Shortage Of Amino Acids Research Articles

Inhibition of nucleo-cytoplasmic proteasome translocation by the aromatic amino acids or silencing Sestrin3-their sensing mediator-is tumor suppressive.

The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response ismediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further showthat the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer.

Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.

The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine.

Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.

Impact of Early Fortification in Very Low Birth Weight Infants on the Incidence of Malnutrition During a Trophamine Shortage.

The devastation of pharmaceutical production facilities from Hurricane Maria caused a national shortage of parenteral amino acids in October 2017. Our institution decreased trophamine in very low birth weight (VLBW) infants and initiated human milk fortification at a lower feeding volume to increase enteral protein intake more quickly. The objective of this study was to assess how protein management during the shortage period affected the incidence of malnutrition. This was a retrospective cohort study of infants admitted to 2 neonatal intensive care units from June 1, 2017 to May 31, 2018. Infants between 23 and 32 weeks' gestation were included in this study. The primary outcome was the incidence of malnutrition at 14 days, defined as a z score decline of ≥0.8 SDs, in the pre-shortage period compared with the shortage period. Clinical data regarding adverse effects associated with early fortification and pharmacy costs were recorded. There were 68 infants prior to and 65 during the shortage who met inclusion criteria. There was no difference in malnutrition between the pre-shortage and shortage groups; however, a significant increase in malnutrition was observed in infants who did not receive early fortification during the shortage. No difference in time to full enteral feeds or necrotizing enterocolitis was observed with early fortification. Early fortification in VLBW infants receiving less trophamine during the shortage was not associated with an increase in malnutrition. Restricting trophamine in neonates during the shortage allowed for distribution to other critically ill patients.

Impact of tumor microenvironment on adoptive T cell transfer activity.

Recent advances in immunotherapy have revolutionized the treatment of cancer. The use of adoptive cell therapies (ACT) such as those based on tumor infiltrating lymphocytes (TILs) or genetically modified cells (transgenic TCR lymphocytes or CAR-T cells), has shown impressive results in the treatment of several types of cancers. However, cancer cells can exploit mechanisms to escape from immunosurveillance resulting in many patients not responding to these therapies or respond only transiently. The failure of immunotherapy to achieve long-term tumor control is multifactorial. On the one hand, only a limited percentage of the transferred lymphocytes is capable of circulating through the bloodstream, interacting and crossing the tumor endothelium to infiltrate the tumor. Metabolic competition, excessive glucose consumption, the high level of lactic acid secretion and the extracellular pH acidification, the shortage of essential amino acids, the hypoxic conditions or the accumulation of fatty acids in the tumor microenvironment (TME), greatly hinder the anti-tumor activity of the immune cells in ACT therapy strategies. Therefore, there is a new trend in immunotherapy research that seeks to unravel the fundamental biology that underpins the response to therapy and identifies new approaches to better amplify the efficacy of immunotherapies. In this review we address important aspects that may significantly affect the efficacy of ACT, indicating also the therapeutic alternatives that are currently being implemented to overcome these drawbacks.

The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost

Intraerythrocytic malaria parasites proliferate bounded by a parasitophorous vacuolar membrane (PVM). The PVM contains nutrient permeable channels (NPCs) conductive to small molecules, but their relevance for parasite growth for individual metabolites is largely untested. Here we show that growth-relevant levels of major carbon and energy sources pass through the NPCs. Moreover, we find that NPCs are a gate for several antimalarial drugs, highlighting their permeability properties as a critical factor for drug design. Looking into NPC-dependent amino acid transport, we find that amino acid shortage is a reason for the fitness cost in artemisinin-resistant (ARTR) parasites and provide evidence that NPC upregulation to increase amino acids acquisition is a mechanism of ARTR parasites invitro and in human infections to compensate this fitness cost. Hence, the NPCs are important for nutrient and drug access and reveal amino acid deprivation as a critical constraint in ARTR parasites.

Oncogene-dependent sloppiness in mRNA translation

mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis. Despite protein quality control mechanisms, amino acid shortage in melanoma induces aberrant proteins by ribosomal frameshifting. The extent and the underlying mechanisms related to this phenomenon are yet unknown. Here, we show that tryptophan depletion-induced ribosomal frameshifting is a widespread phenomenon in cancer. We termed this event sloppiness and strikingly observed its association with MAPK pathway hyperactivation. Sloppiness is stimulated by RAS activation in primary cells, suppressed by pharmacological inhibition of the oncogenic MAPK pathway in sloppy cells, and restored in cells with acquired resistance to MAPK pathway inhibition. Interestingly, sloppiness causes aberrant peptide presentation at the cell surface, allowing recognition and specific killing of drug-resistant cancer cells by T lymphocytes. Thus, while oncogenes empower cancer progression and aggressiveness, they also expose a vulnerability by provoking the production of aberrant peptides through sloppiness.

Placental trophoblast syncytialization potentiates macropinocytosis via mTOR signaling to adapt to reduced amino acid supply

During pregnancy, the appropriate allocation of nutrients between the mother and the fetus is dominated by maternal-fetal interactions, which is primarily governed by the placenta. The syncytiotrophoblast (STB) lining at the outer surface of the placental villi is directly bathed in maternal blood and controls feto-maternal exchange. The STB is the largest multinucleated cell type in the human body, and is formed through syncytialization of the mononucleated cytotrophoblast. However, the physiological advantage of forming such an extensively multinucleated cellular structure remains poorly understood. Here, we discover that the STB uniquely adapts to nutrient stress by inducing the macropinocytosis machinery through repression of mammalian target of rapamycin (mTOR) signaling. In primary human trophoblasts and in trophoblast cell lines, differentiation toward a syncytium triggers macropinocytosis, which is greatly enhanced during amino acid shortage, induced by inhibiting mTOR signaling. Moreover, inhibiting mTOR in pregnant mice markedly stimulates macropinocytosis in the syncytium. Blocking macropinocytosis worsens the phenotypes of fetal growth restriction caused by mTOR-inhibition. Consistently, placentas derived from fetal growth restriction patients display: 1) Repressed mTOR signaling, 2) increased syncytialization, and 3) enhanced macropinocytosis. Together, our findings suggest that the unique ability of STB to undergo macropinocytosis serves as an essential adaptation to the cellular nutrient status, and support fetal survival and growth under nutrient deprivation.

Coordinated control of adiposity and growth by anti-anabolic kinase ERK7.

Energy storage and growth are coordinated in response to nutrient status of animals. How nutrient‐regulated signaling pathways control these processes in vivo remains insufficiently understood. Here, we establish an atypical MAP kinase, ERK7, as an inhibitor of adiposity and growth in Drosophila. ERK7 mutant larvae display elevated triacylglycerol (TAG) stores and accelerated growth rate, while overexpressed ERK7 is sufficient to inhibit lipid storage and growth. ERK7 expression is elevated upon fasting and ERK7 mutant larvae display impaired survival during nutrient deprivation. ERK7 acts in the fat body, the insect counterpart of liver and adipose tissue, where it controls the subcellular localization of chromatin‐binding protein PWP1, a growth‐promoting downstream effector of mTOR. PWP1 maintains the expression of sugarbabe, encoding a lipogenic Gli‐similar family transcription factor. Both PWP1 and Sugarbabe are necessary for the increased growth and adiposity phenotypes of ERK7 loss‐of‐function animals. In conclusion, ERK7 is an anti‐anabolic kinase that inhibits lipid storage and growth while promoting survival on fasting conditions.

Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.

Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions, they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast. It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.

Low-protein/high-carbohydrate diet induces AMPK-dependent canonical and non-canonical thermogenesis in subcutaneous adipose tissue

Low-protein/high-carbohydrate (LPHC) diet has been suggested to promote metabolic health and longevity in adult humans and animal models. However, the complex molecular underpinnings of how LPHC diet leads to metabolic benefits remain elusive. Through a multi-layered approach, here we observed that LPHC diet promotes an energy-dissipating response consisting in the parallel recruitment of canonical and non-canonical (muscular) thermogenic systems in subcutaneous white adipose tissue (sWAT). In particular, we measured Ucp1 induction in association with up-regulation of actomyosin components and several Serca (Serca1, Serca2a, Serca2b) ATPases. In beige adipocytes, we observed that AMPK activation is responsible for transducing the amino acid lowering in an enhanced fat catabolism, which sustains both Ucp1-and Serca-dependent energy dissipation. Limiting AMPK activation counteracts the expression of brown fat and muscular genes, including Ucp1 and Serca, as well as mitochondrial oxidative genes. We observed that mitochondrial reactive oxygen species are the upstream molecules controlling AMPK-mediated metabolic rewiring in amino acid-restricted beige adipocytes. Our findings delineate a novel metabolic phenotype of responses to amino acid shortage, which recapitulates some of the benefits of cool temperature in sWAT. In conclusion, this highlights LPHC diet as a valuable and practicable strategy to prevent metabolic diseases through the enhancement of mitochondrial oxidative metabolism and the recruitment of different energy dissipating routes in beige adipocytes.

Domain II of the translation elongation factor eEF1A is required for Gcn2 kinase inhibition.

The signalling pathway governing general control nonderepressible (Gcn)2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eukaryotic translation initiation factor (eIF)2α, triggering downstream events that ultimately allow cells to cope with starvation. Under nutrient-replete conditions, the translation elongation factor eEF1A binds Gcn2 to contribute to keeping Gcn2 inactive. Here, we aimed to map the regions in eEF1A involved in binding and/or regulating Gcn2. We find that eEF1A amino acids 1-221 and 222-315, containing most of domains I and II, respectively, bind Gcn2 invitro. Overexpression of eEF1A lacking or containing domain III impairs eIF2α phosphorylation. While the latter reduces growth under starvation similarly to eEF1A lacking domain I, the former enhances growth in a Gcn2-dependent manner. Our studies suggest that domain II is required for Gcn2 inhibition and that eEF1A lacking domain III mainly affects the Gcn2 response pathway downstream of Gcn2.

Maternal Protein Restriction in Rats Alters the Expression of Genes Involved in Mitochondrial Metabolism and Epitranscriptomics in Fetal Hypothalamus.

Fetal brain development is closely dependent on maternal nutrition and metabolic status. Maternal protein restriction (PR) is known to be associated with alterations in the structure and function of the hypothalamus, leading to impaired control of energy homeostasis and food intake. The objective of this study was to identify the cellular and molecular systems underlying these effects during fetal development. We combined a global transcriptomic analysis on the fetal hypothalamus from a rat model of maternal PR with in vitro neurosphere culture and cellular analyses. Several genes encoding proteins from the mitochondrial respiratory chain complexes were overexpressed in the PR group and mitochondrial metabolic activity in the fetal hypothalamus was altered. The level of the N6-methyladenosine epitranscriptomic mark was reduced in the PR fetuses, and the expression of several genes involved in the writing/erasing/reading of this mark was indeed altered, as well as genes encoding several RNA-binding proteins. Additionally, we observed a higher number of neuronal-committed progenitors at embryonic day 17 (E17) in the PR fetuses. Together, these data strongly suggest a metabolic adaptation to the amino acid shortage, combined with the post-transcriptional control of protein expression, which might reflect alterations in the control of the timing of neuronal progenitor differentiation.

The effect of amino acid supplementation in an industrial Chinese Hamster Ovary process.

The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.

Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells.

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.

The Stringent Response And Its Involvement In The Reactions Of Bacterial Cells To Stress

Abstract The stringent response is a form of bacterial response to adverse environmental conditions. Its effectors are guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp], which are synthetized by RelA, SpoT and their homologs (RSH). RelA, a (p)ppGpp synthase, is activated when there is a shortage of amino acids, whereas SpoT, which has the ability to synthetize and hydrolyze (p)ppGpp, responds to fatty acids, iron and carbon limits. Accumulation of (p)ppGpp causes an inhibition of translation, replication, a decrease in the transcription of many genes, e.g. rRNA, tRNA, encoding ribosomal proteins, and an increase in the transcription of genes whose proteins are important in bacterial stress response. The stringent response alarmones are crucial for bacterial resistance to oxidative stress and antibiotics. They also regulate the production of specific molecules, the so-called quorum sensing autoinducers, which help bacteria communicate the density of their own population, which enables them to adjust their metabolism to the prevailing conditions, to form a biofilm – a community of microorganisms attached to a certain surface, ensuring them appropriate conditions to survive in an unfavourable environment, and to colonize new niches. (p)ppGpp has a positive impact on biofilm formation not only via the regulation of quorum sensing, but also by stimulating the synthesis of potential elements of the biofilm. It also appears that the stringent response alarmones decrease the ability of Agrobacterium tumefaciens bacteria to transform plants and thus their potential to cause disease. (p)ppGpp enables the bacteria to perform swarming motility, a movement that increases their resistance to adverse environmental factors. 1. Introduction. 2. RelA, SpoT and RSH proteins – enzymes that metabolize the alarmones of the stringent response. 2.1. The regulation of transcription via stringent response alarmones in Gram-negative bacteria. 2.2. The regulation of transcription via (p)ppGpp in Gram-positive bacteria. 2.3. The influence of stringent response alarmones on translation and replication. 3. The role of the stringent response in the regulation of other physiological processes. 3.1. The role of the stringent response in the production of siderophores and antibiotics. 4. Bacterial cell resistance to stress and the stringent response. 4.1. The participation of the stringent response in quorum sensing regulation. 4.2. The regulation of exopolysacharide production and biofilm formation dependent on the stringent response. 4.3. The role of the stringent response in the regulation of bacterial swarming motility. 5. Summary

Tryptophan Deprivation Promotes an Adaptive Response and Contributes to Bioenergetics in Multiple Myeloma

Background and Objective:There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy.In multiple myeloma (MM) disease progression depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth.Our previous work showed that i) MM cells have metabolic plasticity due to availability of external nutrients; ii) aminoacid shortage due to the tryptohan (trp) degrading enzyme IDO-1 was associated to clinical MM progression. Thus, we tested bio-energetic changes in MM upon trp deprivation.Experimental design:We evaluated the trascriptome changes in the adaptive response in four human MM cell lines (MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c-myc and primary BM samples of MGUS and MM to trp shortage.The pathway was confirmed looked at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293).ResultsTrp deprivation induced an adaptive response through increased expression, time and dose-dependent, of ATF4-ASNS-CHOP-GADD34, part of GCN2 signaling, associated to increased expression of p62 (the main autophagic receptor in MM) followed by autophagy flux induction, as shown by flow cytometry and immunofluorescence evaluations.In MM cell lines, trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation, inducing in vitro increase of lactate dehydrogenase (LDH) and intracellular lactate and NAD/NADH with down-regulation of surface expression of the NAD-ase CD38. Similarly, short-term trp deprivation in CD138+ plasma cells isolated by magnetic sorting from MM patients, downregulated surface expression of CD38 and intra-cellular amount of c-myc, as detected by flow cytometry.This phenomenon was associated to increased isocitrate dehydrogenase (IDH-1), enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production.In the GSE66293 MM progression was associated to increased expression of ENO-1, PGK-1, and DLD, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant.ConclusionOverall, our study reveals that trp deprivation promotes an adaptive response of MM offering new putative targets in the bio-energetic pathway for synthetic lethality. DisclosuresPalumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria.

Starvation induces rapid degradation of selective autophagy receptors by endosomal microautophagy.

It is not clear to what extent starvation-induced autophagy affects the proteome on a global scale and whether it is selective. In this study, we report based on quantitative proteomics that cells during the first 4 h of acute starvation elicit lysosomal degradation of up to 2-3% of the proteome. The most significant changes are caused by an immediate autophagic response elicited by shortage of amino acids but executed independently of mechanistic target of rapamycin and macroautophagy. Intriguingly, the autophagy receptors p62/SQSTM1, NBR1, TAX1BP1, NDP52, and NCOA4 are among the most efficiently degraded substrates. Already 1 h after induction of starvation, they are rapidly degraded by a process that selectively delivers autophagy receptors to vesicles inside late endosomes/multivesicular bodies depending on the endosomal sorting complex required for transport III (ESCRT-III). Our data support a model in which amino acid deprivation elicits endocytosis of specific membrane receptors, induction of macroautophagy, and rapid degradation of autophagy receptors by endosomal microautophagy.

Molecular Mechanisms of Signal Transduction in Heme-Containing Sensor Proteins

Heme (the iron protoporphyrin IX complex) is one of the most important and extensively characterized cofactors required for proper functioning of many proteins and enzymes. Heme is involved in O2 storage (myoglobin), O2 transfer (hemoglobin), oxygenation (hydroxylation) reactions (cytochromes P450, NO synthase), electron transfer (cytochrome c) and many other functions. In prototype heme-containing proteins, heme itself forms the catalytic or functional center. These proteins are usually composed of one compact structure (one domain). However, recently it was found that heme itself can regulate many physiological processes in many organisms including humans. The novel role of heme is mediated by a group of hemoproteins called heme-containing sensor proteins. Generally, it is known that the heme can act as (i) a preliminary signal of heme sensor proteins and (ii) a gas sensing site of heme-based gas sensor proteins. Conceptually, these proteins are always composed of at least two domains: one is a sensor domain and the other is a functional domain. However, the structure-function relationship and mechanisms of communication between these domains have not been fully understood. Therefore, we selected model systems from each subfamily of heme-containing sensor proteins - for example, a heme regulated inhibitor, HRI (subfamily i) and a globin-coupled oxygen sensor histidine kinase, AfGcHK (subfamily ii) - in order to study the signal transduction in the novel group of hemoproteins (i.e. heme-containing sensor proteins). The AfGcHK is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Once the oxygen molecule (as the first signal) binds to the heme iron complex in the sensor domain of AfGcHK, the functional domain is stimulated, leading to autophosphorylation at a conserved His residue in the functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. The HRI seems to be one of the most important existing heme sensor proteins for eukaryote survival in response to cell emergency states involved in clinical conditions important for human health. The functional domain of HRI exhibits the kinase activity towards its substrate - eukaryotic initiation factor 2α (eIF2α) depending on the heme availability. The enzyme is active in the absence of heme. The heme binding to the HRI protein induces a global structural change, leading to the heme-induced catalytic inhibition. This sensor protein is important for regulation of eukaryotic proteosynthesis. Eukaryotic cells decrease their overall rates of protein synthesis for survival in response to a variety of stress conditions, such as shortage of amino acids, UV light illumination, viral infection, accumulation of denatured proteins, and shortage of heme. The decrease (inhibition) in protein synthesis is caused by phosphorylation of eIF2α at Ser-51 by eIF2α kinases that respond specifically to stress. HRI is a member of the eIF2α kinase family that controls globin synthesis in response to the heme concentration in reticulocytes to balance the molar ratio of heme and globin. In addition to globin, HRI controls the synthesis of tryptophan 2,3-dioxygenase and cytochrome P450 2B in liver upon acute porphyria. Several biochemical approaches were utilized in order to study the signal transduction between the sensor and function domains of the model systems: (i) enzyme kinetic study, (ii) hydrogen/deuterium exchange experiments associated with mass spectrometry, (iii) X-ray crystallography and others. Overall our results indicated that the coordination and oxidation state of the sensor domain heme iron affect the enzyme’s catalytic activity of the function domain. The possible contact area between sensor and function domains was also reveled. All these data together will be discussed in order to illustrate the mechanism of signal transduction in the heme-containing sensor proteins.

Abstract 2639: Development of small molecule selective inhibitors of GCN2 as an immunotherapy aimed at preventing immune escape of tumor cells

Abstract GCN2 is a protein kinase capable of sensing amino acid (AA) shortage. It gets activated by binding of unloaded tRNAs and subsequently phosphorylates translation initiation factor 2 alpha (eIF2α) eventually leading to induction of ATF4-mediated integrated stress response (ISR). Elevated catabolism of one of the essential amino acids - tryptophan (Trp), driven by overexpression of critical enzymes in Trp metabolism - IDO and TDO, leads to immunosuppressive microenvironment in many types of cancer. GCN2 is a key effector signaling component for IDO/TDO and is considered as a metabolic checkpoint of highly Trp-dependent T-cells. GCN2 activation through accumulation of unloaded tRNAs leads to inhibition of CD8+ effector T-cells and increase in generation and activation of regulatory T-cells. According to these findings, selective inhibition of GCN2 may be an effective strategy for targeting Trp-dependent immunosurveillance of tumor cells. In this study, we report the results for a series of novel small molecule GCN2 kinase inhibitors that have been developed at Selvita and, to our best knowledge, are the most potent GCN2 inhibitors reported so far. Newly synthesized compounds exert low nanomolar potency for GCN2, good selectivity as well as ADME profiles. Profound assessment of the compounds potency and efficacy using a selection of in vitro activity/binding assays will be presented. Citation Format: Michał Gałęzowski, Kamil Sitarz, Eliza Majewska, Stefan Chmielewski, Kinga Michalik, Magdalena Masiejczyk, Agnieszka Adamus, Arkadiusz Białas, Joanna Fogt, Marcin Bień, Mateusz Świrski, Maciej Mikulski, Julian Zachmann, Krzysztof Brzózka. Development of small molecule selective inhibitors of GCN2 as an immunotherapy aimed at preventing immune escape of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2017-2639