Intracellular Amino Acid Levels Research Articles

The γ-glutamyl cycle serves as an amino acids supply system in colorectal cancer organoids under chronic hypoxia

Malignant tumors are characterized by a hypoxic microenvironment, and metabolic reprogramming is necessary to ensure energy production and oxidative stress resistance. Although the microenvironmental properties of tumors vary under acute and chronic hypoxia, studies on chronic hypoxia-induced metabolic changes are limited. In the present study, we performed a comprehensive metabolic analysis in a chronic hypoxia model using colorectal cancer (CRC) organoids, and identified an amino acid supply system through the γ-glutamyl cycle, a glutathione recycling pathway. We analyzed the metabolic changes caused by hypoxia over time and observed that chronic hypoxia resulted in an increase in 5-oxoproline and a decrease in oxidized glutathione (GSSG) compared to acute hypoxia. These findings suggest that chronic hypoxia induces metabolic changes in the γ-glutamyl cycle. Moreover, inhibition of the γ-glutamyl cycle via γ-glutamyl cyclotransferase (GGCT) and γ-glutamyl transferase 1 (GGT1) knockdown significantly reversed chronic hypoxia-induced upregulation of 5-oxoproline and several amino acids. Notably, GGT1 knockdown downregulated the intracellular levels of γ-glutamyl amino acids. Conclusively, these results indicate that the γ-glutamyl cycle serves as an amino acid supply system in CRC under chronic hypoxia, which provides fresh insight into cancer metabolism under chronic hypoxia.

Abstract 3067: SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway

Abstract SLC7A5/LAT1 is a cross-membrane transporter that functions in uptake of large neutral amino acids into cells. Many pathological studies have shown that LAT1 is highly expressed in cancer tissue from various origins such as lung, biliary tract, breast, prostate, bone, and their metastatic legions. High LAT1 expression has been recognized as a significant prognostic marker in various cancers. Our TCGA data analysis showed that the expression level of LAT1 is higher in TNBC tumors compared with normal breast tissues. The higher expression level of LAT1 is linked with poor overall and progression-free survival. Dysregulation of amino acid transporters lead to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer. However, it remains elusive whether and how LAT1 re-wires the cellular metabolic programs to promote TNBC tumor proliferation. In this study, we showed that inhibition of SLC7A5/LAT1 significantly reduced TNBC cell proliferation, viability, migration capability while promoting apoptosis. Mechanistically, we found significant positive correlations of LAT1 with PKM2 and LDHA in breast cancer tissues, which are key enzymes in mediating glycolysis and lactate fermentation or the Warburg Effect. Knock down LAT1 via siRNA decreased the expression levels of p-PKM2 and p-LDHA in TNBC cells. Tryptophan, a major substrate amino acid transported into cytosol by LAT1, is the precursor for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). The enzyme quinolinic acid phosphoribosyltransferase (QPRT) catalyzes the conversion from quinolinate, a product of tryptophan degradation, to NAD+. We showed LAT1 inhibition reduced the expression level of QPRT, cytosolic NAD+/NADH ratio as well as lactate production. Addition of L-Tryptophan increased the levels of p-PKM2 and p-LDHA while knockdown of LAT1 reversed tryptophan-mediated increase of PKM2 and LDHA activities, suggesting that LAT1 may regulate PKM2/LDHA through Trp/QPRT/NAD+ pathway for promoting the Warburg Effect. Additionally, we found upregulations of SLC7A5/LAT1, p-PKM2 and p-LDHA in doxorubicin-resistant cells and PDX mouse model compared with their corresponding controls. Collectively, these results provide evidence that overexpression of LAT1 in TNBC drives cancer progression and drug resistance by reinforcing the Warburg Effect, suggesting LAT1 as a promising therapeutic target for addressing TNBC. Our ongoing work will evaluate whether inhibiting LAT1 genetically or pharmacologically reduces xenograft tumor growth and enhances the sensitivities of TNBC resistant cells to chemotherapy in vivo. Our investigation will elucidate the underlying mechanisms involved in these processes. Citation Format: Margot Y. Fedoroff, Lei Zhao, Leili Saeednejad Zanjani, Jun He. SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway [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 3067.

Abstract 4424: Differential metabolic response of triple-negative and estrogen receptor-positive breast cancer cells to abemaciclib, a CDK4/6 inhibitor

Abstract Background: Inhibition of the cell cycle kinases CDK4/6 is the standard-of-care for advanced hormone receptor-positive breast cancer. The clinical efficacy of CDK4/6 inhibitors in triple-negative breast cancer remains uncertain. Mechanisms of sensitivity and resistance are not fully understood. This study comprehensively investigated the metabolic response of triple-negative and estrogen receptor-positive breast cancer cells to abemaciclib (Abe), with the aim of providing metabolic insights for resistance mechanisms. Methods: MCF-7 (MCF) and MDA-MB-231 (MDA) cells were treated with Abe (0, 100, and 500 nM) for 1, 2, or 7 days. Cell cycle and cell senescence analyses were performed to assess drug effects. Cellular metabolism was investigated using the Seahorse assay, LC-MS/MS based targeted metabolomics, and stable isotope-assisted metabolic flux analyses with [U-13C]glutamine, [1,2-13C]glucose, and [U-13C]glucose as the tracers. Results: Abe induced cell cycle arrest at G0/G1 in both cell lines, more pronounced in MCF. Dose- and time-dependent cell senescence occurred in both treated cell lines, more pronounced in MDA. The Seahorse assay indicated that energy metabolism (assessed by oxygen consumption rate and extracellular acidification rate) was reduced in both treated cell lines. Targeted metabolomics revealed that post Abe treatment, intracellular lactate significantly decreased in both cell lines; intracellular TCA intermediates significantly decreased in MCF while being unchanged in MDA; intracellular deoxynucleotides decreased in both cell lines, to a lesser extent in MDA; cellular energy levels (assessed as the ratios of ATP/AMP, ATP/ADP, CTP/CMP, CTP/CDP) were significantly lower in MCF while remaining unchanged or increased in MDA; intracellular levels of many amino acids significantly increased in MDA while decreasing in MCF. [U-13C]glutamine flux revealed a significant decrease of intracellular glutamine-derived TCA intermediates in both treated cell lines. [1,2-13C]glucose flux indicated an enhanced oxidate pentose phosphate pathway (PPP) activity and increased de novo syntheses of purine nucleotides (ATP and GTP) in treated MDA (but not MCF). [U-13C]glucose flux suggested that while both cell lines responded to Abe with decreased anaerobic glycolysis (pyruvate converting to lactate), MDA (but not MCF) showed an enhanced capability of utilizing glucose-derived carbon to replenish TCA intermediates and increased amino acid synthesis. Conclusion: Adaptive cellular metabolism, characterized by enhanced utilization of glucose-derived carbon to replenish TCA intermediates and increased PPP activity, thus promoting biosynthesis of amino acids and purine nucleotides to maintain cellular energy and redox balance, may contribute to the intrinsic resistance of triple-negative breast cancer to CDK4/6 inhibitors. Citation Format: Jun Jiang, Xun Bao, Yuanyuan Jiang, Yang Yue, Maik Huettemann, Jing Li. Differential metabolic response of triple-negative and estrogen receptor-positive breast cancer cells to abemaciclib, a CDK4/6 inhibitor [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 4424.

Distinct Short-Term Response of Intracellular Amino Acids in Saccharomyces cerevisiae and Pichia pastoris to Oxidative and Reductive Stress

Despite being frequently encountered, the effect of oxidative or reductive stress on the intracellular metabolism and the response of the intracellular metabolome of yeasts is severely understudied. Non-conventional yeasts are attracting increasing attention due to their large substrate portfolio of non-canonical pathways as well as their production and secretion of proteins. To understand the effects of both stresses on yeast, the conventional model yeast S. cerevisiae and the non-conventional model yeast P. pastoris were perturbed with 5 mM of hydrogen peroxide for oxidative stress and 20 mM of dithiothreitol for reductive stress in well-defined chemostat cultures at a steady state, and fermentation profiles, intracellular amino acid levels, and intracellular glutathione levels were measured. Although stable profiles of extracellular metabolites were observed, significant changes were measured in intracellular amino acid levels within the first five minutes. Collectively, the amino acids ranged from 0.5 to 400 µmol/gDW, with the most significant increase upon the induction of oxidative stress being seen in cysteine (up to 90%) for S. cerevisiae and in aspartate (up to 80%) for P. pastoris. Upon the induction of reductive stress, asparagine nearly halves in S. cerevisiae, while tryptophan decreases by 60% in P. pastoris. By inspecting the time traces of each amino acid, possible mechanisms of pathway kinetics are speculated. This work furthers our understanding of the response of metabolism to oxidative stress in two model yeasts.

Insights into the complementation potential of the extreme acidophile's orthologue in replacing Escherichia coli hfq gene-particularly in bacterial resistance to environmental stress.

Acidithiobacillus caldus is a typical extreme acidophile widely used in the biohydrometallurgical industry, which often experiences extreme environmental stress in its natural habitat. Hfq, an RNA-binding protein, typically functions as a global regulator involved in various cellular physiological processes. Yet, the biological functions of Hfq derived from such extreme acidophile have not been extensively investigated. In this study, the recombinant strain Δhfq/Achfq, constructed by CRISPR/Cas9-mediated chromosome integration, fully or partially restored the phenotypic defects caused by hfq deletion in Escherichia coli, including impaired growth performance, abnormal cell morphology, impaired swarming motility, decreased stress resistance, decreased intracellular ATP and free amino acid levels, and attenuated biofilm formation. Particularly noteworthy, the intracellular ATP level and biofilm production of the recombinant strain were increased by 12.2% and 7.0%, respectively, compared to the Δhfq mutant. Transcriptomic analysis revealed that even under heterologous expression, AcHfq exerted global regulatory effects on multiple cellular processes, including metabolism, environmental signal processing, and motility. Finally, we established a potential working model to illustrate the regulatory mechanism of AcHfq in bacterial resistance to environmental stress.

The extracellular matrix supports breast cancer cell growth under amino acid starvation by promoting tyrosine catabolism.

Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.

Comparative transcriptome analysis reveals the contribution of amino acid transporters to acid tolerance in Lactococcus lactis

AbstractAmino acid transporters are promising targets for engineering acid‐tolerant strains of Lactococcus lactis. However, the simple overexpression of transporters alone is insufficient to achieve a highly acid‐resistant phenotype. This study investigated the effects of amino acid transporters on the acid‐stress tolerance of L. lactis. Here, we first verified the contribution of amino acid transporters to acid tolerance by overexpressing the ctrA, glnP, and glnQ genes in L. lactis. Transcriptome analysis revealed that most genes associated with specific amino acid transport, pyrimidine metabolism, and functional proteins, were upregulated in the overexpression strains. Among them, arginine biosynthesis (argG and argH), amino acid transport (yjeM and azlC), and pyrimidine metabolism were considered to be the most important regulatory mechanisms. Importantly, metabolite profiling revealed that the overexpression strains had higher intracellular levels of amino acids, particularly aspartate, glutamate, and arginine at low pH, as well as higher intracellular ATP levels, which was consistent with the corresponding gene‐expression levels. Finally, the simultaneous overexpression of glnP and glnQ led to a further improvement of acid tolerance in L. lactis. This study reveals the regulatory mechanisms of amino acid transporters, and provides a novel strategy for achieving higher acid tolerance via positive tandem expression approaches.

Effects of the Toxic Non-Protein Amino Acid β-Methylamino-L-Alanine (BMAA) on Intracellular Amino Acid Levels in Neuroblastoma Cells.

The cyanobacterial non-protein amino acid (AA) β-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.

L-Type Amino Acid Transporter 1 (LAT1) Promotes PMA-Induced Cell Migration through mTORC2 Activation at the Lysosome.

The mTOR signaling pathway integrates signaling inputs from nutrients, including glucose and amino acids, which are precisely regulated by transporters depending on nutrient levels. The L-type amino acid transporter 1 (LAT1) affects the activity of mTORC1 through upstream regulators that sense intracellular amino acid levels. While mTORC1 activation by LAT1 has been thoroughly investigated in cultured cells, the effects of LAT1 expression on the activity of mTORC2 has scarcely been studied. Here, we provide evidence that LAT1 recruits and activates mTORC2 on the lysosome for PMA-induced cell migration. LAT1 is translocated to the lysosomes in cells treated with PMA in a dose- and time-dependent manner. Lysosomal LAT1 interacted with mTORC2 through a direct interaction with Rictor, leading to the lysosomal localization of mTORC2. Furthermore, the depletion of LAT1 reduced PMA-induced cell migration in a wound-healing assay. Consistent with these results, the LAT1 N3KR mutant, which is defective in PMA-induced endocytosis and lysosomal localization, did not induce mTORC2 recruitment to the lysosome, with the activation of mTORC2 determined via Akt phosphorylation or the LAT1-mediated promotion of cell migration. Taken together, lysosomal LAT1 recruits and activates the mTORC2 complex and downstream Akt for PMA-mediated cell migration. These results provide insights into the development of therapeutic drugs targeting the LAT1 amino acid transporter to block metastasis, as well as disease progression in various types of cancer.

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production.

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.

GPRC5C drives branched-chain amino acid metabolism in leukemogenesis

AbstractLeukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C–NF-κB-SLC7A5–BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML.

The aminopeptidase LAP3 suppression accelerates myogenic differentiation via the AKT-TFE3 pathway in C2C12 myoblasts.

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.

The Levels of DAHP Synthase, the First Enzyme of the Shikimate Pathway, Are Related to Free Aromatic Amino Acids and Glutamine Content in Nicotiana plumbaginifolia Cell Cultures.

Aromatic amino acid homeostasis was investigated in cell suspension cultures of Nicotiana plumbaginifolia and was related to the activity of the first enzyme in aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. An inverse relationship was found between the intracellular content of free phenylalanine, tyrosine and tryptophan and enzyme specific activity levels, suggesting the occurrence of end-product control mechanisms. Two DAHP synthase isogenes are present in wild tobacco that showed a different expression pattern during the culture growth cycle. Intracellular levels of aromatic amino acids were increased or decreased by adding the culture medium with phenylalanine, tyrosine and tryptophan, or with sublethal doses of the shikimate pathway inhibitor glyphosate, respectively. As a consequence, enzyme levels varied in the opposite direction. The concomitant exogenous supply of glutamine further reduced enzyme activity in mid-log cells, suggesting induction by both aromatic amino acid depletion and nitrogen starvation.

Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension

Background: Pulmonary arterial hypertension (PAH) is characterized by narrowing of the pulmonary arteries, endothelial cell dysfunction, and an increased vascular cell proliferation. Increased proliferation is also accompanied by glycolytic shift, where glucose is shunted towards anaerobic respiration and production of critical macromolecules. This decreases the available glucose that can be used as a fuel in the tricarboxylic acid (TCA) cycle. To compensate for the lack of TCA cycle metabolites, there is an increase in the use of non-glucose fuels - such as amino acids and fatty acids. However, the effect of this shift on other metabolic pathways is still unknown. In this study, we investigated the levels of metabolites across the pulmonary circulation to define the metabolic shift that cells undergo during PAH. Material & Methods: We carried out metabolomic analysis on serum samples taken from the right and left ventricles (RV and LV) of rats with PAH (SU5416/Hypoxia model: SuHx) and normoxic controls. We also isolated MVECs from each group and performed both targeted and untargeted metabolomics on the cell lysates. Results: Induction of PAH resulted in increased pulmonary artery pressures and RV hypertrophy. Metabolomics of the RV serum were different in PAH versus control rats. This was driven by lower metabolite levels of several amino acids, including glutamine, phenylalanine, and valine. Normoxia- and SuHx-MVEC lysates samples were also distinct, with increased levels of lactic acid contributing significantly to the differences between animals with PAH and controls. Importantly, and in contrast to the serum, where glutamine levels were decreased, we observed increased levels of intracellular glutamine in our analysis. Similarly, intracellular levels of amino acids like phenylalanine, tyrosine and leucine were increased in these lysates. This also contrasted with the serum, where levels of these amino acids were decreased. Interestingly, LV levels of amino acids like valine, glutamine and lysine were similar in normoxic and SuHx samples. However, we did observe increased citrulline levels in SuHx LV serum samples. Comparing the LV/RV ratio in our samples revealed that increased oxidized glutathione was a major determinant of the difference between the SuHx and control groups. Conclusion: Our data suggests that decreased RV serum levels of amino acids in SuHx rats may be driven by increased consumption in the pulmonary vasculature. Given that glutamine levels in the RV are decreased, while being increased intracellularly in MVECs, we hypothesize that glutamine consumption in the lung microvasculature may be driven in part, to produce reduced glutathione. This is also supported by the oxidized glutathione gradient across the pulmonary vasculature in our experiments. These results provide the basis for a mechanistic study aimed at understanding how interrupting glutathione and/or branched-chain amino acid metabolism in MVECs contributes to PAH. NHLBI grant K08HL145132 (J.S.), NHLBI grant R01HL148112 01 (L.S), NHLBI grant R01HL151530 (K.S) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

OGT controls mammalian cell viability by regulating the proteasome/mTOR/ mitochondrial axis

O-GlcNAc transferase (OGT) modifiesserine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We performeda genome-wide CRISPR-Cas9 screen in mouse embryonic stem cells (mESCs) to identify candidates whose depletion rescued the block in cell proliferationinduced by OGT deficiency. We show that the block in cell proliferation in OGT-deficient cellsstems from mitochondrial dysfunction secondary to mTOR (mechanistic target of rapamycin) hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phospho-proteomic screen. Our study highlights a unique series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness, and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.

Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension.

In pulmonary artery hypertension(PAH), emerging evidence suggests that metabolic abnormalities may be contributing to cellular dysfunction in PAH. Metabolic abnormalities such as glycolytic shift have been observed intracellularly in several cell types in PAH, including microvacular endothelial cells (MVECs). Concurrently, metabolomics of human PAH samples has also revealed a variety of metabolic abnormalities; however the relationship between the intracellular metabolic abnormalities and the serum metabolome in PAH remains under investigation. In this study, we utilize the sugen/hypoxia (SuHx) rodent model of PAH to examine the RV, LV and MVEC intracellular metabolome (using targeted metabolomics) in normoxic and SuHx rats. We additionally validate key findings from our metabolomics experiments with data obtained from cell culture of normoxic and SuHx MVECs, as well as metabolomics of human serum samples from two different PAH patient cohorts. Taken together, our data, spanning rat serum, human serum and primary isolated rat MVECs reveal that: (1) key classes of amino acids (specifically, branched chain amino acids-BCAA) are lower in the pre-capillary (i.e.,RV) serum of SuHx rats (and humans); (2) intracellular amino acid levels (in particular BCAAs) are increased in SuHx-MVECs;(3) there may be secretion rather than utilization of amino acids across the pulmonary microvasculature in PAH and (4) an oxidized glutathione gradient is present across the pulmonary vasculature, suggesting a novel fate for increased glutamine uptake (i.e., as a source of glutathione). in MVECs in PAH. In summary, these data reveal new insight into the shifts in amino acid metabolism occurring across the pulmonary circulation in PAH.

Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l-type amino acid transporter 1

BackgroundCancer-upregulated l-type amino acid transporter 1 (LAT1; SLC7A5) supplies essential amino acids to cancer cells. LAT1 substrates are not only needed for cancer rapid growth, but involved in cellular signaling. LAT1 has been proposed as a potential target for cancer treatment—its inhibitor, JPH203, is currently in clinical trials and targets biliary tract cancer (BTC). Here, we revealed to what extent LAT1 inhibitor affects intracellular amino acid content and what kind of cellular signals are directly triggered by LAT1 inhibition.MethodsLiquid chromatography assay combined with o-phthalaldehyde- and 9-fluorenyl-methylchloroformate-based derivatization revealed changes in intracellular amino acid levels induced by LAT1 inhibition with JPH203 treatment in three BTC cell lines. Tandem mass tag-based quantitative phosphoproteomics characterized the effect of JPH203 treatment on BTC cells, and suggested key regulators in LAT1-inhibited cells. We further studied one of the key regulators, CK2 protein kinase, by using Western blot, enzymatic activity assay, and co-immunoprecipitation. We evaluated anticancer effects of combination of JPH203 with CK2 inhibitor using cell growth and would healing assay.ResultsJPH203 treatment decreased intracellular levels of LAT1 substrates including essential amino acids of three BTC cell lines, immediately and drastically. We also found levels of some of these amino acids were partially recovered after longer-time treatment. Therefore, we performed phosphoproteomics with short-time JPH203 treatment prior to the cellular compensatory response, and revealed hundreds of differentially phosphorylated sites. Commonly downregulated phosphorylation sites were found on proteins involved in the cell cycle and RNA splicing. Our phosphoproteomics also suggested key regulators immediately responding to LAT1 inhibition. Focusing on one of these regulators, protein kinase CK2, we revealed LAT1 inhibition decreased phosphorylation of CK2 substrate without changing CK2 enzymatic activity. Furthermore, LAT1 inhibition abolished interaction between CK2 and its regulatory protein NOLC1, which suggests regulatory mechanism of CK2 substrate protein specificity controlled by LAT1 inhibition. Moreover, we revealed that the combination of JPH203 with CK2 inhibitor resulted in the enhanced inhibition of proliferation and migration of BTC cells.ConclusionThis study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.

A Dynamic rRNA Ribomethylome Drives Stemness in Acute Myeloid Leukemia.

We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247.

Metabolic characteristics of intracellular trehalose enrichment in salt-tolerant Zygosaccharomyces rouxii.

The salt-tolerant flavor yeast Zygosaccharomyces rouxii is an important food flavor microorganism, but its intracellular stress-resistant trehalose synthesis efficiency has been shown to be low, resulting in its weak high-temperature resistance. The intracellular and extracellular levels of carbohydrates, organic acids, and amino acids of Z. rouxii in a 20-L mechanically stirred ventilated fermenter were analyzed using metabolomics research methods. Our results showed that glucose supplementation could promote the growth of yeast cells, but high temperatures (> 35°C) significantly prevented cell growth. Under three different growth strategies, extracellular glucose was continuously utilized and intracellular glucose was continuously metabolized, but glucose overflow metabolism was inhibited by high temperature, which showed that the level of intracellular/extracellular ethanol was stable. High temperature stimulated significant intracellular trehalose accumulation (c20.5h = 80.78 mg/g Dry Cell Weight (DCW)) but not efflux, as well as xylitol accumulation (c20.5h = 185.97 mg/g DCW) but with efflux (c20.5h = 29.78 g/L). Moreover, heat resistance evaluation showed that xylitol and trehalose had heat-protective effects on Z. rouxii. In addition, a large amount of propionic acid and butyric acid accumulated inside and outside these cells, showing that the conversion of glucose to acid in yeast cells becomes the main pathway of glucose overflow metabolism in high temperatures. In addition, the increased demand of yeast cells for phenylalanine, threonine, and glycine at high temperatures suggested that these metabolites participated in the temperature adaptation of Z. rouxii in different ways. Valine and leucine/isoleucine [branched-chain amino acids (BCAAs)] were mainly affected by the addition of glucose, while glucose, sucrose, aspartic acid/asparagine, and glutamate/glutamine were not affected by this temperature regulation as a whole. This study could help deepen our understanding of the high-temperature adaptation mechanism of salt-tolerant Z. rouxii, and has theoretical significance for the application of highly tolerant yeast to food brewing.

Analysis of Growth Phases of Enterotoxigenic Escherichia coli Reveals a Distinct Transition Phase before Entry into Early Stationary Phase with Shifts in Tryptophan, Fucose, and Putrescine Metabolism and Degradation of Neurotransmitter Precursors.

ABSTRACTEnterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to early stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition from logarithmic to stationary growth. The transient phase was found to be different from the subsequent induction of early stationary phase-induced genes. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence from mid-logarithmic to the start of the stationary phase. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., l-tyrosine, l-tryptophan, l-phenylalanine, l-leucine, and l-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire combined transcriptome and metabolome revealed that significant pathways during progression from logarithmic to early stationary phase are involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli.IMPORTANCE We show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of certain amino acids. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described “indole peak,” rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolome is short-lived and disappears when bacteria enter the early stationary phase. We suggest that these changes mainly prepare bacteria for ceased growth, but based on the pathways involved, we could suggest that this transient phase substantially influences survival and virulence.