Role In Protein Synthesis Research Articles

β-Hydroxy β-Methyl Butyric Acid (HMB) and Its Potential Doping Relevance: A Pilot Study on Its Urinary Excretion Profile.

β-hydroxy β-methyl butyric acid (HMB), either as the free acid or in the form of its calcium salt, is a component of several dietary supplements marketed to enhance sports performance, due to its role in protein synthesis. In this pilot study, we investigated the urinary excretion profile of HMB, an endogenous metabolite of the essential amino acid leucine. The endogenous reference ranges of HMB, in human urine samples, were monitored by collecting samples from 20 volunteers. Data obtained were compared with those from controlled excretion studies, following the intake of a 3-g oral dose of HMB. Urine samples were analyzed through a targeted procedure employing a conventional GC-MS system operating in SIM mode. Our results show that the excreted urinary concentration of HMB significantly exceeds the range of endogenous concentrations for at least 24 h, making possible to detect its exogenous origin.

B-328 A Novel Pathway of Akt-Dependent PARP1 Activation Through the Nuclear Localization of EPRS1

Abstract Background Glutamyl-prolyl-tRNA synthetase (EPRS1) is the only bifunctional aminoacyl-tRNA synthetase (aaRS) and is essential for interpreting the genetic code. EPRS1, along with seven other aaRSs, forms the multi-tRNA synthetase complex (MSC). EPRS1 consists of two catalytic domains, GluRS and ProRS, connected by a linker region. Several aaRSs within the MSC, including EPRS1, can dissociate from the MSC in response to specific stimuli. This dissociation enables non-canonical activities distinct from their primary role in protein synthesis. Breast cancer cells harboring a loss-of-function mutation in phosphatase and tensin homolog (PTEN) are known to have more activity in the serine/threonine kinase Akt. Poly-(ADP)-ribose polymerase 1 (PARP1), plays a vital role in DNA damage repair (DDR). PARP1 deposits one or more ADP-Ribose moieties onto damaged DNA or other proteins in a process referred to as PARylation. We hope to connect Akt activation and PARP1 activity through the nuclear localization and noncanonical function of EPRS1. We hypothesize, that when Akt is activated, either through the loss of PTEN function or through stimuli such as heat shock or H2O2 treatment, will stimulate the nuclear localization of EPRS1 and modulate PARP1 activity. Methods To test our hypothesis, we evaluate Akt activation, EPRS1 localization, and PARP1 activity. Using pharmacological activators and inhibitors of Akt, we test if inhibitors and activators alone are sufficient to cause the nuclear localization of EPRS1. Biochemically, we isolate nuclear and cytosolic fractions used in various western blot analysis. We use a variety of assays for PARP1 activity and DDR that include immobilized histones, calculating IC50s of PARP1 inhibitors, comet assay, and confocal microscopy. To ascertain the connection between PARP1 and EPRS1 we use LC-MS/MS analysis and immunoprecipitation. Results We show, in both patient-derived and cultured breast cancer cells, elevated levels of EPRS1 not only in the cytoplasm, but also within the nucleus. We find that nuclear localization EPRS1 is facilitated by a nuclear localization sequence (NLS) located within the linker region of the protein. Breast cancer cells harboring a loss-of-function in PTEN exhibit higher levels of EPRS1 in the nucleus compared to PTEN-positive cells. In EPRS1 knockdown cells, there is diminished expression of multiple tumor-related transcription factors, DNA-damage repair, tumor cell survival, and PARP1 auto-PARylation. We demonstrate that nuclear EPRS1 binds to and shares an interactome with PARP1. Conclusions EPRS1-mediated regulation of PARP1 activity provides a new mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the non-canonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, might provide a new therapeutic approach in breast and other forms of cancer, potentially supplementing existing cancer therapeutics.

Elongation factor 1A1 inhibition elicits changes in lipid droplet size, the bulk transcriptome, and cell type-associated gene expression in MASLD mouse liver.

Eukaryotic elongation factor 1A1 (EEF1A1), originally identified for its role in protein synthesis, has additional functions in diverse cellular processes. Of note, we previously discovered a role for EEF1A1 in hepatocyte lipotoxicity. We also demonstrated that a 2-wk intervention with the EEF1A1 inhibitor didemnin B (DB) (50 µg/kg) decreased liver steatosis in a mouse model of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) [129S6/SvEvTac mice fed Western diet (42% fat) for 26 wk]. Here, we further characterized the hepatic changes occurring in these mice by assessing lipid droplet (LD) size, bulk differential expression, and cell type-associated alterations in gene expression. Consistent with the previously demonstrated decrease in hepatic steatosis, we observed decreased median LD size in response to DB. Bulk RNA sequencing (RNA-Seq) followed by gene set enrichment analysis revealed alterations in pathways related to energy metabolism and proteostasis in DB-treated mouse livers. Deconvolution of bulk data identified decreased cell type association scores for cholangiocytes, mononuclear phagocytes, and mesenchymal cells in response to DB. Overrepresentation analyses of bulk data using cell type marker gene sets further identified hepatocytes and cholangiocytes as the primary contributors to bulk differential expression in response to DB. Thus, we show that chemical inhibition of EEF1A1 decreases hepatic LD size and decreases gene expression signatures associated with several liver cell types implicated in MASLD progression. Furthermore, changes in hepatic gene expression were primarily attributable to hepatocytes and cholangiocytes. This work demonstrates that EEF1A1 inhibition may be a viable strategy to target aspects of liver biology implicated in MASLD progression.NEW & NOTEWORTHY Chemical inhibition of EEF1A1 decreases hepatic lipid droplet size and decreases gene expression signatures associated with liver cell types that contribute to MASLD progression. Furthermore, changes in hepatic gene expression are primarily attributable to hepatocytes and cholangiocytes. This work highlights the therapeutic potential of targeting EEF1A1 in the setting of MASLD, and the utility of RNA-Seq deconvolution to reveal valuable information about tissue cell type composition and cell type-associated gene expression from bulk RNA-Seq data.

Maintenance of magnesium homeostasis by NUF2 promotes protein synthesis and anaplastic thyroid cancer progression

Thyroid cancer is the most frequently observed endocrine-related malignancy among which anaplastic thyroid cancer (ATC) is the most fatal subtype. The synthesis of protein is active to satisfy the rapid growth of ATC tumor, but the mechanisms regulating protein synthesis are still unknown. Our research revealed that kinetochore protein NUF2 played an essential role in protein synthesis and drove the progression of ATC. The prognosis of patients with thyroid carcinoma was positively correlated with high NUF2 expression. Depletion of NUF2 in ATC cells notably inhibited the proliferation and induced apoptosis, while overexpression of NUF2 facilitated ATC cell viability and colony formation. Deletion of NUF2 significantly suppressed the growth and metastasis of ATC in vivo. Notably, knockdown of NUF2 epigenetically inhibited the expression of magnesium transporters through reducing the abundance of H3K4me3 at promoters, thereby reduced intracellular Mg2+ concentration. Furthermore, we found the deletion of NUF2 or magnesium transporters significantly inhibited the protein synthesis mediated by the PI3K/Akt/mTOR pathway. In conclusion, NUF2 functions as an emerging regulator for protein synthesis by maintaining the homeostasis of intracellular Mg2+, which finally drives ATC progression.

A potential role for RNA aminoacylation prior to its role in peptide synthesis

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis.

TRNA-derived fragments in T lymphocyte-beta cell crosstalk and in type 1 diabetes pathogenesis in NOD mice.

tRNAs play a central role in protein synthesis. Besides this canonical function, they were recently found to generate non-coding RNA fragments (tRFs) regulating different cellular activities. The aim of this study was to assess the involvement of tRFs in the crosstalk between immune cells and beta cells and to investigate their contribution to the development of type 1 diabetes. Global profiling of the tRFs present in pancreatic islets of 4- and 8-week-old NOD mice and in extracellular vesicles released by activated CD4+ T lymphocytes was performed by small RNA-seq. Changes in the level of specific fragments were confirmed by quantitative PCR. The transfer of tRFs from immune cells to beta cells occurring during insulitis was assessed using an RNA-tagging approach. The functional role of tRFs increasing in beta cells during the initial phases of type 1 diabetes was determined by overexpressing them in dissociated islet cells and by determining the impact on gene expression and beta cell apoptosis. We found that the tRF pool was altered in the islets of NOD mice during the initial phases of type 1 diabetes. Part of these changes were triggered by prolonged exposure of beta cells to proinflammatory cytokines (IL-1β, TNF-α and IFN-γ) while others resulted from the delivery of tRFs produced by CD4+ T lymphocytes infiltrating the islets. Indeed, we identified several tRFs that were enriched in extracellular vesicles from CD4+/CD25- T cells and were transferred to beta cells upon adoptive transfer of these immune cells in NOD.SCID mice. The tRFs delivered to beta cells during the autoimmune reaction triggered gene expression changes that affected the immune regulatory capacity of insulin-secreting cells and rendered the cells more prone to apoptosis. Our data point to tRFs as novel players in the crosstalk between the immune system and insulin-secreting cells and suggest a potential involvement of this novel class of non-coding RNAs in type 1 diabetes pathogenesis. Sequences are available from the Gene Expression Omnibus (GEO) with accession numbers GSE242568 and GSE256343.

Photochemically Driven Peptide Formation in Supersaturated Aerosol Droplets

The condensation of amino acids into peptides plays a crucial role in protein synthesis and is thus essential for understanding the origins of life. However, the spontaneous formation of peptides from amino acids in bulk aqueous media is energetically unfavorable, posing a challenge for elucidating plausible abiotic mechanisms. In this study, we investigate the formation of amide bonds between amino acids within highly supersaturated aerosol droplets containing dicyandiamide (DCD), a cyanide derivative potentially present on primordial Earth. Metastable states, i.e. supersaturation, within individual micron‐sized droplets are studied using both an optical trap and a linear quadrupole electrodynamic balance. When irradiated with intense visible light, amide bond formation is observed to occur and can be monitored using vibrational bands in Raman spectra. The reaction rate is found to be strongly influenced by droplet size and kinetic modelling suggests that it is driven by the photochemical product of a DCD self‐reaction. Our results highlight the potential of atmospheric aerosol particles as reaction environments for peptide synthesis and have potential implications for the prebiotic chemistry of early Earth.

TSC2 loss in neural progenitor cells suppresses translation of ASD/NDD-associated transcripts in an mTORC1- and MNK1/2-reversible fashion.

Tuberous sclerosis complex (TSC) is an inherited neurodevelopmental disorder (NDD) with frequent manifestations of epilepsy and autism spectrum disorder (ASD). TSC is caused by inactivating mutations in TSC1 or TSC2 tumor suppressor genes, with encoded proteins hamartin (TSC1) and tuberin (TSC2) forming a functional complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. This has led to treatment with allosteric mTORC1 inhibitor rapamycin analogs ("rapalogs") for TSC tumors; however, rapalogs are ineffective for treating neurodevelopmental manifestations. mTORC1 signaling controls protein synthesis by regulating formation of the eIF4F complex, with further modulation by MNK1/2 kinases via phosphorylation of the eIF4F subunit eIF4E. While both these pathways modulate translation, comparing their impact on transcriptome-wide mRNA translation, as well as effects of inhibiting these pathways in TSC has not been explored. Here, employing CRISPR-modified, isogenic TSC2 patient-derived neural progenitor cells (NPCs), we have examined transcriptome-wide changes in mRNA translation upon TSC2 loss. Our results reveal dysregulated translation in TSC2 -Null NPCs, which significantly overlaps with the translatome from TSC1 -Null NPCs. Interestingly, numerous non-monogenic ASD-, NDD-and epilepsy-associated genes identified in patients harboring putative loss-of-function mutations, were translationally suppressed in TSC2 -Null NPCs. Importantly, translation of these ASD- and NDD-associated genes was reversed upon inhibition of either mTORC1 or MNK1/2 signaling using RMC-6272 or eFT-508, respectively. This study establishes the importance of mTORC1-eIF4F- and MNK-eIF4E-sensitive mRNA translation in TSC, ASD and other neurodevelopmental disorders laying the groundwork for evaluating drugs in clinical development that target these pathways as a treatment strategy for these disorders.

Photochemically Driven Peptide Formation in Supersaturated Aerosol Droplets.

The condensation of amino acids into peptides plays a crucial role in protein synthesis and is thus essential for understanding the origins of life. However, the spontaneous formation of peptides from amino acids in bulk aqueous media is energetically unfavorable, posing a challenge for elucidating plausible abiotic mechanisms. In this study, we investigate the formation of amide bonds between amino acids within highly supersaturated aerosol droplets containing dicyandiamide (DCD), a cyanide derivative potentially present on primordial Earth. Metastable states, i.e. supersaturation, within individual micron-sized droplets are studied using both an optical trap and a linear quadrupole electrodynamic balance. When irradiated with intense visible light, amide bond formation is observed to occur and can be monitored using vibrational bands in Raman spectra. The reaction rate is found to be strongly influenced by droplet size and kinetic modelling suggests that it is driven by the photochemical product of a DCD self-reaction. Our results highlight the potential of atmospheric aerosol particles as reaction environments for peptide synthesis and have potential implications for the prebiotic chemistry of early Earth.

Synthesis and Application of Titanium Carbide (Ti3C2)-Cobalt Sulfide (Co3S4) Nanocomposites in Amino Acid Biosensing.

Background The fabrication of titanium carbide (Ti3C2)-cobalt sulfide (Co3S4)-based biosensors with high sensitivity and selectivity can change the biosensor manufacturing industry completely. Molecular and clinical diagnostics, disease progression monitoring, and drug discovery could utilize these sensors for early biomarker detection. MXene (Ti3C2) is a two-dimensional material with exceptional electrical conductivity, hydrophilicity, great thermal stability, large interlayer spacing, and a high surface area. Ti3C2'sremarkable characteristics make it well-suited for biomolecule immobilization and target analyte detection. Co3S4is a transition metal chalcogenide that has shown great potential in biosensors. Co3S4 nanoparticles (NPs) can potentially enhance Ti3C2 electrocatalytic activity, particularly in amino acid detection. L-arginine is a semi-essential amino acid, and the body frequently uses it to support healthy circulation and plays a crucial role in protein synthesis. We fabricated the Ti3C2-Co3S4 biosensor for L-arginine detection. Aim This study aims to synthesize and apply Ti3C2-Co3S4 nanocomposites in amino acid biosensing. Materials and methods The Ti3C2 nanosheets were synthesized by the selective removal of an aluminum (Al) layer from the precursor (Ti3AlC2) using hydrofluoric acid (HF). The resulting mixture serves as an etchant, especially targeting the Al layers on Ti3AlC2while protecting the desired MXene layers at room temperature. Cobalt nitrate hexahydrate was dissolved in deionized water. Sodium hydroxide was added to the cobalt solution and stirred. Thioacetamide was added to the above solution and stirred (Solution B). A mixture of Solution A and Solution B was stirred for 30 minutes. The mixture is transferred to a hydrothermal reactor and maintained at a temperature of 180°C for 12 hours. Once the reaction completes, we cool the resultant mixture to room temperature and then filter it using the washing technique. The sample underwent a 12-hour drying process at 80°C. Results This study investigated the use of a biosensor that employed Ti3C2-Co3S4 NPs to detect the concentration of L-arginine. The X-ray diffraction (XRD) shows clear and distinct peaks, which means that the synthesized Ti3C2-Co3S4 nanostructures have a crystalline structure.Scanning electron microscopy (SEM) analysis revealed that the sheetlike structure of synthesized Ti3C2-Co3S4 nanostructuresrevealedthe crystalline morphology. The results of this study show that the Ti3C2-Co3S4NP-based biosensor can be used to detect L-arginine in a sensitive and selective way. Conclusion This study investigated the synthesis of Ti3C2-Co3S4NPs and their ability to detect L-arginine levels and show a distinct correlation between the L-arginine concentration and the fluorescence intensity, demonstrating the biosensor's effectiveness in detecting L-arginine levels.

Flaring Inflammation and ER Stress by an Organelle-Specific Fluorescent Cage.

The endoplasmic reticulum (ER) plays an important role in protein synthesis and its disruption can cause protein unfolding and misfolding. Accumulation of such proteins leads to ER stress, which ultimately promotes many diseases. Routine screening of ER activity in immune cells can flag serious conditions at early stages,but the current clinically used bio-probes have limitations. Herein, an ER-specific fluorophore based on abiocompatiblebenzothiadiazole-imine cage (BTD-cage) with excellent photophysical properties is developed. The cage outperforms commercially available ER stains in long-term live cell imaging with no fading or photobleaching over time.The cageisresponsive to different levels of ER stress whereitsfluorescence increases accordingly. Incorporating the bio-probe into an immune disorder model, a6-, 21-, and 48-foldincrease in intensity is shown in THP-1, Raw 246.7, and Jurkat cells, respectively (within 15min).Theseresults strongly support that this system can be used for rapid visual and selective detection of ER stress. It is envisaged that tailoring molecular interactions and molecular recognition usingsupramolecular improved fluorophorescan expand the library of biological probes for enhanced selectivity and targetability toward cellular organelles.

Molecular dynamics simulation studies and dynamic network analysis of Bacillus subtilis YsxC in GDP and GTP-Mg 2+ bound states

Ribosomes are essential cellular organelles made up of rRNA and rproteins found in both prokaryotic and eukaryotic cells, and play an important role in protein synthesis. The functional ribosomes are synthesized through the process called ribosome biogenesis, which proceeds with the help of certain factors such as chaperones, ribosomal proteins, and GTPases. GTPases bind to the premature ribosomal subunit and function as a checkpoint, ensuring the proper assembly of other proteins. To aid this process, GTPase undergoes conformational changes, alternating between an active GTP-bound state and an inactive GDP bound state. YsxC is a GTPase that functions this way, to help in the maturation of 50S subunit. Although YsxC plays an important role in ribosome biogenesis, a detailed knowledge of how the GDP and GTPMg2+ states of YsxC assist in the switching process is yet to be realized. Therefore, a study on the GDP and GTP-Mg2+ bound states is done for YsxC GTPase of Bacillus subtilis by all-atom molecular dynamics simulation for a period of 500 ns. The simulations aided the analysis of the RMSD from which it was noticed that both the GDP and GTP-Mg2+ states of YsxC attained equilibration after 200ns. The analysis of the Rg for the systems showed that the GTP-Mg2+ system showed higher values, in comparison to the GDP system, indicating conformational changes in the two systems. Thus, a residue-wise calculation for the difference in the SASA (solvent accessible surface area) of the GTP-Mg2+ and GDP system is done, and it is noticed that the GTP-Mg2+ system, especially in the regions recognized as Switch I and Switch II had a higher difference in SASA value. In which Switch I’s Lys55 (positive residue) and Switch II’s Arg89 (positive residue) showed a distinct difference, indicating that Switch I has more accessibility to solvent. Giving rise to a supposition that the residue Lys55 attracts the negative rRNA and aids in the binding of YsxC with the premature 50S subunit in the GTP-Mg2+ bound state by associating with the rRNA. To fathom the role of the GTP-Mg2+ bound state in increasing the SASA value of Switch I region, a community network is constructed to extract the shortest path. More number of paths are observed between the selected residue in the GTP-Mg2+ bound state inferring that the increased SASA in the Lys55 is due to the stronger connection with the nucleotide. Overall this study suggest that enhanced surface exposure of YsxC’s Sw-I in the GTP-Mg2+ bound state facilitate the GTP-Mg2+ bound system to involve in ribosome biogenesis by interacting with ribosomal constituents (rRNA and rproteins).

The β-subunit of tryptophan synthase is a latent tyrosine synthase.

Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the β-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine.

34 DPP Lecture: Evidence of methionine effects on health and immune response of animals

Abstract Methionine is an essential amino acid that is commonly supplemented in livestock diets, including for swine and poultry. As with other amino acids, requirements for methionine have largely been based on maximizing growth performance; however, there is evidence that nutrient requirements, including for sulfur amino acids, are altered by immune status of the animal. As such, there has been significant interest in exploring the non-proteinogenic functions of amino acids and how these work to support animal health in addition to growth performance. While methionine has a key role in protein synthesis, it is involved in numerous biological functions, including participation in transsulfuration and transmethylation pathways, as a precursor of intermediates in metabolic pathways (e.g., carnitine, cystine), and in components of the immune response (e.g., acute-phase proteins), and in antioxidant status (i.e. glutathione, taurine). While undersupply and oversupply of methionine can lead to deficiency and toxicity, respectively, it is likely that supplementation with methionine above the current recommendations for growth would be beneficial for animal health, especially during times of stress or immune stimulation.

Screening of Amino Acids as a Safe Energy Source for Isolated Rat Pancreatic Acini.

Amino acids play an essential role in protein synthesis, metabolism, and survival of pancreatic acini. Adequate nutritional support is important for acute pancreatitis treatment. However, high concentrations of arginine and lysine may induce acute pancreatitis. The study aimed to identify the most suitable l -amino acids as safe energy sources for pancreatic acinar cells. Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1-20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell death was evaluated with fluorescent microscopy and DNA gel electrophoresis. Among the tested amino acids, glutamate, glutamine, alanine, lysine, and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, whereas arginine, histidine, and asparagine were not. Lysine, arginine, and glutamine (20 mM) caused complete loss of plasma membrane integrity of acinar cells after 24 hours of incubation. Glutamine also caused early (2-4 hours) cell swelling and blebbing. Aspartate, asparagine, and glutamate only moderately decreased the number of viable cells, whereas alanine and histidine were not toxic. DNA fragmentation assay and microscopic analysis of nuclei showed no evidence of apoptosis in cells treated with amino acids. Alanine and glutamate are safe and effective energy sources for mitochondria of pancreatic acinar cells.

Exploration of the Amino Acid Metabolic Profiling and Pathway in Clonorchis sinensis-Infected Rats Revealed by the Targeted Metabolomic Analysis.

Background: Clonorchiasis remains a serious public health problem. However, the molecular mechanism underlying clonorchiasis remains largely unknown. Amino acid (AA) metabolism plays key roles in protein synthesis and energy sources, and improves immunity in pathological conditions. Therefore, this study aimed to explore the AA profiles of spleen in clonorchiasis and speculate the interaction between the host and parasite. Methods: Here targeted ultrahigh performance liquid chromatography multiple reaction monitoring mass spectrometry was applied to discover the AA profiles in spleen of rats infected with Clonorchis sinensis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (KEGG) was performed to characterize the dysregulated metabolic pathways. Results: Pathway analysis revealed that phenylalanine, tyrosine, and tryptophan biosynthesis and β-alanine metabolism were significantly altered in clonorchiasis. There were no significant correlations between 14 significant differential AAs and interleukin (IL)-1β. Although arginine, asparagine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were positively correlated with IL-6, IL-10, tumor necrosis factor (TNF)-α as well as aspartate aminotransferase and alanine aminotransferase; β-alanine and 4-hydroxyproline were negatively correlated with IL-6, IL-10, and TNF-α. Conclusion: This study reveals the dysregulation of AA metabolism in clonorchiasis and provides a useful insight of metabolic mechanisms at the molecular level.

Abstract 4125: Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis

Abstract Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. LIN28B, an RNA-binding protein that regulates mRNA translation, has garnered significant interest in recent cancer research. Our lab has established that LIN28B is an oncogene and promotes CRC metastasis. However, the exact mechanisms through which LIN28B exerts these effects remain elusive. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is altered in 50-70% of CRCs and is characterized by PIK3CA gene mutations in 20-30% of CRC cases. PIK3CA encodes the catalytic subunit p110α of the PI3Kα enzyme, which is critical for PI3K/AKT signaling. Activation of this pathway is associated with worse CRC prognosis. Among the components of this signaling cascade, the ribosomal protein S6 kinase (S6K) is a crucial downstream effector, playing a pivotal role in protein synthesis and cell growth. Despite the frequent occurrence of PIK3CA mutations in CRC, targeted therapies for PIK3CA-mutant CRC are lacking. Alpelisib, a PI3Kα-specific inhibitor approved by the FDA for a subset of PIK3CA-mutant breast cancers, remains largely unassessed in CRC. We hypothesize that LIN28B-mediated CRC metastasis is facilitated by the activation of the PI3K/AKT pathway, representing a potential target for Alpelisib or other related inhibitors. Results: Our data indicate that LIN28B expression in CRC cells promotes liver metastasis in mouse models of metastatic CRC (mCRC). Furthermore, LIN28B expression activates the PI3K/AKT pathway, as measured by an AKT phosphorylation array. Treatment of CRC cells with SC79, a pan-AKT activator, enhanced cell migration and invasiveness. Similarly, VillinCre;R26Pik3ca transgenic mice, expressing a constantly active mutant form of PI3Kα in the intestinal epithelium, demonstrated crypt hyperplasia and an amplified propensity for tumorigenesis, mirroring the effects of LIN28B expression in CRC. Importantly, Alpelisib effectively hinders LIN28B-driven CRC progression in cell lines, transgenic mice-derived organoids, and mCRC mouse models, as well as the growth rate of CRC patient-derived organoids (PDOs). The effects observed with Alpelisib treatment were replicated with inhibition of S6K using either LY25284702 or PF4708671. Moreover, a combination of low-dose Alpelisib with low-dose LY25284702 or PF4708671 synergistically restricted the growth of PDOs. Conclusion: Our data provide compelling evidence that the PI3K/AKT pathway regulates LIN28B-mediated CRC metastasis, and its inhibition by Alpelisib is feasible. This underscores the potential for precision medicine in CRC, offering targeted therapies to PIK3CA-mutant CRC patients who lack specific treatment options. Furthermore, our study highlights the clinical advantages of combined inhibition strategies for improved anti-metastatic outcomes, a field in dire need of interventions in mCRC. Citation Format: Alice E. Shin, Kensuke Sugiura, Secunda W. Kariuki, David A. Cohen, Christopher J. Lengner, Peter A. Sims, Joel T. Gabre, Anil K. Rustgi. Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4125.

N-acetyl-l-methionine dietary supplementation improves meat quality by oxidative stability of finishing Angus heifers

Methionine plays a vital role in protein synthesis, and regulation of antioxidant response in ruminants. This study aimed to assess the effects of dietary supplementation with N-acetyl-l-methionine (NALM), which serves a source of rumen-protected methionine, on growth performance, carcass traits, meat quality, and oxidative stability. Sixty Angus heifers (initial body weight = 408 ± 51.2 kg, 15–18 months) were stratified by body weight and randomly assigned to four dietary treatments: a control group (0% NALM), and experimental groups receiving diets containing 0.125%, 0.25%, and 0.50% NALM (dry matter (DM) basis), respectively. The experiment included a 2-week adaptation and a 22-week data and sample collection period. Results indicated that blood urea nitrogen in the plasma of the 0.25% NALM group was lower compared to the control and the 0.50% NALM groups (P = 0.02). The plasma methionine (P = 0.04), proline (P < 0.01), and tryptophan (P = 0.05) were higher in the 0.25% and 0.50% NALM groups, as well as the methionine and proline in the muscle of the 0.25% NALM group (P < 0.01). The muscle pH (P < 0.01) was increased by supplementing 0.25% and 0.50% NALM in diets but decreased the lactate (P < 0.01). The 0.25% NALM group also increased a* (P = 0.05), decreased L* (P = 0.05), drip loss (P = 0.01), and glycolytic potential in the muscle (P < 0.01). The total antioxidant capacity, superoxide dismutase, glutathione peroxidase, catalase, and glutathione in muscle of 0.25% NALM group were higher than that of the control (P < 0.01), and the malondialdehyde and protein carbonyl were lower (P < 0.01). In conclusion, the dietary supplement with NALM improves meat quality by enhancing the antioxidant effect of lipids and proteins.

Abstract P288: Sex Differences in Krebs Cycle Metabolites May Explain Differential Predisposition to Cardiovascular Disease

Introduction: Although there have been numerous observational studies in the literature describing differential predisposition to cardiovascular disease between sexes, there is relatively little in the literature that provides a biochemical account suitable to explain these disparities. We hypothesized that there may be a fundamental biochemical distinction between sexes, productive first of differential metabolic contexts, and thence ultimately of the differential predisposition to disease states described in the epidemiologic literature. Methods: We employed metabolomic analysis together with an unsupervised learning approach to uncover sex-specific alterations in the abundance of several polar metabolites. Blood was collected from healthy volunteers (5 female, 4 male). Polar metabolites were extracted and analyzed using targeted liquid chromatography coupled with tandem mass spectrometry; we then utilized nonnegative matrix factorization (NMF) to search for metabolomic differences. Results: The female and male signatures differ in metabolites related to Krebs Cycle (KC). In males, we observed statistically significant or nearly significant decreases in citrate (P=0.054), isocitrate (P=0.006), and malate (P=0.076); while aconitate, succinate, fumarate, oxaloacetate, and glyoxylate, an intermediate in an alternative KC pathway, were non-significantly decreased. GABA P=0.0008) was also strikingly decreased in males. The KC product GTP (P=0.014) was significantly decreased as well. Conclusion: We observed significant decreases in several KC intermediates in males when compared with female counterparts, in GABA, and in GTP, which plays important roles in protein synthesis, gluconeogenesis, and cellular signal transduction. The status of the KC as the central hub of metabolism confers wide-ranging implications on this data: the reduced KC functionality observed in our study may confer disproportionate exposure to processes requiring efficient energy production in males.

Cell competition: emerging signaling and unsolved questions.

Multicellular communities have an intrinsic mechanism that optimizes their structure and function via cell-cell communication. One of the driving forces for such self-organization of the multicellular system is cell competition, the elimination of viable unfit or deleterious cells via cell-cell interaction. Studies in Drosophila and mammals have identified multiple mechanisms of cell competition caused by different types of mutations or cellular changes. Intriguingly, recent studies have found that different types of "losers" of cell competition commonly show reduced protein synthesis. In Drosophila, the reduction in protein synthesis levels in loser cells is caused by phosphorylation of the translation initiation factor eIF2α via a bZip transcription factor Xrp1. Given that a variety of cellular stresses converge on eIF2α phosphorylation and thus global inhibition of protein synthesis, cell competition may be a machinery that optimizes multicellular fitness by removing stressed cells. In this review, we summarize and discuss emerging signaling mechanisms and critical unsolved questions, as well as the role of protein synthesis in cell competition.