Essential Arginine Research Articles

Effect of an essential arginine mutation (R473C) on the catalytic reaction of cytochrome c oxidase from Paracoccus denitrificans. Characterization through UV/Vis and electrochemical-FTIR spectroscopy

Effect of an essential arginine mutation (R473C) on the catalytic reaction of cytochrome c oxidase from Paracoccus denitrificans. Characterization through UV/Vis and electrochemical-FTIR spectroscopy

Abstract A005: A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors (such as MRTX1719) have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET™ Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Kelly A. Teske, Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Kaitlin Dunn Hoffman, Jennifer Wilkinson, Michael Beck, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A005.

Abstract 2044: A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Michael Beck, Jennifer Wilkinson, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition [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 2044.

Effects of organic trace minerals chelated with oligosaccharides on growth performance, blood parameters, slaughter performance and meat quality in sheep.

The present study assessed the effects of oligosaccharide-chelated organic trace minerals (OTM) on the growth performance, digestive enzyme activity, blood parameters, slaughter performance, and meat quality indexes of mutton sheep. A total of 60 East Ujumuqin × small-tailed Han crossbred mutton sheep were assigned to two groups (10 duplicates per group) by body weight (26.12 ± 3.22 kg) according to a completely randomized design. Compared to the CON group, the results of the OTM group showed: (1) no significant changes in the initial body weight, final body weight, dry matter intake, average daily gain, and feed conversion ratio (p > 0.05); (2) the activities of trypsin, lipase, and amylase in the jejunum were significantly increased (p < 0.05); (3) serum total protein, albumin, and globulin of the blood were significantly increased (p < 0.05), and the growth factor interleukin IL-10 was significantly higher (p < 0.05), while IL-2, IL-6, and γ-interferon were significantly lower (p < 0.05). Immunoglobulins A, M, and G were significantly higher (p < 0.05); (4) the live weight before slaughter, carcass weights, dressing percentage, eye muscle areas, and GR values did not differ significantly (p > 0.05); (5) shear force of mutton was significantly lower (p < 0.05), while the pH45min, pH24h, drip loss, and cooking loss did not show a significant difference (p > 0.05). The content of crude protein was significantly higher (p < 0.05), while the ether extract content was significantly reduced (p < 0.05), but no significant difference was detected between moisture and ash content; (6) the total amino acids, essential amino acids, semi-essential amino acids, and umami amino acids were significantly increased (p < 0.05). Although umami amino acids were not significant, the total volume increased (p > 0.05). Among these, the essential amino acids, threonine, valine, leucine, lysine in essential amino acids and arginine were significantly increased (p < 0.05). Also, non-essential amino acids, glycine, serine, proline, tyrosine, cysteine, and aspartic acid, were significantly higher (p < 0.05). The content of alanine, aspartate, glutamic acid, phenylalanine, and tyrosine in umami amino acids was significantly higher (p < 0.05).

Mechanism of proton-powered c-ring rotation in a mitochondrial ATP synthase

Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of adenosine triphosphate (ATP). Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.

Inclusion of guanidinoacetic acid in a low metabolizable energy diet improves broilers growth performance by elevating energy utilization efficiency through modulation serum metabolite profile.

This study was aimed to explore the elevating energy utilization efficiency mechanism for the potentially ameliorative effect of guanidinoacetic acid (GAA) addition on growth performance of broilers fed a low metabolizable energy (LME) diet. A total of 576 d old broilers were randomly allocated to one of the six treatments: a basal diet (normal ME, positive control, PC), or an LME diet (50 kcal/kg reduction in ME, negative control, NC) supplemented with 0.02%, 0.04%, 0.06%, and 0.08% GAA from 1 to 42 d of age, respectively. The GAA fortification in LME diet linearly or quadratically dropped (P < 0.05) the feed conversion ratio (FCR) from 22 to 42 and 1 to 42 d of age, abdominal fat rate on day 42, serum alanine aminotransferase (ALT) on day 21, and serum creatinine (CREAN) on days 21 and 42, elevated (P < 0.05) breast muscle rate and leg muscle rate on day 42, serum creatine kinase (CK) on days 21 and 42, as well as alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) on day 21. The dietary optimal GAA levels were 0.03%-0.08% based on the best-fitted quadratic models (P < 0.03) of the above parameters. Thus, the PC, LME, and 0.04% GAA-LME groups were selected for further analysis. Serum essential amino acids (EAA) tryptophan, histidine and arginine, non-essential amino acids (NEEA) serine, glutamine and aspartic acid were significantly decreased (P < 0.05), compared to PC diet by LME or 0.04% GAA-LME diet. 0.04% GAA-LME group reversed (P < 0.05) the reduction of arginine, 3-methyhistidine, and 1-methylhistidine by LME diet. Besides, six birds at 28 d of age from LME and 0.04% GAA-LME groups were selected for energy utilization observation in calorimetry chambers. The results demonstrated that 0.04% GAA-LME group significantly improved (P < 0.05) the ME intake (MEI) and net energy (NE) compared to the LME diet. Overall, these findings suggest that 0.04% GAA is the ideal dose of broilers fed the LME diet, which can significantly improve the growth performance and carcass characteristics by modulation of creatine metabolism through elevating serum CK activity and arginine concentration.

RBM39: A druggable metabolic sensor linking RNA splicing, transcriptional regulation, and metabolic reprogramming in cancer

In a recent issue of Cell, Mossmann etal.1 describe a novel role for an emerging cancer target, RNA-binding motif protein 39, as a metabolic sensor of the conditionally essential amino acid arginine.

Nutrient supplementation by genome-eroded Burkholderia symbionts of scale insects

Hemipterans are known as hosts to bacterial or fungal symbionts that supplement their unbalanced diet with essential nutrients. Among them, scale insects (Coccomorpha) are characterized by a particularly large diversity of symbiotic systems. Here, using microscopic and genomic approaches, we functionally characterized the symbionts of two scale insects belonging to the Eriococcidae family, Acanthococcus aceris and Gossyparia spuria. These species host Burkholderia bacteria that are localized in the cytoplasm of the fat body cells. Metagenome sequencing revealed very similar and highly reduced genomes (<900KBp) with a low GC content (~38%), making them the smallest and most AT-biased Burkholderia genomes yet sequenced. In their eroded genomes, both symbionts retain biosynthetic pathways for the essential amino acids leucine, isoleucine, valine, threonine, lysine, arginine, histidine, phenylalanine, and precursors for the semi-essential amino acid tyrosine, as well as the cobalamin-dependent methionine synthase MetH. A tryptophan biosynthesis pathway is conserved in the symbiont of G. spuria, but appeared pseudogenized in A. aceris, suggesting differential availability of tryptophan in the two host species’ diets. In addition to the pathways for essential amino acid biosynthesis, both symbionts maintain biosynthetic pathways for multiple cofactors, including riboflavin, cobalamin, thiamine, and folate. The localization of Burkholderia symbionts and their genome traits indicate that the symbiosis between Burkholderia and eriococcids is younger than other hemipteran symbioses, but is functionally convergent. Our results add to the emerging picture of dynamic symbiont replacements in sap-sucking Hemiptera and highlight Burkholderia as widespread and versatile intra- and extracellular symbionts of animals, plants, and fungi.

Involvement of insulin-like growth factor binding proteins (IGFBPs) and activation of insulin/IGF-like signaling (IIS)-target of rapamycin (TOR) signaling cascade in pacific white shrimp, Litopenaeus vannamei exposed to acute low-salinity

Acute low-salinity induces multiple stress responses and significantly impact the health of aquatic animals. However, the molecular regulation network and metabolic adaptation of low salinity stress in aquatic organisms remain unclear. The insulin-like growth factor binding proteins (IGFBPs) and insulin/IGF-like signaling (IIS)-target of rapamycin (TOR) signaling cascade are evolutionarily conserved regulators known to play crucial roles in response to various stressors. In this study, we used the euryhaline Pacific white shrimp (Litopenaeus vannamei) as a model organism to investigate the involvement of IGFBP and IIS-TOR signaling cascade in acute low-salinity response. We found seven LvIGFBPs and evaluated 18s as one of most stable internal reference genes under acute low salinity stress. The expression of LvIGFBP4, LvSIBD1, and LvSIBD2 was significantly increased after 6-hours 5‰ salinity treatment. LvSIBD2 was specifically expressed on the surface of gill branch axis. Acute low-salinity significantly induced the phosphorylation of Akt and S6K in the gill but not in the muscle. We found acute low-salinity increased the levels of total essential amino acids, including leucine and arginine, while decreasing the total non-essential amino acid levels in the gill. Furthermore, glucose and key metabolites in glycolysis and tricarboxylic acid cycle (TCA cycle) were increased, indicating remodeling of energy metabolism occurred. These results demonstrate the involvement of IGFBP and IIS-TOR signaling cascade in acute low-salinity response and provide new insights into understanding the mechanism of osmoregulation in euryhaline crustacean species.

Amino Acid Content of Some Species from Trigonella L. Genus Collected from Turkey

A study was undertaken to evaluate the amino acid composition of the seeds of Trigonella cylindracea Desv., Trigonella mesopotamica Hub.-Mor. and Trigonella smyrnea Boiss. which are growing wild in Turkey. The seeds of three species contain both essential (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, arginine, and tryptophan) and non-essential (alanine, aspartic acid, glycine, glutamic acid, proline, serine, and tyrosine) amino acids. The major amino acid was glutamic acid with a value of 4819 mg/100 g (T. cylindracea), 5888 mg/100 g (T. mesopotamica) and 4146 mg/100 g (T. smyrnea). Among the essential amino acids, lysine is found at the highest rate in all three species. The highest amount of lysine was found in the seeds of T. mesopotamica, followed by the seeds of T. cylindracea and T. smyrnea (3352±0.02 mg/100 g, 3059±0.03 mg/100 g and 2947±0.04 mg/100 g, respectively). The literature review showed that the amino acid composition of the studied seeds were evaluated for the first time in this study, therefore; our study provides important preliminary data to the literature in terms of evaluating the chemical compositions of three Trigonella species.

A novel score system based on arginine metabolism-related genes to predict prognosis, characterize immune microenvironment, and forecast response to immunotherapy in IDH-wildtype glioblastoma.

Introduction: Glioblastoma is one of the most lethal cancers and leads to more than 200,000 deaths annually. However, despite lots of researchers devoted to exploring novel treatment regime, most of these attempts eventually failed to improve the overall survival of glioblastoma patients in near 20 years. Immunotherapy is an emerging therapy for cancers and have succeeded in many cancers. But most of its application in glioblastoma have been proved with no improvement in overall survival, which may result from the unique immune microenvironment of glioblastoma. Arginine is amino acid and is involved in many physiological processes. Many studies have suggested that arginine and its metabolism can regulate malignancy of multiple cancers and influence the formation of tumor immune microenvironment. However, there is hardly study focusing on the role of arginine metabolism in glioblastoma. Methods: In this research, based on mRNA sequencing data of 560 IDH-wildtype glioblastoma patients from three public cohorts and one our own cohort, we aimed to construct an arginine metabolism-related genes signature (ArMRS) based on four essential arginine metabolism-related genes (ArMGs) that we filtered from all genes with potential relation with arginine metabolism. Subsequently, the glioblastoma patients were classified into ArMRS high-risk and low-risk groups according to calculated optimal cut-off values of ArMRS in these four cohorts. Results: Further validation demonstrated that the ArMRS was an independent prognostic factor and displayed fine efficacy in prediction of glioblastoma patients' prognosis. Moreover, analyses of tumor immune microenvironment revealed that higher ArMRS was correlated with more immune infiltration and relatively "hot" immunological phenotype. We also demonstrated that ArMRS was positively correlated with the expression of multiple immunotherapy targets, including PD1 and B7-H3. Additionally, the glioblastomas in the ArMRS high-risk group would present with more cytotoxic T cells (CTLs) infiltration and better predicted response to immune checkpoint inhibitors (ICIs). Discussion: In conclusion, our study constructed a novel score system based on arginine metabolism, ArMRS, which presented with good efficacy in prognosis prediction and strong potential to predict unique immunological features, resistance to immunotherapy, and guide the application of immunotherapy in IDH-wild type glioblastoma.

Free-Choice Feeding of Whole Grains Improves Meat Quality and Intestinal Development of Pigeon Squabs Compared with Complete Pelleted Feed.

Effects of different feeding strategies on meat quality and intestinal development in pigeon squabs were investigated. 120 pairs of pigeons with two squabs each were assigned to five groups (four free-choice feeding systems and one complete feeding system): T1 (corn, pea, wheat, and pelleted feed), T2 (corn, wheat, and pelleted feed), T3 (corn, pea, and pelleted feed), T4 (corn and pelleted feed), and T5 (complete pelleted feed). Compared with T5, the diet in T4 made the breast meat redder and more yellow (p < 0.05). T2 and T4 resulted in an enhanced total superoxide dismutase activity of meat. Breast muscle in T1 and T2 was determined to have higher contents of nonessential amino acids, glycine, alanine, and glutamic acid (p < 0.05). The contents of the essential amino acids, lysine, threonine, valine, histidine, and arginine were also higher in T1 (p < 0.05). Villus height, surface area, and alkaline phosphatase activity of the duodenum and jejunum in T2 were the highest among the treatments (p < 0.05). In conclusion, free-choice feeding system can improve the meat quality and intestinal development of pigeon squabs, but which combination method of whole grains to use in the production depends on the feeding purpose.

Functional food ingredient - chromium complex with protein fermentolysate of bivalve Mactra chinensis for the prevention of hyperlipidemia and obesity

The creation of specialized food systems capable of correcting body weight and influencing lipid metabolism is an urgent task at the present stage. The main way to create such food systems is to modify product profiles (lipid, protein, carbohydrate, mineral, vitamin, etc.). The aim of the study was to obtain and characterize a new food source of an organic form of chromium for the prevention of hyperlipidemia and obesity in the form of a complex with peptide fractions of fermentolysate proteins of the Mactra chinensis bivalve mollusk from the Far Eastern region. Material and methods. Soft food parts of the Mactra chinensis bivalve mollusk from the Far Eastern region (the motor muscle and mantle) were used as objects of research. Mollusk specimens were collected in the Amur Bay in June - September 2022. Biotechnological modification of soft tissue proteins was carried out using alkaline protease protozyme B with the following parameters: the duration of the process - 12 and 24 hours, pH - 7.0-7.2, temperature 55 °C. The fractional composition of proteins and peptides was determined by medium pressure gel permeation chromatography, the molecular weight (MW) of proteins and peptides was calculated using MW markers by comparing retention volumes. Extraction of free amino acids was carried out with 70% ethanol for 24 hours at a temperature of 20 °C, the composition and quantitative content of amino acids were determined by HPLC. The complexation reaction was carried out by adding to the fermentolysate with stirring a 10% aqueous solution of CrCl3×6H2O in a mass ratio of 6.25 × nitrogen in the liquid fraction : chromium (III) chloride = 20:1. Process conditions: temperature 20-25 °C, time 60 min, pH 7.0-7.1. The chromium content was determined by the atomic absorption method using spectrophotometer. Results. The content of low-molecular fractions increased more significantly over time in the mantle fermentolysate. The content of the high-molecular fraction of peptides weighing more than 160 kDa was minimal for both the muscle and the mantle and didn't exceed 1.1%. Low-molecular fractions weighing no more than 18 kDa in the 24-hour fermentolysate accounted for 79.6% for the muscle and 86.9% for the mantle. The obtained fermentolysates were characterized by a high content of the biologically active amino acid taurine - 25.9% (muscle) and 30.05% (mantle) of the total amino acid content. The high content of such essential amino acids as glycine, alanine, leucine, lysine, and the conditionally essential amino acid arginine was also determined. The obtained fermentolysates of soft tissue proteins of the Mactra chinensis were characterized by a fairly balanced amino acid composition. It has been experimentally established that more than 84% of the total chromium content in muscle fermentolysate and 80.9% in mantle fermentolysate was associated with peptide fractions with molecular weights from 24 to 1.4 kDa, and the highest specific chromium content - 1.67 mg/g protein (muscle) and 1.58 mg/g protein (mantle) was determined in the interval fractions with molecular weights of 18.0-12.5 kDa. The minimum specific content of the trace element was established for high-molecular fractions of 160-67 kDa and low-molecular fraction weighing less than 1.4 kDa. Conclusion. Biotechnological modification of soft tissue proteins of the Mactra chinensis bivalve mollusk from the Far Eastern region made it possible to obtain a balanced amino acid composition fermentolysate with a high content of the free biologically active amino acid taurine. Fortification of the obtained fermentolysate with chromium demonstrated high efficiency of its binding to the amino acid and peptide matrices of the protein hydrolysate. The chromium complex with fermentolysate of proteins of the Mactra chinensis bivalve mollusk can be used as a food source of chromium and taurine, including as a functional ingredient in special food systems for the prevention of hyperlipidemia and obesity.

Genome-wide association study for the free amino acid and nucleotide components of breast meat in an F2 crossbred chicken population

Flavor is an important sensory trait of chicken meat. The free amino acid (FAA) and nucleotide (NT) components of meat are major factors affecting meat flavor during the cooking process. As a genetic approach to improve meat flavor, we performed a genome-wide association study (GWAS) to identify the potential candidate genes related to the FAA and NT components of chicken breast meat. Measurements of FAA and NT components were recorded at the age of 10 weeks from 764 and 767 birds, respectively, using a White leghorn and Yeonsan ogye crossbred F2 chicken population. For genotyping, we used 60K Illumina single-nucleotide polymorphism (SNP) chips. We found a total of nine significant SNPs for five FAA traits (arginine, glycine, lysine, threonine content, and the essential FAAs and one NT trait (inosine content), and six significant genomic regions were identified, including three regions shared among the essential FAAs, arginine, and inosine content traits. A list of potential candidate genes in significant genomic regions was detected, including the KCNRG, KCNIP4, HOXA3, THSD7B, and MMUT genes. The essential FAAs had significant gene regions the same as arginine. The genes related to arginine content were involved in nitric oxide metabolism, while the inosine content was possibly affected by insulin activity. Moreover, the threonine content could be related to methylmalonyl-CoA mutase. The genes and SNPs identified in this study might be useful markers in chicken selection and breeding for chicken meat flavor.

Optimizing heterotrophic production of Chlorella sorokiniana SU-9 proteins potentially used as a sustainable protein substitute in aquafeed

Alternative protein sources for the reduction/replacement of fish meal in aqua-feeds are in urgent demand. Microalgae are considered sustainable protein sources for aquaculture due to their high-quality proteins with a complete profile of essential amino acids. This study examined the heterotrophic production of proteins from Chlorella sorokiniana SU-9. Culture parameters for maximal biomass and protein production are as follows: glucose – 10 g/L glucose, sodium nitrate – 1.5 g/L, and iron – 46 μM iron in BG-11 medium. Under optimal conditions, biomass content, protein content and protein productivity of SU-9 reached 4.14 ± 0.20 g/L, 403 ± 33 mg/g and 382 ± 36 mg/L/d, respectively. The protein profile of Chlorella sorokiniana SU-9 is comparable to fishmeal and soybean meal. The essential amino acids arginine, lysine and cysteine, along with glutamine and glutamate, were high. The production cost of SU-9 can be significantly reduced under heterotrophic cultivation conditions, making it a potential protein substitute in aquafeed.

Replacement of dietary fish meal with Clostridium autoethanogenum meal on growth performance, intestinal amino acids transporters, protein metabolism and hepatic lipid metabolism of juvenile turbot (Scophthalmus maximus L.)

Clostridium autoethanogenum meal (CAM) is a novel single-cell protein, which is produced from bacteria using carbon monoxide (CO) as sole carbon source. To evaluate the efficiency of CAM as an alternative for dietary fish meal, a 56-days growth experiment was performed on juvenile turbot (Scophthalmus maximus L.) with initial average weight of 9.13 ± 0.02 g. Six iso-nitrogenous (crude protein, 51.0%) and iso-lipidic (crude lipid, 11.5%) diets were formulated with 0%, 15%, 30%, 45%, 60% and 80% dietary fish meal protein substituted by CAM protein, which were designated as CAM0 (the control group), CAM15, CAM30, CAM45, CAM60 and CAM80, respectively. Results showed that no significant differences were observed in survival rate (over 97.50%) among different dietary treatments (p > 0.05). The specific growth rate (SGR) was not significantly affected when replacement levels of dietary fish meal with CAM were less than 45% (p > 0.05). The feed intake (FI) was significantly linear reduced with increasing dietary CAM (p < 0.05), whereas no significant differences were observed in feed efficiency ratio (FER), protein efficiency ratio (PER) and protein retention (PR) among different dietary treatments (p > 0.05). With increasing dietary CAM, lipid retention (LR) and carcass lipid tended to be increased in both significantly linear and quadratic patterns (p < 0.05). The apparent digestibility coefficient (ADC) of crude protein and some essential amino acids, including threonine, valine, lysine, histidine and arginine, showed significantly linear increase with increasing dietary CAM (p < 0.05). Furthermore, with the increase of dietary CAM, the gene expression of intestinal peptide and amino acids transporters was first up-regulated and then down-regulated with significantly quadratic pattern (p < 0.05), peaking in fish fed with diets CAM30 or CAM45, which was similar to the expression of genes related protein degradation in muscle. For genes related to protein metabolism in liver and muscle, the expression of mammalian target of rapamycin (mtor) was not significantly affected by dietary CAM, while the general control nonderepressible 2 (gcn2) tended to be first up-regulated and then down-regulated with significantly quadratic pattern (p < 0.05). Apart from that, the lipid metabolism of turbot was also affected by high dietary CAM, evidenced by increased expression of hepatic genes related to lipogenesis as well as reduced expression of genes related to lipid oxidation and lipid transport. In conclusion, CAM can replace up to 45% fish meal protein in diet for juvenile turbot without significantly adverse effects on growth performance. But excessive dietary CAM would result in significant growth reduction, and excessive lipid deposition may also occur in fish fed diets with high levels of CAM.

F1FO ATP synthase molecular motor mechanisms.

The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of c-subunits that is attached to subunit-γ. This c-ring rotates relative to the subunit-a stator in response to transmembrane proton flow driven by a pH gradient, which drives subunit-γ rotation in the opposite direction to force ATP synthesis in F1. Single-molecule studies of F1FO embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F1-ATPase-driven rotation every 36° (at each c-subunit in the c10-ring of E. coli F1FO) and was able to rotate 11° in the direction of ATP synthesis. Protonation and deprotonation of the conserved carboxyl group on each c-subunit is facilitated by separate groups of subunit-a residues, which were determined to have different pKa’s. Mutations of any of any residue from either group changed both pKa values, which changed the occurrence of the 11° rotation proportionately. This supports a Grotthuss mechanism for proton translocation and indicates that proton translocation occurs during the 11° steps. This is consistent with a mechanism in which each 36° of rotation the c-ring during ATP synthesis involves a proton translocation-dependent 11° rotation of the c-ring, followed by a 25° rotation driven by electrostatic interaction of the negatively charged unprotonated carboxyl group to the positively charged essential arginine in subunit-a.

Abstract 94: PRMT5 substrate recruitment as a therapeutic target in CDKN2A/MTAP null cancers

Abstract PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5. Current efforts are focused on understanding whether PRMT5-substrate adaptor disruption synergizes with immune therapies or new classes of PRMT5 inhibitors in CDKN2A/MTAP null cancer. Citation Format: Kathleen M. Mulvaney, Brian McMillan, Christa Blomquist, Nischal Acharya, Matthew Ranaghan, Devishi Kesar, Alessandra Ianari, William Sellers. PRMT5 substrate recruitment as a therapeutic target in CDKN2A/MTAP null cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 94.

Liquid chromatography-tandem mass spectrometry based quantification of arginine metabolites including polyamines in different sample matrices

The conditionally essential amino acid arginine and its metabolic products play an important role in different biological processes, such as metabolic regulation of the immune response, including macrophage activation and polarization and regulation of T cell function. Furthermore, the polyamine spermidine has a role in aging and age-related diseases. Additionally, altered polyamine metabolism may be associated with neurodegenerative diseases, while polyamine levels may present useful biomarkers associated with severity of Parkinson's disease or with progression of non-alcoholic fatty liver disease. In the present study, a simple, derivatization-free hydrophilic interaction liquid chromatography based tandem mass spectrometry (LC-MS/MS) method is described, that allows the accurate quantification of arginine and related amine, polyamine and acetylated polyamine metabolites in different experimental sample matrices, such as cell lysates, cell culture supernatants and tissues. Ten arginine metabolites, including citrulline, agmatine, ornithine, putrescine, spermidine, spermine, N1-acetylspermidine, N1-acetylspermine, N1,N12-diacetylspermine and arginine in conjunction with the metabolic cofactors S-adenosylhomocysteine and S-adenosylmethionine are simultaneously analyzed within a total LC-MS/MS run time of 9.5 min. The assay is suitable to quantify concentration ranges over multiple orders of magnitude for all metabolites with averaged accuracies observed at 103.2% ± 6.8%, 99.0% ± 4.2% and 100.4% ± 4.3% in cell lysates, cell culture supernatant and tissue extracts, respectively. Inter-day coefficients of variation ranged from 5.9 to 14.8% in cell lysates, 6.7 to 14.6% in cell culture supernatants and 5.3 to 12.0% in tissue extracts. The method was successfully applied to cell culture systems of different origin as well as different murine tissues and organs. The herein described LC-MS/MS method provides a simple tool for a fast and simultaneous analysis of arginine metabolites, including polyamines and their respective metabolic cofactors. Assay performance characteristics demonstrate suitability for applications in different experimental and preclinical settings.

GAP positions catalytic h-Ras residue Q61 for GTP hydrolysis in MD simulations

Ras binds to a GTPase activating protein (GAP) to turn off the cell propagation signal. Together, they form a transition state that has a very precise configuration that catalyzes GTP hydrolysis. GAP provides an essential arginine residue to the transition state. Fluorescence experiments show that an arginine analogue alone cannot restore Ras function. We hypothesize that the shared interface between the proteins plays a critical role in the Ras-GAP transition state. We perform molecular dynamics simulations of the Ras-GAP complex and of Ras to do a comparison of Ras structural features and conformational freedom.