Amino Acid Availability Research Articles

Blastocyst trophectoderm endocytic activation, a marker of adverse developmental programming.

The mouse preimplantation embryo is sensitive to its environment, including maternal dietary protein restriction, which can alter the developmental programme and affect lifetime health. Previously, we have shown maternal low-protein diet (LPD) causes a reduction in blastocyst mTORC1 signalling coinciding with reduced availability of branched-chain amino acids (BCAAs) in surrounding uterine fluid. BCAA deficiency leads to increased endocytosis and lysosome biogenesis in blastocyst trophectoderm (TE), a response to promote compensatory histotrophic nutrition. Here, we first investigated the induction mechanism by individual variation in BCAA deficiency in an in vitro quantitative model of TE responsiveness. We found isoleucine (ILE) deficiency as the most effective activator of TE endocytosis and lysosome biogenesis, with less potent roles for other BCAAs and insulin; cell volume was also influential. TE response to low ILE included upregulation of vesicles comprising megalin receptor and cathepsin-B, and the response was activated from blastocyst formation. Secondly, we identified the transcription factor TFEB as mediating the histotrophic response by translocation from cytoplasm to nucleus during ILE deficiency and in response to mTORC1 inhibition. Lastly, we investigated whether a similar mechanism responsive to maternal nutritional status was found in human blastocysts. Blastocysts from women with high body-mass index, but not the method of fertilisation, revealed stimulated lysosome biogenesis and TFEB nuclear migration. We propose TE lysosomal phenotype as an early biomarker of environmental nutrient stress that may associate with long-term health outcomes.

Knockout of the Amino Acid Transporter SLC6A19 and Autoimmune Diabetes Incidence in Female Non-Obese Diabetic (NOD) Mice.

High protein feeding has been shown to accelerate the development of type 1 diabetes in female non-obese diabetic (NOD) mice. Here, we investigated whether reducing systemic amino acid availability via knockout of the Slc6a19 gene encoding the system B(0) neutral amino acid transporter AT1 would reduce the incidence or delay the onset of type 1 diabetes in female NOD mice. Slc6a19 gene deficient NOD mice were generated using the CRISPR-Cas9 system which resulted in marked aminoaciduria. The incidence of diabetes by week 30 was 59.5% (22/37) and 69.0% (20/29) in NOD.Slc6a19+/+ and NOD.Slc6a19−/− mice, respectively (hazard ratio 0.77, 95% confidence interval 0.41–1.42; Mantel-Cox log rank test: p = 0.37). The median survival time without diabetes was 28 and 25 weeks for NOD.Slc6a19+/+ and NOD.Slc6a19−/− mice, respectively (ratio 1.1, 95% confidence interval 0.6–2.0). Histological analysis did not show differences in islet number or the degree of insulitis between wild type and Slc6a19 deficient NOD mice. We conclude that Slc6a19 deficiency does not prevent or delay the development of type 1 diabetes in female NOD mice.

Combination of osmotic stress and sugar stress response mechanisms is essential for Gluconacetobacter diazotrophicus tolerance to high-sucrose environments.

Sugar-rich environments represent an important challenge for microorganisms. The osmotic and molecular imbalances resulting from this condition severely limit microbial metabolism and growth. Gluconacetobacter diazotrophicus is one of the most sugar-tolerant prokaryotes, able to grow in the presence of sucrose concentrations up to 30%. However, the mechanisms that control its tolerance to such conditions remain poorly exploited. The present work investigated the key mechanisms of tolerance to high sugar in G. diazotrophicus. Comparative proteomics was applied to investigate the main functional pathways regulated in G. diazotrophicus when cultivated in the presence of high sucrose. Among 191 proteins regulated by high sucrose, regulatory pathways related to sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system were highlighted. The role of these pathways on high-sucrose tolerance was investigated by mutagenesis analysis, which revealed that the knockout mutants zwf::Tn5 (sugar metabolism), tbdr::Tn5 (nutrient uptake), mtlK::Tn5 (compatible solute synthesis), pepN::Tn5 (proteolytic system), metH::Tn5 (amino acid metabolism), and ilvD::Tn5 (amino acid metabolism) became more sensitive to high sucrose. Together, our results identified mechanisms involved in response to high sugar in G. diazotrophicus, shedding light on the combination of osmotolerance and sugar-tolerance mechanisms. KEY POINTS: • G. diazotrophicus intensifies glycolysis to metabolize the excess of sugar. • G. diazotrophicus turns down the uptake of nutrients in response to high sugar. • G. diazotrophicus requires amino acid availability to resist high sugar.

Crystal structure of Aspergillus fumigatus AroH, an aromatic amino acid aminotransferase.

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5′‐phosphate‐dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L‐kynurenine and α‐aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus‐related diseases. We have solved the crystal structure of Af‐AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH.

The Combinatorial Fusion Cascade to Generate the Standard Genetic Code.

Combinatorial fusion cascade was proposed as a transition stage between prebiotic chemistry and early forms of life. The combinatorial fusion cascade consists of three stages: eight initial complimentary pairs of amino acids, four protocodes, and the standard genetic code. The initial complimentary pairs and the protocodes are divided into dominant and recessive entities. The transitions between these stages obey the same combinatorial fusion rules for all amino acids. The combinatorial fusion cascade mathematically describes the codon assignments in the standard genetic code. It explains the availability of amino acids with the even and odd numbers of codons, the appearance of stop codons, inclusion of novel canonical amino acids, exceptional high numbers of codons for amino acids arginine, leucine, and serine, and the temporal order of amino acid inclusion into the genetic code. The temporal order of amino acids within the cascade is congruent with the consensus temporal order previously derived from the similarities between the available hypotheses. The control over the combinatorial fusion cascades would open the road for a novel technology to develop artificial microorganisms.

Nitrogen Balance after the Administration of a Prolonged-Release Protein Substitute for Phenylketonuria as a Single Dose in Healthy Volunteers.

Nitrogen balance is the difference between nitrogen excreted as urea and nitrogen ingested, mainly in proteins. Increased circulating concentrations of amino acids (AA) in the bloodstream are usually associated with proportional increases in the production and excretion of urea. Previously, we reported results from a randomized, controlled, single-dose, crossover trial in healthy adult volunteers (n = 30) (Trial Registration: ISRCTN11016729), in which a Test product (prolonged-release AA mixture formulated with Physiomimic Technology™ (PT™)) significantly slowed down the release and reduced the peak plasma concentrations of essential AAs compared with a free AA mixture (Reference product) while maintaining essential AA bioavailability. Here, we report an assessment of the nitrogen balance from the same study. The amount of nitrogen contained in plasma AAs, levels of blood urea nitrogen (BUN) (p < 0.0001) and changes in BUN (p < 0.0001) were smaller after the Test product compared with the Reference product. These findings suggest that the production of urea in proportion to systemic AA availability was significantly smaller after the administration of the Test product compared with the Reference product and that the test product conferred the increased utilization of AAs for protein synthesis and reduced their oxidation and conversion to urea. In the clinical setting, it is possible that the effects of PT™ observed on the disposition of free AAs in this study may translate to health benefits in terms of physiological body composition and growth if used for the treatment of subjects with phenylketonuria (PKU). Further investigation in patients with PKU is warranted.

C-MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency.Graphical c-MYC promotes anabolic metabolism, mitochondrial fitness and lipid remodelling early in cell reprogramming.A high rate of aerobic glycolysis is crucial to provide intermediaries for biosynthetic pathways. To ensure the availability of nucleotides, amino acids and lipids for cell proliferation, cells must provide with a constant flux of the elemental building blocks for macromolecule assembly and fulfil the anabolic demands to reach the critical cellular mass levels to satisfactorily undergo cell division.A high rate of aerobic glycolysis is induced by c-MYC, increasing the amounts of intracellular Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and glyceraldehyde-3-phosphate (GA3P), which can all enter pentose phosphate pathway (PPP) to produce Ribose-5-Phosphate (R5P) and NADPH, which are necessary for the biosynthesis of biomolecules such as proteins, nucleic acids, or lipids. C-MYC-dependent activation of glucose-6-phosphate dehydrogenase (G6PD) may play a critical role in the shunting of G6P to PPP and generation of NADPH. High glycolytic flux increases the amounts of dihydroxyacetone phosphate (DHAP), which is crucial for biosynthesis of phospholipids and triacylglycerols, and pyruvate (Pyr), which can be converted to citrate (Cit) in the mitochondria and enter the biosynthesis of fatty acids (FA). During cell reprogramming, c-MYC-dependent lipid remodelling leads to Polyunsaturated Fatty Acid (PUFA) downregulation and Monounsaturated Fatty Acid (MUFA) upregulation, which may play critical roles in cytoarchitectural remodelling of cell membrane or non-canonical autophagy, respectively. Cardiolipin (pink dots) rise early in cell reprogramming correlates with an increase in mitochondrial fitness, suggesting that c-MYC may restore proper levels of cardiolipins and antioxidant proteins, such as UCP2, to guarantee an optimal mitochondrial function while upholding ROS levels, reinforcing the idea of cell rejuvenation early in cell reprogramming.

Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

Amino Acid Transport and Metabolism in Myeloid Function.

Regulation of amino acid availability and metabolism in immune cells is essential for immune system homeostasis and responses to exogenous and endogenous challenges including microbial infection, tumorigenesis and autoimmunity. In myeloid cells the consumption of amino acids such as arginine and tryptophan and availability of their metabolites are key drivers of cellular identity impacting development, functional polarization to an inflammatory or regulatory phenotype, and interaction with other immune cells. In this review, we discuss recent developments and emerging concepts in our understanding of the impact amino acid availability and consumption has on cellular phenotype focusing on two key myeloid cell populations, macrophages and myeloid derived suppressor cells (MDSCs). We also highlight the potential of myeloid-specific of amino acid transporters and catabolic enzymes as immunotherapy targets in a variety of conditions such as cancer and autoimmune disease discussing the opportunities and limitations in targeting these pathways for clinical therapy.

The Dynamic Conversion of Dietary Protein and Amino Acids into Chicken-Meat Protein.

Simple SummaryThe conversion of dietary protein and amino acids into chicken-meat is a dynamic and complex process. Dietary protein is transferred to protein in a chicken carcass at a conversion ratio in the order of 2.50:1, which leaves scope for improvement. Nevertheless, this conversion ratio in broiler chickens cannot be matched by other terrestrial food-producing animals. The quest for sustainable chicken-meat production would be greatly facilitated by enhancing the efficiency of this conversion. Therefore, this review explores the various pathways and processes involved with the objective of identifying approaches and strategies whereby the transition from dietary protein to chicken-meat protein can be advanced.This review considers the conversion of dietary protein and amino acids into chicken-meat protein and seeks to identify strategies whereby this transition may be enhanced. Viable alternatives to soybean meal would be advantageous but the increasing availability of non-bound amino acids is providing the opportunity to develop reduced-crude protein (CP) diets, to promote the sustainability of the chicken-meat industry and is the focus of this review. Digestion of protein and intestinal uptakes of amino acids is critical to broiler growth performance. However, the transition of amino acids across enterocytes of the gut mucosa is complicated by their entry into either anabolic or catabolic pathways, which reduces their post-enteral availability. Both amino acids and glucose are catabolised in enterocytes to meet the energy needs of the gut. Therefore, starch and protein digestive dynamics and the possible manipulation of this ‘catabolic ratio’ assume importance. Finally, net deposition of protein in skeletal muscle is governed by the synchronised availability of amino acids and glucose at sites of protein deposition. There is a real need for more fundamental and applied research targeting areas where our knowledge is lacking relative to other animal species to enhance the conversion of dietary protein and amino acids into chicken-meat protein.

Amino Acid Removal During Hemodialysis Is Compensated By Protein Ingestion And Not Compromised By Exercise

PURPOSE: Hemodialysis (HD) is a life-saving treatment for end-stage renal disease (ESRD) patients. However, it removes amino acids (AAs) from the circulation, thereby lowering plasma AA concentrations and stimulating muscle catabolism. To prevent deterioration in the nutritional status of HD patients, protein ingestion and exercise during HD are increasingly implemented in clinical practice. Nevertheless, it is not known whether these interventions modulate the removal of AAs from the circulation. METHODS: Therefore, we recruited ten ESRD patients (age: 65 ± 16 y, BMI: 24.2 ± 4.8 kg·m-2) undergoing HD to participate in this double-blind, crossover trial. During four 4-h HD sessions each patient ingested, in a randomized order, 40 g protein (PRO) or a placebo (PLA) both at rest, as well as following 30 min of intradialytic exercise (PRO+EX and PLA + EX, respectively). Blood and spent dialysate samples were collected every 30 min throughout HD to assess AA concentrations and, as such, to quantify plasma AA availability and AA removal. RESULTS: Baseline plasma AA concentrations averaged 2.93 ± 0.40 mmol·L-1, with no differences between interventions (P = 0.66). During PLA and PLA + EX interventions, subsequent plasma AA concentrations decreased over time to 1.84 ± 0.18 and 1.83 ± 0.16 mmol·L-1, respectively (time effect P < 0.001), with no differences between interventions (P = 0.94). Plasma AA concentrations increased following protein ingestion up to peak values of 4.40 ± 0.45 and 4.37 ± 0.73 mmol·L-1 during PRO and PRO+EX interventions, respectively (time effect P < 0.001), with no differences between interventions (P = 0.18). Accordingly, a significant effect of protein ingestion (P < 0.001), but no effect of intradialytic exercise (P = 0.22) was observed in AA availability during PLA, PLA + EX, PRO, and PRO+EX interventions (incremental AUC: -227 ± 54, -208 ± 68, 49 ± 87, and 70 ± 34 mmol·L-1·240 min-1, respectively). AA removal was greater during PRO and PRO+EX interventions (16.6 ± 2.2 and 17.3 ± 2.3 g, respectively) when compared to PLA and PLA + EX interventions (9.8 ± 2.0 and 10.2 ± 1.6 g, respectively; protein effect P < 0.001; intradialytic exercise effect P = 0.32). CONCLUSIONS: Removal of AAs during HD can be compensated for by protein ingestion and is not compromised by intradialytic exercise in ESRD patients.

Pineapple stem-derived bromelain based priming improves pepper seed protein reserve mobilization, germination, emergence and plant growth.

Pepper seeds are slow to germinate and emergence is often non-uniform and incomplete, reducing gains from this cash crop. This study investigated the effects of pineapple stem- derived protease (stem bromelain) based priming on pepper seed germination in relation to reserve mobilization (specifically, proteins and aminoacids), germination, emergence and plant growth. These parameters were compared across two controls, (1) unsoaked seeds and (2) seeds soaked in deionized water, and seeds soaked in pineapple stem bromelain crude extract (treatment). Seeds were soaked in bromelain crude extract possessing a proteolytic activity of 6.25 tU or deionized water (first control) for 3h at 35°C. Light microscopy revealed an abundance of protein bodies in the endosperm of the seeds prior to imbibition. When observed for a period of 96h, these bodies were progressively degraded, with the rate of this degradation being fastest in bromelain-treated seeds. Quantitative analysis of protein levels confirmed this observation: 17.2mg proteins/g FW at 120h after priming in bromelain-treated seeds compared with 22.1mg/g FW in controls (average). The bromelain treatment also increased levels of free amino acids from 3.9mg/g FW in the controls to 4.6mg/g FW after 120h of imbibition. Germination and emergence percentages were initially higher in bromelain-treated seeds: 92.0% germination in bromelain-treated seeds vs. ~ 52.2% in the controls at 18 d; 100% emergence in protease-treated seeds vs. ~ 72.2% in the controls at 18 d. However, these parameters were comparable across the treatment and the controls at 28 d. Importantly, plant fresh and dry weights were significantly higher when seeds were primed with bromelain. The results suggest the use of bromelain extracts for priming pepper seeds based on their proteolytic activity, since germination is dependent on the availability of crude protein and essential aminoacids. The benefits of bromelain seed priming appear to translate into improved seedling growth as well.

GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri

N-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC–MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.

Corn substitution by mesquite bean flour (Prosopis juliflora) maintains growth and improves protein metabolism of Nile tilapia juveniles (Oreochromis niloticus).

The objective of this study was to evaluate the potential of mesquite bean flour (Prosopis juliflora) as an energy ingredient in extruded diets for juvenile Nile tilapia (Oreochromis niloticus). Two experiments were carried out: the first consisted of a study to evaluate the chemical composition and digestibility of the energy and nutrients of MBF; the second consisted of a growth test, in which juveniles consumed diets containing different proportions of corn substitution by MBF, in which zootechnical, hematological, physiological, and metabolic variables were evaluated. It was observed that MBF has a chemical composition similar to corn, as well as the apparent digestibility of energy and nutrients. The higher sucrose/starch ratio of the ingredient stands out, as well as the difference in digestibility (p < 0.05) between the predominant carbohydrates 87.63 and 99.25% for starch and sucrose, respectively. In the growth assay, no difference was observed between zootechnical variables (p > 0.05), and sucrase and alkaline phosphatase activities were increased (p < 0.05), which was not observed for amylase and lipase (p > 0.05). The hematological variables did not change (p > 0.05). Metabolic variables indicate a reduction in gluconeogenesis from amino acids, as can be seen by the reduction in liver transaminase levels (ALT and AST) and glutamate dehydrogenase (GDH), as well as the greater availability of free amino acids in plasmas (p < 0.05). Thus, it can be said that MBF has a high nutritional value and can totally replace corn in diets for juvenile tilapia and the metabolic findings indicate a potential protein-sparing effect.

Gut bacterial gene changes following pegaspargase treatment in pediatric patients with acute lymphoblastic leukemia

Treatment of pediatric acute lymphoblastic leukemia (ALL) with pegaspargase exploits ALL cells dependency on asparagine. Pegaspargase depletes asparagine, consequentially affecting aspartate, glutamine and glutamate. The gut as a confounding source of these amino acids (AAs) and the role of gut microbiome metabolism of AAs has not been examined. We examined asparagine, aspartate, glutamine and glutamate in stool samples from patients over pegaspargase treatment. Microbial gene-products, which interact with these AAs were identified. Stool asparagine declined significantly, and 31 microbial genes changed over treatment. Changes were complex, and included genes involved in AA metabolism, nutrient sensing, and pathways increased in cancers. While we identified changes in a gene (iaaA) with limited asparaginase activity, it lacked significance after correction leaving open other mechanisms for asparagine decline, possibly including loss from gut to blood. Understanding pathways that change AA availability, including by microbes in the gut, could be useful in optimizing pegaspargase therapy.

Collagen peptide provides Streptomyces coelicolor CGMCC 4.7172 with abundant precursors for enhancing undecylprodigiosin production

Effective and ecofriendly converting biomass to chemicals is important for sustainable engineering based on the foreseeable shortage of fossil resources. Undecylprodigiosin (UP) is a promising antibiotic, but the direct feeding of pure precursor amino acids makes it costly for large-scale production. Here, collagen peptide (CP), a renewable animal-derived biomass contains abundant precursor amino acids of UP. CP can act as carbon and nitrogen source for the growth of Streptomyces coelicolor CGMCC 4.7172. The plant biomasses including soybean meal, wheat bran, and malt extract were unsuitable for UP prodution. However, 365.40 µg/L UP was detected after 24 h in the media containing CP, and its highest concentration reached 1198.01 µg/L. UP was also detected in the media containing meat hydrolysates of domestic animals, but its initial production time was delayed, and final concentration was lower than that in the medium containing CP only. Compared the fermentation performances of CP and other proteins, CP has a special superiority for UP production. These results revealed that UP biosynthesis may be dependent on amino acid availability of substrates and CP is beneficial for UP production because of its specific amino acid composition.Graphical abstract

Resetting amino acid metabolism of cancer cells by ATB0,+-targeted nanoparticles for enhanced anticancer therapy

Reprogramed cellular metabolism is one of the most significant hallmarks of cancer. All cancer cells exhibit increased demand for specific amino acids, and become dependent on either an exogenous supply or upregulated de novo synthesis. The resultant enhanced availability of amino acids supports the reprogramed metabolic pathways and fuels the malignant growth and metastasis of cancers by providing energy and critical metabolic intermediates, facilitating anabolism, and activating signaling networks related to cell proliferation and growth. Therefore, pharmacologic blockade of amino acid entry into cancer cells is likely to have a detrimental effect on cancer cell growth. Here we developed a nanoplatform (LJ@Trp-NPs) to therapeutically target two transporters, SLC6A14 (ATB0,+) and SLC7A5 (LAT1), that are known to be essential for the sustenance of amino acid metabolism in most cancers. The LJ@Trp-NPs uses tryptophan to guide SLC6A14-targeted delivery of JPH203, a high-affinity inhibitor of SLC7A5. In the process, SLC6A14 is also down-regulated. We tested the ability of this strategy to synergize with the anticancer efficacy of lapatinib, an inhibitor of EGFR/HER1/HER2-assocated kinase. These studies show that blockade of amino acid entry amplifies the anticancer effect of lapatinib via interference with mTOR signaling, promotion of apoptosis, and suppression of cell proliferation and metastasis. This represents the first study to evaluate the impact of amino acid starvation on the anticancer efficacy of widely used kinase inhibitor.

Response: Commentary: An In Silico-In Vitro Pipeline Identifying an HLA-A*02:01+ KRAS G12V+ Spliced Epitope Candidate for a Broad Tumor-Immune Response in Cancer Patients.

Proteasomes are the central cytosolic and nuclear proteases in eukaryotic cells. They degrade a broad range of proteins, mainly cytoplasmic, though they are also active in other cell compartments (1). The peptides produced during protein degradation can be further processed by cytosolic aminopeptidases to preserve amino acid availability for new protein synthesis. Not all proteasomal products are destinated for full degradation. For example, p105 is cleaved by proteasomes, thereby generating a component of the transcription factor NF-κB. Similarly, osteopontin seems to be processed by these proteases, which produce peptides that promote cell migration (2–4). However, the best studied peptide products produced by proteasomes are peptides destined for binding HLA-I complexes (i.e. the HLA-I immunopeptidome) for presentation at the cell surface to CD8+ T cells (5). HLA-I immunopeptidomes have widely been investigated in the context of cancer, radiotherapy, infection, autoimmunity and other conditions. These peptides can be used for detection of specific T cell immune responses and for vaccination purposes against infections and cancer. It is long established that proteases such as trypsin can both cleave peptide bonds but also ligate (repair) these peptide bonds (6, 7). The proteasome is no exception and can produce so-called spliced peptides through proteasome-catalyzed peptide splicing (PCPS). This latter process can occur by combining non-contiguous peptide fragments of either the same molecule – i.e. cis-PCPS – or of two distinct proteins - i.e. trans-PCPS ( Figure 1A ). This results in peptide sequences that are not defined by the genetic code. Cis-spliced peptide identification is challenging: depending on the method of identification used, their estimated frequency in HLA-I immunopeptidomes ranged from 1% to 34% (8). PCPS is not a random process and seems to be ruled by sequence motif preferences and driving forces, which may differ from peptide hydrolysis, although the exact rules are largely unknown (7, 9–11). Cis-spliced peptides seem to be routinely produced and presented by various cells, depicting peptide splicing as a standard activity of proteasomes (7, 9, 12–17). On average, they are produced and presented by HLA-I complexes in smaller amount than non-spliced peptides although exceptions have been described (10, 12, 15, 17). Cis-spliced peptides can target in vivo CD8+ T cell response against bacterial antigens otherwise neglected in a mouse model of Listeria monocytogenes infection (18). They can activate ex vivo CD8+ T cells specific to Listeria monocytogenes or HIV through cross-recognition (19, 20). Cis-spliced epitopes derived from melanoma-associated antigens are recognized by CD8+ T cells in peripheral blood of melanoma patients (13, 17). A melanoma patient with metastasis was cured through adoptive T cell therapy using an autologous tumor-infiltrating lymphocyte (TIL) clone, which was proved, in a later study, to be specific to a cis-spliced epitope rather than any non-spliced peptides derived from the melanoma-associated antigen tyrosinase (21, 22). Despite their theoretically vast sequence variability, cis-spliced peptides might not play a destabilizing role in central and peripheral tolerance and in pruning T cell repertoire (23), although they may be involved in autoreactive CD8+ T cell response in autoimmune diseases such as Type 1 Diabetes (T1D) (24, 25). Open in a separate window Figure 1 Proteasome-catalyzed peptide splicing and the investigated KRAS G12V cis-spliced and non-spliced peptides. (A) Proteasome-generated cis-spliced peptides can be formed by cis-PCPS, when the two splice-reactants, i.e. the non-contiguous peptide fragments ligated by proteasomes, derive from the same polypeptide molecule; the ligation can occur in normal order, i.e. following the orientation from N- to C-terminus of the parental protein (normal cis-PCPS), or in the reverse order (reverse cis-PCPS). (B) The KRAS5-6/8-14 G12V [KL][VVGAVGV] cis-spliced peptide and the KRAS5-13 G12V [KLVVVGAVG] non-spliced peptide, which are investigated in this study and derived from the KRAS2-35 G12V synthetic substrate polypeptide. The G12V mutation is marked in bold.

Amino acid transporters as emerging therapeutic targets in cancer

Amino acids are indispensable nutrients for both normal and cancer cells. Cancer cells are unable to synthesize essential amino acids as well as some non‐essential amino acids adequately to support rapid proliferation, and must take up amino acids from the surroundings. To meet the increased demand for the amino acid needed for proliferation, high levels of amino acid transporters are expressed on the surface of cancer cells. Cancer cells utilize amino acids to synthesize proteins and nucleotides, as well as to obtain energy. In addition, amino acids are known to play pathological roles in cancer cells. Interestingly, breast cancer cells limit the use of amino acids for cell proliferation based on amino acid availability, which depends on estrogen receptor status. Here, we present a summarized literature review of novel amino acid functions in cancer cells. This review organizes the available knowledge on 2 amino acid transporters, SLC7A5 and SLC7A11, which are considered essential for breast cancer cell growth in a cell‐dependent manner. In particular, we propose the glutamine recycling model to clarify the mechanism underlying aberrant SLC7A5 activation. Finally, we overview the pathological significances of SLC7A5 and SLC7A11 in cancer tissues.

Lysine and Threonine Restriction Reproduced the Lower Synthesis but Not the Higher Catabolism of Liver and Muscle Protein of Severely Protein Restricted Growing Rats

Abstract Objectives The availability of indispensable amino acids (IAA) modulates protein turnover. More particularly AAI deficiency reduces protein synthesis while the consequence on proteolysis remains unclear. The present study aims to evaluate the specific response of both protein synthesis and proteolysis to a diet restricted on one strictly indispensable IAA, either lysine or threonine Methods Sixty-four growing rats were divided into 8 groups (n = 8/group). They were fed for 3 weeks isocaloric diets composed with different levels of lysine or threonine (L or T), 15, 25, 40, 60, 75, 100 or 170% of the theoretical lysine/threonine requirements. At the end of the experiment, rats were injected with valine13C and tissues and biological fluids were collected for gene expression measurement and blood amino acids (AA). Protein synthesis rate (Fractional and Absolute rate synthesis, ie FSR, ASR) were determined in liver and muscle. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Results Severe (L/T15, L/T25) and moderate (T40) lysine or threonine deficiency resulted in a decrease in body weight gain due to a decrease in lean body mass. Severe restriction (L15, T15, T25) decreased the muscle FSR whereas no effect was observed in the liver. When the rate of protein synthesis was expressed per tissue, the ASR was decreased by severe restriction of lysine and threonine in liver and muscle and by moderate threonine deficiency (T40, T60, T75) in muscle. In liver, no effect of lysine and threonine on proteolysis was observed. In muscle, only severe lysine (L15) deficiency increased proteolysis. Dietary lysine deficiency induced a decrease in lysine concentration in the portal vein and in the vena cava whereas for threonine deficiency, all AAIs except threonine were decreased in the portal vein and vena cava. Conclusions These results indicate that the decreased protein synthesis is the primary mechanism involved in decreased lean body mass in response to the severe deficiency in a single AAI. Deficiency of a single AAI reproduce the effect of the low protein diet on protein synthesis. Lysine and threonine deficiency differently affect for a part protein turnover probably in relation with the tissue where they are metabolized. Funding Sources This study was funded by the doctoral school ABIES and AlimH-INRAE department.