Elevated Protein Synthesis Research Articles

FGF23 and Cell Stress in SaOS-2 Cells-A Model Reflecting X-Linked Hypophosphatemia Dynamics.

Our study investigates the impact of FGF23 overexpression on SaOS-2 cells to elucidate its role in cellular stress and morphology, contributing to the understanding of skeletal pathologies like X-linked hypophosphatemia (XLH). Using transmission electron microscopy and protein analysis (Western blot), we analyzed the rough endoplasmic reticulum (rER) and mitochondria in SaOS-2 cells with FGF23 overexpression compared to controls. We found significant morphological changes, including enlarged and elongated rER and mitochondria, with increased contact zones, suggesting enhanced interaction and adaptation to elevated protein synthesis and secretion demands. Additionally, we observed higher apoptosis rates of the cells after 24-72 h in vitro and upregulated proteins associated with ER stress and apoptosis, such as CHOP, XBP1 (spliced and unspliced), GRP94, eIF2α, and BAX. These findings indicate a robust activation of the unfolded protein response (UPR) and apoptotic pathways due to FGF23 overexpression. Our results highlight the critical role of ER and mitochondrial interactions in cellular stress responses and provide new insights into the mechanistic link between FGF23 signaling and cellular homeostasis. In conclusion, our study underscores the importance of analyzing UPR-related pathways in the development of therapeutic strategies for skeletal and systemic diseases and contributes to a broader understanding of diseases like XLH.

Costs of ribosomal RNA stabilization affect ribosome composition at maximum growth rate

Ribosomes are key to cellular self-fabrication and limit growth rate. While most enzymes are proteins, ribosomes consist of 1/3 protein and 2/3 ribonucleic acid (RNA) (in E. coli).Here, we develop a mechanistic model of a self-fabricating cell, validated across diverse growth conditions. Through resource balance analysis (RBA), we explore the variation in maximum growth rate with ribosome composition, assuming constant kinetic parameters.Our model highlights the importance of RNA instability. If we neglect it, RNA synthesis is always cheaper than protein synthesis, leading to an RNA-only ribosome at maximum growth rate. Upon accounting for RNA turnover, we find that a mixed ribosome composed of RNA and proteins maximizes growth rate. To account for RNA turnover, we explore two scenarios regarding the activity of RNases. In (a) degradation is proportional to RNA content. In (b) ribosomal proteins cooperatively mitigate RNA instability by protecting it from misfolding and subsequent degradation. In both cases, higher protein content elevates protein synthesis costs and simultaneously lowers RNA turnover expenses, resulting in mixed RNA-protein ribosomes. Only scenario (b) aligns qualitatively with experimental data across varied growth conditions.Our research provides fresh insights into ribosome biogenesis and evolution, paving the way for understanding protein-rich ribosomes in archaea and mitochondria.

SRC family kinase inhibition rescues molecular and behavioral phenotypes, but not protein interaction network dynamics, in a mouse model of Fragile X syndrome.

Glutamatergic synapses encode information from extracellular inputs using dynamic protein interaction networks (PINs) that undergo widespread reorganization following synaptic activity, allowing cells to distinguish between signaling inputs and generate coordinated cellular responses. Here, we investigate how Fragile X Messenger Ribonucleoprotein (FMRP) deficiency disrupts signal transduction through a glutamatergic synapse PIN downstream of NMDA receptor or metabotropic glutamate receptor (mGluR) stimulation. In cultured cortical neurons or acute cortical slices from P7, P17 and P60 FMR1-/y mice, the unstimulated protein interaction network state resembled that of wildtype littermates stimulated with mGluR agonists, demonstrating resting state pre-activation of mGluR signaling networks. In contrast, interactions downstream of NMDAR stimulation were similar to WT. We identified the Src family kinase (SFK) Fyn as a network hub, because many interactions involving Fyn were pre-activated in FMR1-/y animals. We tested whether targeting SFKs in FMR1-/y mice could modify disease phenotypes, and found that Saracatinib (SCB), an SFK inhibitor, normalized elevated basal protein synthesis, novel object recognition memory and social behavior in FMR1-/y mice. However, SCB treatment did not normalize the PIN to a wild-type-like state in vitro or in vivo, but rather induced extensive changes to protein complexes containing Shank3, NMDARs and Fyn. We conclude that targeting abnormal nodes of a PIN can identify potential disease-modifying drugs, but behavioral rescue does not correlate with PIN normalization.

Phenotype and energy metabolism differ between osteoarthritic chondrocytes from male compared to female patients: Implications for sexual dimorphism in osteoarthritis development?

ObjectivesThe prevalence and severity of knee osteoarthritis (OA) are greater in females than males. The purpose of this study was to determine whether there is an underlying difference in the biology of OA chondrocytes between males and females. MethodsChondrocytes were obtained following knee arthroplasty from male and female patients with primary OA. Phenotype marker expression, glucose and fat consumption, and rates of glycolysis and oxidative phosphorylation were compared between females and males. RNAi was used to determine the consequences of differential expression of Sry-box transcription factor 9 (SOX9) and PGC1α between males and females. ResultsOA chondrocytes from male donors showed elevated RNA and protein levels of SOX9, elevated COL2A1 protein synthesis, higher glucose consumption, and higher usage of glycolysis compared to females. OA chondrocytes from females had higher PGC1α protein levels, higher fat consumption, and higher oxidative energy metabolism than males. Knockdown of SOX9 reduced expression of COL2A1 to a greater extent in male OA chondrocytes than females whereas knockdown of PGC1α reduced COL2A1 expression in females but not males. Expression of ACAN and the glycolytic enzyme PGK1 was also reduced in males but not females following SOX9 knockdown. ConclusionsOA chondrocyte phenotype and energy metabolism differ between males and females. Our results indicate transcriptional control of COL2A1 differs between the two. Differences in chondrocyte biology between males and females imply the underlying mechanisms involved in OA may also differ, highlighting the need to consider sex and gender when investigating pathogenesis and potential treatments for OA.

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

The translation initiating factor eIF4E and arginine methylation underlie G3BP1 function in dendritic spine development of neurons

Communication between neurons relies on neurotransmission that takes place at synapses. Excitatory synapses are located primarily on dendritic spines that possess diverse morphologies, ranging from elongated filopodia to mushroom-shaped spines. Failure in the proper development of dendritic spines has detrimental consequences on neuronal connectivity, but the molecular mechanism that controls the balance of filopodia and mushroom spines is not well understood. G3BP1 is the key RNA-binding protein that assembles the stress granules in non-neuronal cells to adjust protein synthesis upon exogenous stress. Emerging evidence suggests that the biological significance of G3BP1 extends beyond its role in stress response, especially in the nervous system. However, the mechanism underlying the regulation and function of G3BP1 in neurons remains elusive. Here we found that G3BP1 suppresses protein synthesis and binds to the translation initiation factor eIF4E via its NTF2-like domain. Notably, the over-production of filopodia caused by G3BP1 depletion can be alleviated by blocking the formation of the translation initiation complex. We further found that the interaction of G3BP1 with eIF4E is regulated by arginine methylation. Knockdown of the protein arginine methyltransferase PRMT8 leads to elevated protein synthesis and filopodia production, which is reversed by the expression of methylation-mimetic G3BP1. Our study, therefore, reveals arginine methylation as a key regulatory mechanism of G3BP1 during dendritic spine morphogenesis and identifies eIF4E as a novel downstream target of G3BP1 in neuronal development independent of stress response.

Ribosome biosynthesis and Hedgehog activity are cooperative actionable signaling mechanisms in breast cancer following radiotherapy

Hyperactivated ribosome biosynthesis is attributed to a need for elevated protein synthesis that accommodates cell growth and division, and is characterized by nucleomorphometric alterations and increased nucleolar counts. Ribosome biogenesis is challenged when DNA-damaging treatments such as radiotherapy are utilized. Tumor cells that survive radiotherapy form the basis of recurrence, tumor progression, and metastasis. In order to survive and become metabolically revitalized, tumor cells need to reactivate RNA Polymerase I (RNA Pol I) to synthesize ribosomal RNA, an integral component of ribosomes. In this study, we showed that following radiation therapy, tumor cells from breast cancer patients demonstrate activation of a ribosome biosynthesis signature concurrent with enrichment of a signature of Hedgehog (Hh) activity. We hypothesized that GLI1 activates RNA Pol I in response to irradiation and licenses the emergence of a radioresistant tumor population. Our work establishes a novel role for GLI1 in orchestrating RNA Pol I activity in irradiated breast cancer cells. Furthermore, we present evidence that in these irradiated tumor cells, Treacle ribosome biogenesis factor 1 (TCOF1), a nucleolar protein that is important in ribosome biogenesis, facilitates nucleolar translocation of GLI1. Inhibiting Hh activity and RNA Pol I activity disabled the outgrowth of breast cancer cells in the lungs. As such, ribosome biosynthesis and Hh activity present as actionable signaling mechanisms to enhance the effectiveness of radiotherapy.

Enhanced protein synthesis is a defining requirement for neonatal B cell development.

The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b. Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.

Heat-Induced Proteotoxic Stress Response in Placenta-Derived Stem Cells (PDSCs) Is Mediated through HSPA1A and HSPA1B with a Potential Higher Role for HSPA1B.

Placenta-derived stem cells (PDSCs), due to unique traits such as mesenchymal and embryonic characteristics and the absence of ethical constraints, are in a clinically and therapeutically advantageous position. To aid in stemness maintenance, counter pathophysiological stresses, and withstand post-differentiation challenges, stem cells require elevated protein synthesis and consequently augmented proteostasis. Stem cells exhibit source-specific proteostasis traits, making it imperative to study them individually from different sources. These studies have implications for understanding stem cell biology and exploitation in the augmentation of therapeutic applications. Here, we aim to identify the primary determinants of proteotoxic stress response in PDSCs. We generated heat-induced dose-responsive proteotoxic stress models of three stem cell types: placental origin cells, the placenta-derived mesenchymal stem cells (pMSCs), maternal origin cells, the decidua parietalis mesenchymal stem cells (DPMSCs), and the maternal–fetal interface cells, decidua basalis mesenchymal stem cells (DBMSCs), and measured stress induction through biochemical and cell proliferation assays. RT-PCR array analysis of 84 genes involved in protein folding and protein quality control led to the identification of Hsp70 members HSPA1A and HSPA1B as the prominent ones among 17 significantly expressed genes and with further analysis at the protein level through Western blotting. A kinetic analysis of HSPA1A and HSPA1B gene and protein expression allowed a time series evaluation of stress response. As identified by protein expression, an active stress response is in play even at 24 h. More prominent differences in expression between the two homologs are detected at the translational level, alluding to a potential higher requirement for HSPA1B during proteotoxic stress response in PDSCs.

Lipid-independent activation of a muscle-specific PKCα splicing variant.

Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes.NEW & NOTEWORTHY PKCα is an important signaling molecule extensively studied in many cellular processes. However, no isoforms have been reported for PKCα except one prototypic isoform. Alternative mRNA splicing of Prkca gene was detected for the first time in rodent and human cardiac tissue, which can produce a previously unknown PKCα-novel exon (NE) isoform. The biochemistry and molecular effects of PKCα-NE are markedly different from PKCα wild type, suggesting potential functional diversity of PKCα signaling in muscle.

Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15–30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry.

The Aminoacyl-tRNA Synthetase and tRNA Expression Levels Are Deregulated in Cancer and Correlate Independently with Patient Survival

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that load amino acids to their cognate tRNA molecules. The expression of certain ARSs and tRNAs has been shown to be deregulated in cancer, presumably to accommodate elevated protein synthesis requirements. In this work, the expression of cytoplasmic ARSs and tRNAs in ten TCGA cancers has been systematically examined. ARSs were found to be mostly upregulated in tumours and their upregulation often correlated with worse patient survival. tRNAs were found to be either upregulated or downregulated in tumours and their expression sometimes correlated to worse survival outcomes. However, although the expression of most ARSs and tRNAs was deregulated in tumours when compared to healthy adjacent tissues, only in a few cases, and independently, did it correlate to patient survival. These data point to the general uncoupling of concomitant ARS and tRNA expression deregulation and patient survival, highlighting the different ARS and tRNA requirements in cancers.

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. While Yki-induced cell proliferation has extensively been studied, the mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. Here, we show that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. Through a genetic screen in Drosophila, we find that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. Our data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells.

Vorinostat Corrects Cognitive and Non-Cognitive Symptoms in a Mouse Model of Fragile X Syndrome.

BackgroundFragile X syndrome (FXS) is caused by mutations in the FMR1 gene. It is a form of heritable intellectual disability and autism. Despite recent advance in elucidating disease mechanisms, there is no efficacious medication. Because de novo drug development is a lengthy process, repurposing the existing FDA-approved drugs offers an opportunity to advance clinical intervention for FXS. Our previous study with transcriptome analysis predicts potential therapeutic effects of vorinostat on FXS.MethodsWe analyzed the vorinostat-induced transcriptome changes and confirmed its similarity to that induced by trifluoperazine, which was previously shown to correct pathological outcomes associated with FXS. To validate the therapeutic efficacy, we examined vorinostat’s effect on correcting the key behavioral and cellular symptoms in a mouse model of FXS.ResultsWe found that vorinostat restores object location memory and passive avoidance memory in the Fmr1 knockout mice. For the non-cognitive behavioral symptoms, vorinostat corrected the autism-associated alterations, including repetitive behavior and social interaction deficits. In the open field test, vorinostat dampened hyperactivity in the center area of the arena. Surprisingly, vorinostat did not correct the abnormally elevated protein synthesis in cultured Fmr1 knockout hippocampal neurons, suggesting that different aspects of pathological outcomes may respond differently to a specific therapeutic intervention.ConclusionsWe used the drug-induced transcriptome signature to predict new application of existing drugs. Our data reveal the therapeutic effects of the FDA-approved drug vorinostat in a mouse model of FXS.

RNase Z Oxidative Degradation Impedes tRNA Maturation and is Involved in Streptococcal Translation Regulation in Response to Oxidative Stress.

ABSTRACTWhen encountering oxidative stress, organisms selectively upregulate antioxidant genes and simultaneously suppress the translation of most other proteins. Eukaryotes employ multiple strategies to adjust translation at both the initiation and elongation stages; however, how prokaryotes modulate translation under oxidative stress remains unclear. Here, we report that upon hydrogen peroxide (H2O2) challenge, Streptococcus oligofermentans reduced translation via RNase Z (So-RNaseZ) oxidative degradation, thus hindering tRNA maturation. S. oligofermentans encodes all CCA-less tRNAs that require So-RNaseZ for 3′ end maturation. A combination of nonreducing SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC/MS-MS) assays demonstrated that H2O2 oxidation induced Cys38-Cys149 disulfide linkages in recombinant So-RNaseZ protein, and serine substitution of Cys38 or Cys149 abolished these disulfide linkages. Consistently, redox Western blotting also determined intramolecular disulfide-linked So-RNaseZ in H2O2-treated S. oligofermentans cells. The disulfide-linked So-RNaseZ and monomer were both subject to proteolysis, whereas C149S mutation alleviated oxidative degradation of So-RNaseZ, suggesting that H2O2-mediated disulfide linkages substantially contributed to So-RNaseZ degradation. Accordingly, Northern blotting determined that tRNA precursor accumulation and mature tRNA species decrease in H2O2-treated S. oligofermentans. Moreover, reduced overall protein synthesis, as indicated by puromycin incorporation, and retarded growth of S. oligofermentans occurred in an H2O2 concentration-dependent manner. Overexpression of So-RNaseZ not only elevated tRNA precursor processing and protein synthesis but also partly rescued H2O2-suppressed S. oligofermentans growth. Moreover, So-RNaseZ oxidative degradation-mediated translation repression elevated S. oligofermentans survival under high H2O2 stress. Therefore, this work found that So-RNaseZ oxidative degradation-impeded tRNA maturation contributes to streptococcal translation repression and provides the oxidative stress adaptability for S. oligofermentans.IMPORTANCE Translation regulation is a common strategy used by organisms to reduce oxidative damage. Catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports a novel translation regulation mechanism employed by Streptococcus oligofermentans in response to H2O2 challenge, in which the key tRNA endonuclease So-RNaseZ is oxidized to form Cys38-Cys149 disulfide linkages and both the disulfide-linked So-RNaseZ and monomers are subject to proteolysis; thus, tRNA maturation, protein translation, and growth are all suppressed. Notably, So-RNaseZ oxidative degradation-mediated translation repression offers oxidative adaptability to S. oligofermentans and enhances its survival against high H2O2 challenge. So-RNaseZ orthologs and H2O2-sensitive cysteines (Cys38 and Cys149) are widely distributed in Streptococcus and Lactococcus species genomes, which also encode all CCA-less tRNAs and lack catalase. Therefore, RNase Z oxidative degradation-based translation regulation could be widely employed by these lactic acid bacteria, including pathogenic streptococci, to cope with H2O2.

Role of the IL-33/ST2 axis in cigarette smoke-induced airways remodelling in chronic obstructive pulmonary disease

BackgroundEfficient therapy and potential prophylaxis are confounded by current ignorance of the pathogenesis of airway remodelling and blockade in COPD.ObjectiveTo explore the role of the IL-33/ST2 axis in cigarette smoke...

Abstract 2689: Investigating the molecular mechanisms linking disrupted growth homeostasis to tumourigenesis in Li-Fraumeni Syndrome

Abstract Li-Fraumeni Syndrome (LFS) is a highly penetrant cancer predisposition syndrome caused by germline TP53 mutations. Patients face an increased lifetime risk to develop a broad spectrum of cancers. Implementation of comprehensive cancer screening protocols has led to improvements in survival for LFS patients. However, these interventions pose a significant burden to both patient quality of life and health resource utilization. While these interventions will always be important, recent evidence has suggested an additional approach which may improve patient outcomes: pharmacological prevention of cancer onset. mTORC1 inhibition selectively targets tumor initiation. While rapamycin, an mTORC1 inhibitor, has not shown efficacy in treatment of established cancers, it is reported to prevent tumor onset in mice that are genetically predisposed to cancer, including mouse models of LFS. mTORC1 is associated with protein synthesis and cellular growth. Hyperactivated cellular growth is associated with gain-of-function (GOF) TP53 mutations that underlie many cases of LFS. Wild-type p53 (wtp53) inhibits mTORC1 and, as a consequence, biosynthesis. We have shown that mutant p53 with oncogenic GOF (mutp53) may promote the opposite effect. Under conditions of inappropriately elevated protein synthesis, as is seen in cancer, stress responses, such as the unfolded protein response (UPR), are activated. UPR activation contributes to the secretion of pro-tumorigenic cytokines. We hypothesized that under conditions of elevated protein synthesis, mutp53 engages in a feedforward activation of biosynthesis which hyperactivates UPR-mediated secretion of pro-tumorigenic cytokines. In order to harness the cancer risk reduction potential of mTORC1 inhibition, we undertook investigation of the stress responses which may underlie tumorigenesis in LFS. We sought to establish whether mTORC1 ablation in the tumor microenvironment could influence growth of tumor xenografts formed from the co-injection of LFS patient-derived mutp53 fibroblasts and RH4 rhabdomyosarcoma cells, a representative LFS spectrum cancer cell line. While both wtp53 fibroblasts and mutp53 fibroblasts supported tumor growth, only the tumor growth reinforced by mutp53 fibroblasts was suppressed by rapamycin. Additionally, we utilized protein arrays to profile conditioned media collected from mutp53 and wtp53 fibroblast cultures. Mutp53 fibroblasts secreted higher levels of various pro-tumorigenic cytokines compared to wtp53 fibroblasts, which was partially negated by rapamycin. UPR target gene expression is elevated in fibroblasts expressing mutp53, and this effect is negated by rapamycin. Altogether, our work supports a role for mutp53-driven, mTORC1-mediated hyperactivated protein biosynthesis and subsequent engagement of the UPR, resulting in production of a pro-tumorigenic secretory milieu. Citation Format: Camilla Giovino, Frank Telfer, Nish Patel, Sangeetha Paramathas, Ran Kafri, David Malkin. Investigating the molecular mechanisms linking disrupted growth homeostasis to tumourigenesis in Li-Fraumeni Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2689.

MTORC1 Activity Occurs Predominantly in the Periphery of Human Skeletal Muscle Fibers Following Anabolic Stimuli

The mechanistic target of rapamycin complex 1 (mTORC1) is regarded as the master regulator of protein synthesis in skeletal muscle and is essential for optimal responses to acute anabolic stimuli. We have shown mTORC1 to translocate toward the cell periphery where it colocalizes with upstream activators, downstream effectors and the microvasculature after protein feeding and muscle contraction. These events occur at similar times to elevated whole muscle mTORC1 kinase activity and protein synthesis; however, whether mTORC1 activity occurs in a region-specific pattern is yet to be studied. Therefore, the aim of the current study was to develop an immunofluorescent staining (IF) protocol to measure mTORC1 activation in a region-specific manner following anabolic stimuli in human skeletal muscle. Fourteen young, healthy males (22±4yrs, 14±4% body fat, mean±SD) participated in the study and were randomized to complete trials where a protein-carbohydrate beverage was ingested at rest (FED, n=7) or following a whole-body resistance exercise session (EXFED, n=7). Vastus lateralis biopsy samples were obtained prior to feeding/exercise (basal) and 120 and 300min following anabolic stimuli. Phosphorylated ribosomal protein S6Ser240/244 (p-RPS6Ser240/244) was chosen as the marker of mTORC1 activation as this phosphorylation event is rapamycin-sensitive. We first optimized and validated an IF protocol for p-RPS6Ser240/244 through the omission of primary and secondary antibodies. Using this protocol, we then showed that RPS6Ser240/244 phosphorylation was elevated at 120min (p<0.04) with EXFED being greater than FED (p=0.02). p-RPS6Ser240/244 remained elevated above basal values at 300min in EXFED only (p=0.04). Importantly, a strong positive correlation was observed between RPS6Ser240/244 phosphorylation measured by immunoblotting and IF (r=0.85, p<0.001) showing our novel IF method is a valid readout of mTORC1 activity. In central regions of skeletal muscle fibers, p-RPS6Ser240/244 phosphorylation was only elevated in EXFED at 120min. In the outer 5.5µm of fibers (periphery), mTORC1 activity increased at 120min in both conditions (p<0.03) but to a greater extent in EXFED (p=0.002), remaining elevated above basal levels at 300min only in EXFED (p=0.02). Similarly, the peripheral-to-central ratio of RPS6Ser240/244 phosphorylation was elevated above basal values in both conditions at 120min and remained elevated only in EXFED at 300min, suggesting mTORC1 activity occurs to a greater extent in the periphery of skeletal muscle fibers. In conclusion, we have optimized and validated an IF technique to investigate mTORC1 activity in a region-specific manner in human skeletal muscle. We show that, following physiological anabolic stimuli, mTORC1 activity occurs to a greater extent in the periphery of fibers, regions where mTORC1 is commonly visualized in humans and protein synthesis is observed in animal models. These findings extend our knowledge regarding the spatial regulation of mTORC1 in human skeletal muscle and provide a novel method to investigate mTORC1 dynamics in various populations.

Responses of the reproduction, population growth and metabolome of the marine rotifer Brachionus plicatilis to tributyl phosphate (TnBP).

The typical alkyl organophosphorus flame retardant tributyl phosphate (TnBP) can leak from common products into the marine environment, with potential negative effects on marine organisms. However, risk assessments for TnBP regarding zooplankton are lacking. In this study, a marine rotifer, Brachionus plicatilis, was used to analyze the effect of TnBP (0.1μg/L, environmental concentration; 1 and 6mg/L) on reproduction, population growth, oxidative stress, mitochondrial function and metabolomics. Mortality increased as the TnBP concentration rose; the 24-h LC50 value was 12.45mg/L. All tested TnBP concentrations inhibited B.plicatilis population growth, with reproductive toxicity at the higher levels. Microstructural imaging showed ovary injury, the direct cause of reproductive toxicity. Despite elevated glutathione reductase activities, levels of reactive oxygen species and malonyldialdehyde increased under TnBP stress, indicating oxidative imbalance. TnBP induced mitochondrial malformation and activity suppression; the ROS scavenger N-acetylcysteine alleviated this inhibition, suggesting an internal connection. Nontargeted metabolomics revealed 398 and 583 differentially expressed metabolites in the 0.1μg/L and 6mg/L treatments relative to control, respectively, which were enriched in the pathways such as biosynthesis of amino acids, purine metabolism, aminoacyl-tRNA biosynthesis. According to metabolic pathway analysis, oxidative stress from purine degradation, mitochondrial dysfunction, disturbed lipid metabolism and elevated protein synthesis were jointly responsible for reproduction and population growth changes. This study echoes the results previously found in rotifer on trade-off among different life processes in response to environmental stress. Our systematic study uncovers the TnBP toxic mode of action.

Carbamazepine Restores Neuronal Signaling, Protein Synthesis, and Cognitive Function in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a leading genetic disorder of intellectual disability caused by the loss of the functional fragile X mental retardation protein (FMRP). To date, there is no efficacious mechanism-based medication for FXS. With regard to potential disease mechanisms in FXS, it is widely accepted that the lack of FMRP causes elevated protein synthesis and deregulation of neuronal signaling. Abnormal enhancement of the ERK½ (extracellular signal-regulated kinase ½) and PI3K-Akt (Phosphoinositide 3 kinase-protein kinase B) signaling pathways has been identified in both FXS patients and FXS mouse models. In this study, we show that carbamazepine, which is an FDA-approved drug and has been mainly used to treat seizure and neuropathic pain, corrects cognitive deficits including passive avoidance and object location memory in FXS mice. Carbamazepine also rescues hyper locomotion and social deficits. At the cellular level, carbamazepine dampens the elevated level of ERK½ and Akt signaling as well as protein synthesis in FXS mouse neurons. Together, these results advocate repurposing carbamazepine for FXS treatment.