manner as per the proforma drafted. VAC dressing was done and all cases were followed up to discharge and subsequently for a follow up on 10th day. Results: In our clinical study of 30 cases managed by VAC dressing, 30% of the cases were of post-operative infected wounds of radius & ulna, 13.3% humerus, 23.3% femur, 23.3% tibia, 3.3% spine, 3.3% calcaneum, 3.3% metatarsals. By the completion of VAC therapy none of the cases required a revision surgery or implant removal. There is a progression of healthy healing in all the cases (100%). Commonest organisms isolated were Staphylococcus, Pseudomonas and Proteus. Interpretation and conclusion: In our study VAC therapy enhanced granulation tissue formation leading to better wound healing, and faster recovery. VAC is thus a promising new technology in the field of wound healing with multiple applications in a variety of wounds and can be used in both acute and chronic wounds, salvage procedures or as an adjuvant therapy to improve the results of various surgical procedures.

Using type III recombinant human collagen to construct a series of highly porous scaffolds for tissue regeneration

As an alternative biopolymer material without the risks of the use of animal-derived collagens in soft tissue engineering applications, recombinant human collagen polypeptide (RHC) was used to construct three-dimensional porous scaffolds. RHC and RHC-chitosan (RHC-CHI) porous scaffolds were fabricated using a freeze-drying method to create highly porous internal structures and then cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). All scaffolds had interconnected porous structures with high porosity (90%), and pore size that ranged from 111 µm to 159 µm. The swelling ability and in vitro degradation of the prepared scaffolds were investigated. The mechanical properties could be tailored to meet the requirements of end-use application by adjusting the concentrations of the polymer or cross-linking agent, and the resulting mechanical strengths were comparable to those of biological soft tissues. The cytocompatibility of the fabricated porous scaffolds was investigated by seeding 3T3 fibroblasts into the porous structures, and then cell proliferation, distribution, morphology, and synthesis of extra cellular matrix-associated proteins were assessed. The results indicated that RHC-based porous scaffolds were non-cytotoxic and promoted the attachment and proliferation of the seeded cells. Finally, the in vivo study proved these porous scaffolds were able to accelerate the cell infiltration and collagen deposition that promoted the wound closure. Overall, the results indicate that RHC-based porous scaffolds show promise for use in soft tissue engineering due to their excellent in vitro cytocompatibility and adjustable mechanical properties.

BAFF receptor polymorphisms and deficiency in humans.

The BAFF-receptor (BAFFR) is a member of the TNF-receptor family. It is expressed only by B cells and binds BAFF as single ligand, which activates key signaling pathways regulating essential cellular functions, including survival, protein synthesis, and metabolic fitness. In humans, BAFFR deficiency interrupts B cell development at the transition from immature to mature B cells and causes B lymphopenia, hypogammaglobulinemia, and impaired humoral immune responses. Polymorphisms in TNFRSF13C gene affecting BAFFR oligomerization and signaling have been described in patients with immunodeficiency, autoimmunity and B cell lymphomas. Despite a uniform expression pattern of BAFFR in peripheral mature B cells, depletion of BAFF with neutralizing antibodies in patients with systemic lupus erythematosus does not affect the survival of switched memory B cells. These findings imply a distinct dependency of mature B cell subsets on BAFF/BAFFR interaction and highlight the contribution of BAFFR-derived signals in peripheral B cell development and homeostasis.

The TOR‐dependent phosphoproteome and regulation of cellular protein synthesis

Cell growth is orchestrated by a number of interlinking cellular processes. Components of the TOR pathway have been proposed as potential regulators of cell growth, but little is known about their immediate effects on protein synthesis in response to TOR‐dependent growth inhibition. Here, we present a resource providing an in‐depth characterisation of Schizosaccharomyces pombe phosphoproteome in relation to changes observed in global cellular protein synthesis upon TOR inhibition. We find that after TOR inhibition, the rate of protein synthesis is rapidly reduced and that notable phosphorylation changes are observed in proteins involved in a range of cellular processes. We show that this reduction in protein synthesis rates upon TOR inhibition is not dependent on S6K activity, but is partially dependent on the S. pombe homologue of eIF4G, Tif471. Our study demonstrates the impact of TOR‐dependent phospho‐regulation on the rate of protein synthesis and establishes a foundational resource for further investigation of additional TOR‐regulated targets both in fission yeast and other eukaryotes.

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides.

Oxidative stress triggered by the Fenton reaction (chemical) or UVR exposure (photo) can damage cellular biomolecules including RNA through oxidation of nucleotides. Besides such xenobiotic chemical modifications, RNA also contains several post-transcriptional nucleoside modifications that are installed by enzymes to modulate structure, RNA-protein interactions, and biochemical functions. We examined the extent of oxidative damage to naturally modified RNA which is required for cellular protein synthesis under two different contexts. The extent of oxidative damage is higher when RNA is not associated with proteins, but the degree of damage is lower when the RNA is presented in the form of a ribonucleoprotein complex, such as an intact ribosome. Our studies also indicate that absence of methylations in ribosomal RNA at specific positions could make it more susceptible to photooxidative stress. However, the extent of guanosine oxidation varied with the position at which the modification is deficient, indicating position-dependent structural effects. Further, an E. coli strain deficient in 5-methylaminomethyl-2-thiouridine (mnm5s2U) (found in lysine and glutamate tRNA anticodon) is more vulnerable to oxidative RNA damage compared to its wildtype strain suggesting an auxiliary function for the mnm5s2U modification. These studies indicate that oxidative damage to RNA is altered by the presence of enzymatic modified nucleosides or protein association inside the cell.

Niche Regulation of Residual Hematopoietic Stem Cell Protein Synthesis By Extracellular Vesicle (EV) Trafficking in the Acute Myeloid Leukemia (AML) Microenvironment

Work by several groups has shown that the presence of Acute Myeloid Leukemia (AML) deregulates critical functions of the bone marrow microenvironment, including hematopoiesis, in part through indirect stroma-secreted factors. We recently reported that Extracellular Vesicles (EV) released from AML blasts directly suppress the clonogenicity of hematopoietic progenitors via the action of miRNA-150 and miR-155 on cMyb, a transcription factor highly expressed in differentiating progenitors. The fate of long-term hematopoietic stem cells (LT- HSC) in the AML niche has not been widely studied. Using a xenograft model of human AML (Molm-14, U937 and HL-60 cell lines), we first ascertained the in vivo uptake of AML-EVs and found different internalization efficiencies among hematopoietic stem and progenitor cell (HSPC) populations, including LT-HSC (CD150+CD48−KSL) by imaging and flow cytometry. Live-cell microscopy studies of LT-HSCs revealed AML-EV binding and uptake within minutes following in vitro exposure. To determine the functional implications of EV trafficking, we next evaluated the relative frequency of the hematopoietic populations in low chimerism xenografts and found a consistent increase in the LT-HSC population, but not multi-potent progenitors, suggestingthe proportional accumulation of LT-HSC population caused by mobilization and lack of expansion and differentiation of other hematopoietic cells. To gain insight into how AML-EVs may regulate cell fate and function following internalization, we performed a proteomic profiling of HSPC cells exposed to EVs from two cell lines representing different AML subtypes. Bioinformatics analysis found significant enrichment for the ribosomal biogenesis pathways and negative cell cycle regulators in the EV-exposed cells. Because LT-HSC have been shown to be particularly susceptible to impairment of ribosome biogenesis, we wished to determine if AML-EVs modulate the LT-HSC fate by suppressing protein synthesis. We therefore injected AML-EVs into the femurs of both immuno-competent and immuno-compromised mice and evaluated the cellular protein synthesis by quantifying O-Propargyl-Puromycin (OPP) incorporation. Results show that AML-EVs significantly suppress the protein synthesis in the LT-HSC, but not in the HSPC pool population. The mTOR signaling pathway is known to be greatly involved in ribosome biogenesis via phosphorylation of the ribosomal protein S6 (S6RP) which in turn regulates several repressors of rRNA and ribosomal proteins. Consistent with the notion of translational suppression, we found that AML-EVs reduced the phosphorylation of S6RP in LT-HSC. In correlation with the observation that ribosomal stress induction via suppression of ribosomal proteins we found upregulation of P53 and its transcriptional target p21, as well as a gain in quiescence (G0) in the LT-HSC population. To address the functional fate of deregulated protein synthesis and quiescence, we transplanted KSL cells purified from male mice engrafted with AML cells into irradiated female recipient mice and measured the donor chimerism via qPCR for Sex-determining Region Y (SRY). Early repopulation capacities between cohorts of mice transplanted with KSL from AML xenograft versus those from control non-engrafted mice are similar (with long-term evaluation ongoing) in the host non-leukemic microenvironment. This suggesting that hematopoietic impairment by AML-EVs is reversible upon transplantation to a different host environment. Collectively, our results provide the first evidence that AML-EV impair LT-HSC via suppressing of ribosomal biogenesis. Understanding the regulation of LT-HSC by AML-EV will help reduce the morbidity and mortality from hematopoietic suppression in patients with leukemia. DisclosuresNo relevant conflicts of interest to declare.

APOBEC3G rescues cells from the deleterious effects of DNA damage.

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (hA3G), a member of the APOBEC family, was described as an anti-HIV-1 restriction factor, deaminating reverse transcripts of the HIV-1 genome. Several types of cancer cells that express high levels of A3G, such as diffuse large B-cell lymphoma cells and glioblastomas, show enhanced cell survival after ionizing radiation and chemotherapy treatments. Previously, we showed that hA3G promotes (DNA) double-strand breaks repair in cultured cells and rescues transgenic mice from a lethal dose of ionizing radiation. Here, we show that A3G rescues cells from the detrimental effects of DNA damage induced by ultraviolet irradiation and by combined bromodeoxyuridine and ultraviolet treatments. The combined treatments stimulate the synthesis of cellular proteins, which are exclusively associated with A3G expression. These proteins participate mainly in nucleotide excision repair and homologous recombination DNA repair pathways. Our results implicate A3G inhibition as a potential strategy for increasing tumor cell sensitivity to genotoxic treatments.

Translation stress and collided ribosomes are co-activators of cGAS

SummaryThe cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to activate the innate immune system. Here, we report the unexpected discovery that cGAS also senses dysfunctional protein production. Purified ribosomes interact directly with cGAS and stimulate its DNA-dependent activity in vitro. Disruption of the ribosome-associated protein quality control (RQC) pathway, which detects and resolves ribosome collision during translation, results in cGAS-dependent ISG expression and causes re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and orthogonal perturbations that result in elevated levels of collided ribosomes and RQC activation cause sub-cellular re-localization of cGAS and ribosome binding in vivo as well. Thus, translation stress potently increases DNA-dependent cGAS activation. These findings have implications for the inflammatory response to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.

Cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles: Toxicity and changes in the proteome of breast, colon and prostate cancer cells

Nanoparticles composed of poly(alkyl cyanoacrylate) (PACA) have shown great promise due to their biodegradability and high drug loading capacity. Development of optimal PACA nanocarriers requires detailed analysis of the overall cellular impact exerted by PACA variants. We here perform a comprehensive comparison of cabazitaxel (CBZ)-loaded nanocarriers composed of three different PACA monomers, i.e. poly(n-butyl cyanoacrylate) (PBCA), poly(2-ethylbutyl cyanoacrylate) (PEBCA) and poly(octyl cyanoacrylate) (POCA). The cytotoxicity of drug-loaded and empty PACA nanoparticles were compared to that of free CBZ across a panel of nine cancer cell lines by assessing cellular metabolism, proliferation and protein synthesis. The analyses revealed that the cytotoxicity of all CBZ-loaded PACAs was similar to that of free CBZ for all cell lines tested, whereas the empty PACAs exerted much lower toxicity. To increase our understanding of the toxic effects of these treatments comprehensive MS-based proteomics were performed with HCT116, MDA-MB-231 and PC3 cells incubated with PACA-CBZ variants or free CBZ. Interestingly, PACA-CBZ specifically led to decreased levels of proteins involved in focal adhesion and stress fibers in all cell lines. Since we recently demonstrated that encapsulation of CBZ within PEBCA nanoparticles significantly improved the therapeutic effect of CBZ on a patient derived xenograft model in mice, we investigated the effects of this PACA variant more closely by immunoblotting. Interestingly, we detected several changes in the protein expression and degree of phosphorylation of SRC-pathway proteins that can be relevant for the therapeutic effects of these substances.

Circadian Clock Control of Translation Initiation Factor eIF2α Activity Requires eIF2γ-Dependent Recruitment of Rhythmic PPP-1 Phosphatase in Neurospora crassa

ABSTRACTThe circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of N. crassa PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of N. crassa eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that N. crassa eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events.

Human IRES Atlas: an integrative platform for studying IRES-driven translational regulation in humans

It is now known that cap-independent translation initiation facilitated by internal ribosome entry sites (IRESs) is vital in selective cellular protein synthesis under stress and different physiological conditions. However, three problems make it hard to understand transcriptome-wide cellular IRES-mediated translation initiation mechanisms: (i) complex interplay between IRESs and other translation initiation–related information, (ii) reliability issue of in silico cellular IRES investigation and (iii) labor-intensive in vivo IRES identification. In this research, we constructed the Human IRES Atlas database for a comprehensive understanding of cellular IRESs in humans. First, currently available and suitable IRES prediction tools (IRESfinder, PatSearch and IRESpy) were used to obtain transcriptome-wide human IRESs. Then, we collected eight genres of translation initiation–related features to help study the potential molecular mechanisms of each of the putative IRESs. Three functional tests (conservation, structural RNA–protein scores and conditional translation efficiency) were devised to evaluate the functionality of the identified putative IRESs. Moreover, an easy-to-use interface and an IRES–translation initiation interaction map for each gene transcript were implemented to help understand the interactions between IRESs and translation initiation–related features. Researchers can easily search/browse an IRES of interest using the web interface and deduce testable mechanism hypotheses of human IRES-driven translation initiation based on the integrated results. In summary, Human IRES Atlas integrates putative IRES elements and translation initiation–related experiments for better usage of these data and deduction of mechanism hypotheses. Database URL: http://cobishss0.im.nuk.edu.tw/Human_IRES_Atlas/

SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-191. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response2,3. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression4-7, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.

Small molecule Y-320 stimulates ribosome biogenesis, protein synthesis, and aminoglycoside-induced premature termination codon readthrough.

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

Sex‐Related Differential Gene Expression Underlies Distinct Responses of Adipose Endothelial Cells Under A High‐Fat Diet

Background Despite similar rates of obesity, the prevalence and severity of obesity-related metabolic diseases is lower in females compared to males. This relates with better preserved function of the adipose tissue in females. Consistent with the importance of capillaries in maintaining tissue functions, insufficient vascularization in expanding adipose tissue leads to adipocyte dysfunction, inflammation and whole-body metabolic disturbances. Previously, we reported that angiogenesis is higher in the perigonadal adipose tissue in female compared to male mice on a high-fat diet, leading to a higher capillary density in female adipose. Endothelial cells line capillaries and coordinate different cellular processes involved in angiogenesis, allowing for the necessary vascular remodelling needed in adipose maintenance. We hypothesize that differential gene networks/pathways in the endothelial cells of females compared to males support the better angiogenic response in female adipose tissue. Methods Male and female (5-7 weeks old; n=4/group) mice were fed a high-fat diet (60% kcal fat) for 7 weeks. White adipose tissue was harvested and used for whole-mount imaging and endothelial cell isolation. Adipose tissue was stained with lectin and imaged to determine the vascular density. RNA extracted from the isolated endothelial cells was analyzed by RNA-sequencing. Transcripts were assessed for differentially expressed genes (DEGs). Gene ontologies and gene set enrichment analysis were used to identify patterns of gene expression. Results Adipose tissue showed greater vascularization in high fat -fed females with vascular areas 27.2 ± 2.2 and 21.0 ± 0.6 % field of view in females and males respectively. RNA-sequencing led to identification of 1225 DEG between male and female endothelial cells (P<0.01). Ontology analyses of the DEGs showed that male endothelial cells expressed higher levels of genes enriched in inflammatory responses and immune processes including chemokine production, complement/coagulation and leukocyte adhesion. In contrast, female endothelial cells lacked an inflammatory signature. Instead, the genes that were higher in female endothelial cells were related to cellular dynamics, such as machinery regulating transcription, translation, DNA replication and axon guidance. Conclusion Adipose endothelial cells from male and female mice have a high number of DEGs that reflect differences in endothelial cell angiogenic phenotype as well as other unique profiles that may be relevant to adipose function. Upregulation of ontologies relating to cellular replication and protein synthesis in females supports the observation of a better angiogenic response in the female adipose tissue and implies that intrinsic differences in male and female endothelial cells contribute to this capacity.

The Sensor IRE1 Couples Stress Detection to Protein Synthesis

Endoplasmic reticulum (ER) stress results in accumulation of misfolded proteins and profoundly regulates cellular protein synthesis. The regulation includes both inhibition of translation of most proteins and upregulation of synthesis of a select group of stress-related polypeptides. This differential regulation has long been attributed to the kinase PERK, but recently the related stress sensor, inositol requiring enzyme 1 (IRE1), was suggested to be linked to translation by virtue of its physical association with both ribosomes1 and the Sec61 translocon2,3. ER stress causes the quiescent IRE1 to dissociate from the inhibitor BiP, self-phosphorylate, oligomerize (an event also called clustering) and activate its RNase activity. Consequently, active IRE1 activates the critical transcription factor XBP1, and also reduces the expression of select other transcripts, to cope with increased ER stress. We previously showed that IRE1 clustering is a response to stress4, but is not related to its RNase activity, raising the question – what is the function of IRE1 clustering? In examining the requirements for IRE clustering we discovered that ongoing protein synthesis and translocation are necessary for both the formation of IRE1 clusters upon ER stress and their maintenance. IRE1 clusters do not develop when cells are treated with either cycloheximide or lactimidomycin, which inhibit nascent chain elongation and ‘freeze’ membrane-bound ribosomes on the ER membrane. If added after clusters were allowed to form under ER stress, both inhibitors caused rapid disappearance of the pre-formed clusters. IRE1 clustering is similarly sensitive to Eeyarestatin I (EEY), an inhibitor of the Sec61 translocon. When translocation is inhibited, IRE1 is dissociated from Sec61 and its interaction with BiP is increased. Concomitantly, more IRE1 is found in high molecular weight species, which perhaps represent ubiquitinylated species. Paradoxically, EEY also induces IRE1 activation (XBP1 splicing). To explain the paradox, we hypothesize the following model: in unstressed conditions, IRE1 binds the translocon and is kept inactive by the interaction with the chaperone BiP. Upon ER stress, different scenarios with distinct IRE1 populations can appear at the same time: 1) IRE1 is released from BiP and clusters on a subset of translocons that are engaged in synthesis of stress-related polypeptides; 2) translocons not engaged in active translocation release IRE1 which is then kept inactive by increased interaction with BiP; 3) some activated IRE1 dimerizes (appearing as non-clustered) and binds the ribosomes that recruit XBP1 mRNA, facilitating its splicing. Acosta-Alvear D et al. eLife 2018 Dec 24;7:e43036. Plumb R et al. eLife 2015 2015 May 20;4:e07426. Sundaram A et al. eLife 2017 May 15;6:e27187. Ricci, D. et al. The FASEB Journal 2019; Sep;33(9):9811-9827.