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Plants possess cell surface-localized immune receptors that detect microbe-associated molecular patterns (MAMPs) and initiate defenses that provide effective resistance against microbial pathogens. Many MAMP-induced signaling pathways and cellular responses are known, yet how pattern-triggered immunity (PTI) limits pathogen growth in plants is poorly understood. Through a combined metabolomics and genetics approach, we discovered that plant-exuded proline is a virulence-inducing signal and nutrient for the bacterial pathogen Pseudomonas syringae, and that MAMP-induced depletion of proline from the extracellular spaces of Arabidopsis leaves directly contributes to PTI against P. syringae. We further show that MAMP-induced depletion of extracellular proline requires the amino acid transporter Lysine Histidine Transporter 1 (LHT1). This study demonstrates that depletion of a single extracellular metabolite is an effective component of plant induced immunity. Given the important role for amino acids as nutrients for microbial growth, their depletion at sites of infection may be a broadly effective means for defense against many pathogens., (© 2024. The Author(s).)

The tumor microenvironment (TME) is restricted in metabolic nutrients including the semi-essential amino acid arginine. While complete arginine deprivation causes T cell dysfunction, it remains unclear how arginine levels fluctuate in the TME to shape T cell fates. Here, we find that the 20-μM low arginine condition, representing the levels found in the plasma of patients with cancers, confers Treg-like immunosuppressive capacities upon activated T cells. In vivo mouse tumor models and human single-cell RNA-sequencing datasets reveal positive correlations between low arginine condition and intratumoral Treg accumulation. Mechanistically, low arginine-activated T cells engage in metabolic and transcriptional reprogramming, using the ATF4-SLC7A11-GSH axis, to preserve their suppressive function. These findings improve our understanding of the role of arginine in human T cell biology with potential applications for immunotherapy strategies., Competing Interests: Declaration of interests M.S. receives consulting fees (unrelated to this work) from Nucleome Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients., (© 2024. The Author(s).)

Amino acids are essential for the survival of all living organisms and living cells. Amino acid transporters mediate the transport and absorption of amino acids, and the dysfunction of these proteins can induce human diseases. Cationic amino acid transporters (CAT family, SLC7A1-4, and SLC7A14) are considered to be a group of transmembrane transporters, of which SLC7A1-3 are essential for arginine transport in mammals. Numerous studies have shown that CAT family-mediated arginine transport is involved in signal crosstalk between malignant tumor cells and immune cells, especially T cells. The modulation of extracellular arginine concentration has entered a number of clinical trials and achieved certain therapeutic effects. Here, we review the role of CAT family on tumor cells and immune infiltrating cells in malignant tumors and explore the therapeutic strategies to interfere with extracellular arginine concentration, to elaborate its application prospects. CAT family members may be used as biomarkers for certain cancer entities and might be included in new ideas for immunotherapy of malignant tumors., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

RNA-binding proteins (RBP) have emerged as essential regulators that control gene expression and modulate multiple cancer traits. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from transformation of T-cell progenitors that normally undergo discrete steps of differentiation in the thymus. The implications of essential RBP during T-cell neoplastic transformation remain largely unclear. Systematic evaluation of RBP identifies RNA helicase DHX15, which facilitates the disassembly of the spliceosome and release of lariat introns, as a T-ALL dependency factor. Functional analysis using multiple murine T-ALL models demonstrates the essential importance of DHX15 in tumor cell survival and leukemogenesis. Moreover, single-cell transcriptomics reveals that DHX15 depletion in T-cell progenitors hinders burst proliferation during the transition from doublenegative to double-positive cells (CD4-CD8- to CD4+CD8+). Mechanistically, abrogation of DHX15 perturbs RNA splicing and leads to diminished levels of SLC7A6 and SLC38A5 transcripts due to intron retention, thereby suppressing glutamine import and mTORC1 activity. We further propose a DHX15 signature modulator drug ciclopirox and demonstrate that it has prominent anti-T-ALL efficacy. Collectively, our data highlight the functional contribution of DHX15 to leukemogenesis through regulation of established oncogenic pathways. These findings also suggest a promising therapeutic approach, i.e., splicing perturbation by targeting spliceosome disassembly, may achieve considerable anti-tumor efficacy.

We investigated the effects of a low-protein diet and feed restriction on the mRNA expression of cationic amino acid transporters (CATs) in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pigs. Eighteen piglets were divided into three groups: a control (CP21%), low-protein diet (LP, CP16%), and feed-restricted diet (FR, CP21%, 76% feed intake of control pigs) groups. The expression levels of CAT-1 in the LD and BF muscles of LP pigs were higher than that of control pigs, whereas that of FR pigs showed no difference. The CAT-2A expression levels in the RH muscle of FR pigs were higher than that of control pigs. The free lysine concentrations in all muscles of LP and FR pigs were lower than that of control pigs. To examine the factors that affect CATs mRNA expression, we evaluated the effects of lysine, arginine, insulin-like growth factor-I, and dexamethasone on the expression of CATs in C2C12 myotubes. CAT-1 expression levels increased in lysine and/or arginine deprivation. We show that CAT-1 and CAT-2A expression levels in skeletal muscles differ in response to dietary treatments and CAT-1 expression in skeletal muscles appears to increase in response to low free lysine concentrations., (© 2023 Japanese Society of Animal Science.)

Metabolic and signaling mechanisms in mammalian cells are facilitated by the transportation of L-arginine (Arg) across the plasma membrane through cationic amino acid transporter (CAT) proteins. Due to a lack of argininosuccinate synthase (ASS) activity in various tumor cells such as acute myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, these tumor entities are arginine-auxotrophic and therefore depend on the uptake of the amino acid arginine. Cationic amino acid transporter-1 (CAT-1) is the leading arginine importer expressed in the aforementioned tumor entities. Hence, in the present study, to investigate the transportation mechanism of arginine in CAT-1, we performed molecular dynamics (MD) simulation methods on the modeled human CAT-1. The MM-PBSA approach was conducted to determine the critical residues interacting with arginine within the corresponding binding site of CAT-1. In addition, we found out that the water molecules have the leading role in forming the transportation channel within CAT-1. The conductive structure of CAT-1 was formed only when the water molecules were continuously distributed across the channel. Steered molecular dynamics (SMD) simulation approach showed various energy barriers against arginine transportation through CAT-1, especially while crossing the bottlenecks of the related channel. These findings at the molecular level might shed light on identifying the crucial amino acids in the binding of arginine to eukaryotic CATs and also provide fundamental insights into the arginine transportation mechanisms through CAT-1. Understanding the transportation mechanism of arginine is essential to developing CAT-1 blockers, which can be potential medications for some types of cancers.Communicated by Ramaswamy H. Sarma.

Exogenous polyamines are able to induce life span and improve glucose homeostasis and insulin sensitivity. However, the effects of exogenous polyamines on adipocyte differentiation and which polyamine transporters mediate them have not been elucidated yet. Here, we identified for the first time that exogenous polyamines can clearly stimulate adipocyte differentiation through polyamine transporters, solute carrier family 3 member A2 (SLC3A2) and SLC7A1. Exogenous polyamines markedly promote 3T3-L1 adipocyte differentiation by increasing the intracellular lipid accumulation and the expression of both adipogenic and lipogenic genes in a concentration-dependent manner. In particular, exogenous putrescine mainly regulates adipocyte differentiation in the early and intermediate stages. Moreover, we have assessed the expression of polyamine transporter genes in 3T3-L1 preadipocytes and adipocytes. Interestingly, the putrescine-induced adipocyte differentiation was found to be significantly suppressed in response to a treatment with a polyamine transporter inhibitor (AMXT-1501). Furthermore, knockdown experiments using siRNA that specifically targeted SLC3A2 or SLC7A2, revealed that both SLC3A2 and SLC7A2 act as important transporters in the cellular importing of exogenous putrescine. Thus, the exogenous putrescine entering the adipocytes via cellular transporters is involved in adipogenesis through a modulation of both the mitotic clonal expansion and the expression of master transcription factors. Taken together, these results suggest that exogenous polyamines (such as putrescine) entering the adipocytes through polyamine transporters, can stimulate adipogenesis.

Amino acid-mediated metabolism is one of the key catabolic and anabolic processes involved in diverse cellular functions. However, the role of the semi-essential amino acid arginine in normal and malignant hematopoietic cell development is poorly understood. Here we report that a continuous supply of exogenous arginine is required for the maintenance/function of normal hematopoietic stem cells (HSCs). Surprisingly, knockout of Slc7a3 (CAT3), a major L-arginine transporter, does not affect HSCs in steady-state or under stress. Although Slc7a3 is highly expressed in naïve and activated CD8 T cells, neither T cell development nor activation/proliferation is impacted by Slc7a3 depletion. Furthermore, the Slc7a3 deletion does not attenuate leukemia development driven by Pten loss or the oncogenic Ptpn11 E76K mutation. Arginine uptake assays reveal that L-arginine uptake is not disrupted in Slc7a3 knockout cells. These data suggest that extracellular arginine is critically important for HSCs, but CAT3 is dispensable for normal hematopoiesis and leukemogenesis., (© 2022. The Author(s).)

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The amino acid transporter protein cationic amino acid transporter 1 (CAT1) is part of the nutrient sensor in the fat body of mosquitoes. A member of the SLC7 family of cationic amino acid transporters, it is paramount for the detection of elevated amino acid levels in the mosquito hemolymph after a blood meal and the subsequent changes in gene expression in the fat body., Methods: We performed a re-annotation of Aedes aegypti cationic amino acid transporters (CATs) and selected the C-terminal tail of CAT1 to perform a yeast two-hybrid screen to identify putative interactors of this protein. One interesting interacting protein we identified was general control nonderepressible 1 (GCN1). We determined the expression pattern of GCN1 in several adult organs and structures using qRT-PCR and western blots. Finally, we knocked down GCN1 using double-stranded RNA and identified changes in downstream signaling intermediates and the effects of knockdown on vitellogenesis and fecundity., Results: In a screen for Ae. aegypti CAT1-interacting proteins we identified GCN1 as a putative interactor. GCN1 is highly expressed in the ovaries and fat body of the mosquito. We provide evidence that eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation changed during vitellogenesis and that RNA interference knockdown of GCN1 in whole mosquitoes reduced egg clutch sizes of treated mosquitoes relative to controls., Conclusions: Aedes aegypti CAT1 and GCN1 are likely interacting partners and GCN1 is likely necessary for proper egg development. Our data suggest that GCN1 is part of a nutrient sensor mechanism in various mosquito tissues involved in vitellogenesis., (© 2022. The Author(s).)

Cystinuria is a genetic disorder of cystine transport, including defective protein b 0,+ AT (encoded by SLC7A9), and/or rBAT (encoded by SLC3A1). Patients present hyperexcretion of cystine in the urine, recurrent cystine lithiasis, and progressive decline in kidney function. Moreover, heterodimer transport is defective. To date, little omics data are accessible regarding this metabolic disease caused by membrane proteins. Since membrane function is closely related to changes in the lipidome, we decided to explore the changes in kidney tissue of a self-established cystinuria rat model by performing lipidomic analysis by LC-MS/MS. Our results demonstrated that Slc7a9 deficiency changed the lipid profile of the renal cortex and induced vital modifications in the lipidome, including major alterations in ChE, LPA, and PA. Among those alterations, this lipidomic study highlights the lipid changes that participate in inflammatory responses during cystinuria. As a result, lipid research, perhaps has great potential, for it may lead to the identification of novel therapeutic targets for the prevention and treatment of cystinuria., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood-brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport., Methods: iPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques., Results: Following a screen of 73 compounds, 17β-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17β-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport., Conclusions: A major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of hormone transport across the BBB., (© 2022. The Author(s).)

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Biofilm formation by yeast is a critical issue in clinical and industrial fields because of the strong adhesion of yeast biofilm to abiotic surfaces and tissues. Here, we clarified the arginine-mediated inhibition of biofilm formation by yeast. First, we showed that arginine inhibits biofilm formation in fungi such as Saccharomyces cerevisiae, Candida glabrata, and Cladosporium cladosporioides, but not in bacteria. In regard to the underlying mechanism, biochemical analysis indicated that arginine inhibits biofilm formation by suppressing Flo11-dependent flocculation. Intriguingly, a strain with deletion of the arginine transporter-encoding CAN1 was insensitive to arginine-mediated inhibition of biofilm formation. Finally, Can1 endocytosis appeared to be required for the inhibitory mechanism of biofilm formation by arginine. The present results could help to elucidate the molecular mechanism of yeast biofilm formation and its control., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The interaction between plants and plant pathogens can have significant effects on ecosystem performance. For their growth and development, both bionts rely on amino acids. While amino acids are key transport forms of nitrogen and can be directly absorbed from the soil through specific root amino acid transporters, various pathogenic microbes can invade plant tissues to feed on different plant amino acid pools. In parallel, plants may initiate an immune response program to restrict this invasion, employing various amino acid transporters to modify the amino acid pool at the site of pathogen attack. The interaction between pathogens and plants is sophisticated and responses are dynamic. Both avail themselves of multiple tools to increase their chance of survival. In this review, we highlight the role of amino acid transporters during pathogen infection. Having control over the expression of those transporters can be decisive for the fate of both bionts but the underlying mechanism that regulates the expression of amino acid transporters is not understood to date. We provide an overview of the regulation of a variety of amino acid transporters, depending on interaction with biotrophic, hemibiotrophic or necrotrophic pathogens. In addition, we aim to highlight the interplay of different physiological processes on amino acid transporter regulation during pathogen attack and chose the LYSINE HISTIDINE TRANSPORTER1 (LHT1) as an example., (© 2022. The Author(s).)

Cystinuria is a genetic disorder of cystine transport that accounts for 1-2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b 0,+ AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated., (© 2022. The Author(s).)

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b 0,+ AT, which constitute system b 0,+ , are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca 2+ mediates higher-order assembly of system b 0,+ . Ca 2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b 0,+ AT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca 2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b 0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases., (© 2022. The Author(s).)

Background: The expression of solute carrier (SLC) 7 family genes is reportedly associated with several malignancies. Here, we focused on SLC7A9 and investigated its expression, function, and clinical significance in esophageal squamous cell carcinoma (ESCC)., Methods: SLC7A9 transcription levels were evaluated in 13 ESCC cell lines, and polymerase chain reaction (PCR) array analysis was conducted to detect coordinately expressed genes with SLC7A9. SLC7A9 contributions to proliferation, invasion, and migration were evaluated in ESCC cells subjected to siRNA-mediated gene knockdown and pCMV6-entry plasmid-mediated overexpression. SLC7A9 expression was detected in 189 ESCC tissues by quantitative reverse-transcription (qRT)-PCR and correlated with clinicopathological parameters., Results: The expression levels of SLC7A9 varied widely in ESCC cell lines and correlated with FGFBP1 expression. Knockdown of SLC7A9 significantly suppressed the proliferation, invasion, and migration of the ESCC cell lines. Moreover, overexpression of SLC7A9 enhanced cell proliferation and migration. In analyses of clinical specimens, SLC7A9 mRNA was overexpressed in the ESCC tissues compared with the adjacent normal esophageal tissues. High mRNA expression was significantly associated with high levels of squamous cell carcinoma-related antigen and carcinoembryonic antigen, advanced disease stage, and lymph node metastasis. High SLC7A9 expression was also significantly associated with poor disease-specific and disease-free survival, and lymph node recurrence after radical surgery, but not with the other recurrence patterns. On multivariate analysis, high SLC7A9 expression was an independent predictor of lymph node recurrence., Conclusions: SLC7A9 influences the malignant behavior of ESCC cells. Tumor SLC7A9 expression may serve as a novel biomarker for predicting lymph node metastasis and recurrence in ESCC patients., (© 2021. Society of Surgical Oncology.)

LysO, a prototypical member of the LysO family, mediates export of L-lysine (Lys) and resistance to the toxic Lys antimetabolite, L-thialysine (Thl) in Escherichia coli. Here, we have addressed unknown aspects of LysO function pertaining to its membrane topology and the mechanism by which it mediates Lys/Thl export. Using substituted cysteine (Cys) accessibility, here we delineated the membrane topology of LysO. Our studies support a model in which both the N- and C-termini of LysO are present at the periplasmic face of the membrane with a transmembrane (TM) domain comprising eight TM segments (TMSs) between them. In addition, a feature of intramembrane solvent exposure in LysO is inferred with the identification of membrane-located solvent-exposed Cys residues. Isosteric substitutions of a pair of conserved acidic residues, one E233, located in the solvent-exposed TMS7 and the other D261, in a solvent-exposed intramembrane segment located between TMS7 and TMS8, abolished LysO function in vivo. Thl, but not Lys, elicited proton release in inside-out membrane vesicles, a process requiring the presence of both E233 and D261. We postulate that Thl may be exported in antiport with H + and that Lys may be a low-affinity export substrate. Our findings are compatible with a physiological scenario wherein in vivo LysO exports the naturally occurring antimetabolite Thl with higher affinity over the essential cellular metabolite Lys, thus affording protection from Thl toxicity and limiting wasteful export of Lys., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this manuscript., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

L-type amino acid transporter 3 (LAT3, SLC43A1) is abundantly expressed in prostate cancer (PC) and is thought to play an essential role in PC progression through the cellular uptake of essential amino acids. Here, we analyzed the expression, function, and downstream target of LAT3 in PC. LAT3 was highly expressed in PC cells expressing androgen receptor (AR), and its expression was increased by dihydrotestosterone treatment and decreased by bicalutamide treatment. In chromatin immunoprecipitation sequencing of AR, binding of AR to the SLC43A1 region was increased by dihydrotestosterone stimulation. Knockdown of LAT3 inhibited cell proliferation, migration, and invasion, and the phosphorylation of p70S6K and 4EBP-1. Separase (ESPL1) was identified as a downstream target of LAT3 by RNA sequencing analysis. In addition, immunostaining of prostatectomy specimens was performed. In the multivariate analysis, high expression of LAT3 was an independent prognostic factor for recurrence-free survival (hazard ratio: 3.24; P = .0018). High LAT3 expression was correlated with the pathological T stage and a high International Society of Urological Pathology grade. In summary, our results suggest that LAT3 plays an important role in the progression of PC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is 'trans-stimulated' by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites., Competing Interests: The authors have declared that no competing interests exist.

Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; "picklock two") is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge (" trans ") compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter's cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners., Competing Interests: The authors declare no competing interest. See online for related content such as Commentaries., (Copyright © 2021 the Author(s). Published by PNAS.)

Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.

Cystinuria is the most common cause of inherited stone disease and is caused by the failure of absorption of filtered dibasic amino acids including cystine in the proximal tubules. It is associated with a very high recurrence rate in affected patients, with the potential for significant morbidity in such patients due to the need for repeated surgical interventions. A multimodal and multispecialty approach in a dedicated centre is the key to improving treatment outcomes and patient adherence to the treatment. This article reviews the latest knowledge on the clinical and diagnostic features and summarises key developments to aid clinicians in diagnosis and management options, together with future directions for the care of these patients., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

In Saccharomyces cerevisiae, Avt4 exports neutral and basic amino acids from vacuoles. Previous studies have suggested that the GATA transcription factors, Gln3 and Gat1, which are key regulators that adapt cells in response to changes in amino acid status, are involved in the AVT4 transcription. Here, we show that mutations in the putative GATA-binding sites of the AVT4 promoter reduced AVT4 expression. Consistently, a chromatin immunoprecipitation (ChIP) assay revealed that Gat1-Myc13 binds to the AVT4 promoter. Previous microarray results were confirmed that gln3∆gat1∆ cells showed a decrease in expression of AVT1 and AVT7, which also encode vacuolar amino acid transporters. Additionally, ChIP analysis revealed that the AVT6 encoding vacuolar acidic amino acid exporter represents a new direct target of the GATA transcription factor. The broad effect of the GATA transcription factors on the expression of AVT transporters suggests that vacuolar amino acid transport is integrated into cellular amino acid homeostasis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

PQLC2, a lysosomal cationic amino acid transporter, also serves as a sensor that responds to scarcity of its substrates by recruiting a protein complex composed of C9orf72, SMCR8, and WDR41 to the surface of lysosomes. This protein complex controls multiple aspects of lysosome function. Although it is known that this response to changes in cationic amino acid availability depends on an interaction between PQLC2 and WDR41, the underlying mechanism for the regulated interaction is not known. In this study, we present evidence that the WDR41-PQLC2 interaction is mediated by a short peptide motif in a flexible loop that extends from the WDR41 β-propeller and inserts into a cavity presented by the inward-facing conformation of PQLC2. The data support a transceptor model wherein conformational changes in PQLC2 related to substrate transport regulate the availability of the WDR41-binding site on PQLC2 and mediate recruitment of the WDR41-SMCR8-C9orf72 complex to the surface of lysosomes., Competing Interests: The authors declare no competing interest.

Background: Glutamine metabolism is considered an important metabolic phenotype of proliferating tumor cells., Objective: The objective of this study was to investigate the expression of glutamine metabolism-related and amino acid transporter proteins in adrenal cortical neoplasms (ACNs) and pheochromocytomas (PCCs) in the adrenal gland., Methods: A tissue microarray was constructed for 132 cases of ACN (115 cases of adrenal cortical adenoma and 17 cases of adrenal cortical carcinoma) and 189 cases of PCC. Immunohistochemical staining for glutamine metabolism-related proteins GLS1 and GDH and amino acid transporter proteins SLC1A5, SLC7A5, and SLC7A11 as well as SDHB was performed and compared with clinicopathologic parameters., Results: The expression levels of GLS ( p < 0.001), SLC7A5 ( p = 0.049), and SDHB ( p = 0.007) were higher in ACN than in PCC, whereas the expression levels of SLC1A5 ( p < 0.001) and SLC7A11 ( p < 0.001) were higher in PCC than in ACN. In ACN, GLS positivity was associated with a higher Fuhrman grade ( p = 0.009), and SLC1A5 positivity was associated with SDHB positivity ( p = 0.004) and a clear cell proportion < 25% ( p = 0.010). SDHB negativity was also associated with tumor cell necrosis ( p = 0.007). In PCC, SLC7A11 positivity was associated with nonnorepinephrine type ( p = 0.008). In Kaplan-Meier analysis, patients with GLS positivity ( p = 0.039) and SDHB negativity ( p = 0.005) had significantly shorter overall survival in ACN. In PCC patients with a GAPP score ≥ 3, GLS positivity ( p = 0.001) and SDHB positivity ( p = 0.001) were associated with shorter disease-free survival, whereas GLS positivity ( p = 0.004) was also associated with shorter overall survival., Conclusions: The expression of glutamine metabolism-related and amino acid transporter proteins in ACN and PCC is distinct and associated with prognosis., Competing Interests: The authors declare that they have no conflict of interest regarding the publication of this article., (Copyright © 2021 Hye Min Kim and Ja Seung Koo.)

DNA damage has been hypothesized to be a driving force of the aging process. At the same time, there exists multiple compounds that can extend lifespan in model organisms, such as yeast, worms, flies, and mice. One possible mechanism of action for these compounds is a protective effect against DNA damage. We investigated whether five of these lifespan-extending compounds, dinitrophenol, metformin, rapamycin, resveratrol, and spermidine, could protect nuclear DNA in the yeast Saccharomyces cerevisiae at the same doses under which they confer lifespan extension. We found that rapamycin and spermidine were able to decrease the spontaneous mutation rate at the CAN1 locus, whereas dinitrophenol, metformin, and resveratrol were able to protect yeast against CAN1 mutations induced by ethyl methanesulfonate (EMS). We also tested whether these compounds could enhance survival against EMS, ultraviolet (UV) light, or hydrogen peroxide (H 2 O 2 ) insult. All five compounds conferred a protective effect against EMS, while metformin and spermidine protected yeast against UV light. Somewhat surprisingly, none of the compounds were able to afford a significant protection against H 2 O 2 , with spermidine dramatically sensitizing cells. We also examined the ability of these compounds to increase lifespan when growth-arrested by hydroxyurea; only spermidine was found to have a positive effect. Overall, our results suggest that lifespan-extending compounds may act in part by protecting nuclear DNA., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The higher alcohols produced by Saccharomyces cerevisiae exert remarkable influence on the taste and flavour of Chinese Baijiu. In order to study the regulation mechanism of amino acid metabolism genes on higher alcohol production, eight recombinant strains with amino acid metabolism gene deletion were constructed. The growth, fermentation performance, higher alcohol production, and expression level of genes in recombinant and original α 5 strains were determined. Results displayed that the total higher alcohol concentration in α 5 ΔGDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α 5. The total content of higher alcohols in α 5 ΔCAN1 and α 5 ΔGAT1 strains increased by 211.44% and 28.36% to 1493.96 and 615.73 mg/L, respectively, compared with that of α 5. This study is the first to report that the CAN1 and GAT1 genes have great influence on the generation of higher alcohols. The results demonstrated that amino acid metabolism plays a substantial role in the metabolism of higher alcohols by S. cerevisiae. Interestingly, we also found that gene knockout downregulated the expression levels of the knocked out gene and other genes in the recombinant strain and thus affected the formation of higher alcohols by S. cerevisiae . This study provides worthy insights for comprehending the metabolic mechanism of higher alcohols in S. cerevisiae for Baijiu fermentation., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Ya-Ping Wang et al.)

Yeast amino acid transporters of the APC superfamily are responsible for the proton motive force-driven uptake of amino acids into the cell, which for most secondary transporters is a reversible process. The l-lysine proton symporter Lyp1 of Saccharomyces cerevisiae is special in that the Michaelis constant from out-to-in transport ( K m out → in ) is much lower than K m in → out , which allows accumulation of l-lysine to submolar concentration. It has been proposed that high intracellular lysine is part of the antioxidant mechanism of the cell. The molecular basis for the unique kinetic properties of Lyp1 is unknown. We compared the sequence of Lyp1 with APC para- and orthologues and find structural features that set Lyp1 apart, including differences in extracellular loop regions. We screened the extracellular loops by alanine mutagenesis and determined Lyp1 localization and activity and find positions that affect either the localization or activity of Lyp1. Half of the affected mutants are located in the extension of extracellular loop 3 or in a predicted α-helix in extracellular loop 4. Our data indicate that extracellular loops not only connect the transmembrane helices but also serve functionally important roles., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Corynebacterium glutamicum has a long and successful history in the biotechnological production of L-lysine. Besides the adjustment of metabolic pathways, intracellular and extracellular transport systems are critical for the cellular metabolism of L-lysine or its by-products. Here, three amino acid transmembrane transporters, namely, GluE, BrnE/BrnF, and LysP, which are widely present in C. glutamicum strains, were each investigated by gene knockout. In comparison with that in the wild-type strain, the yield of L-lysine increased by 9.0%, 12.3%, and 10.0% after the deletion of the gluE, brnE/brnF, and lysP genes, respectively, in C. glutamicum 23,604. Moreover, the amount of by-product amino acids decreased significantly when the gluE and brnE/brnF genes were deleted. It was also demonstrated that there was no effect on the growth of the strain when the gluE or lysP gene was deleted, whereas the biomass of C. glutamicum WL1702 (ΔbrnE/ΔbrnF) in the fermentation medium was significantly reduced in comparison with that of the wild type. These results also provide useful information for enhancing the production of L-lysine or other amino acids by C. glutamicum.

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b [0,+] AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b (0,+) AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid-binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters., Competing Interests: The authors declare no competing interest.

l-Histidine is a functional amino acid with numerous therapeutic and ergogenic properties. It is one of the few amino acids that is not produced on a large scale by microbial fermentation due to the lack of an efficient microbial cell factory. In this study, we demonstrated the engineering of wild-type Escherichia coli to overproduce histidine from glucose. First, removal of transcription attenuation and histidine-mediated feedback inhibition resulted in 0.8 g/L histidine accumulation. Second, chromosome-based optimization of the expression levels of histidine biosynthesis genes led to a 4.75-fold increase in histidine titer. Third, strengthening phosphoribosyl pyrophosphate supply and rerouting the purine nucleotide biosynthetic pathway improved the histidine production to 8.2 g/L. Fourth, introduction of the NADH-dependent glutamate dehydrogenase from Bacillus subtilis and the lysine exporter from Corynebacterium glutamicum enabled the final strain HW6-3 to produce 11.8 g/L histidine. Finally, 66.5 g/L histidine was produced under fed-batch fermentation, with a yield of 0.23 g/g glucose and a productivity of 1.5 g/L/h. This is the highest titer and productivity of histidine ever reported from an engineered strain. Additionally, the metabolic strategies utilized here can be applied to engineering other microorganisms for the industrial production of histidine and related bioproducts.

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5-fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine-Histidine-type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in 15 N-amino acid-fed mutants versus wild-type, a higher percentage of 15 N remained in roots instead of being allocated to the shoot. 15 N-ammonium uptake and subsequently the delivery of root-synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Slc7a11 (xCT) and Slc3a1 (rBAT) are cystine uptake transporters that maintain intracellular concentrations of cysteine, the rate-limiting amino acid in glutathione synthesis. This study was conducted to first determine the tissue distribution of the two transporters in male and female mice. Because Slc3a1 was the primary cystine transporter in liver, its sex-divergent expression, ontogeny, diurnal rhythm and whether its mRNA expression is altered by transcription factors (AhR, CAR, PXR, PPARα, and Nrf2) was also investigated. Slc7a11 was expressed highest in brain and gonads. Slc3a1 was expressed highest in kidney and intestine, followed by liver. Duodenal and hepatic Slc3a1 was higher in females than males. Hepatic Slc3a1 was high during darkness and low during daytime. Hepatic Scl3a1 was lowest pre-birth, increased to near maximal levels at birth, decreased back to pre-birth levels between Days 3-10, and then returned to peak levels by Day 45. Except for CAR, activation of transcription factors did not increase hepatic mRNA expression of Slc3a1. Chemical activation of CAR significantly induced Slc3a1 1.4-fold in wild-type but not CAR-null mice. Slc3a1 mRNA was higher in livers of AhR- and Nrf2-null mice compared to wild-type mice. High doses of diquat but not acetaminophen induced Slc3a1, suggesting Slc3a1 may respond to oxidative stress but not necessarily to GSH depletion. Overall, Slc7a11 is mainly expressed in brain and gonads, whereas Slc3a1 is mainly expressed in kidney, small intestine and liver, and its hepatic expression is regulated by diurnal rhythm and certain xenobiotic treatments.

Maintenance of cell surface residency and function of glycoproteins by lectins are essential for regulating cellular functions. Galectins are β-galactoside-binding lectins and form a galectin-lattice, which regulates stability, clustering, membrane sub-domain localization and endocytosis of plasmalemmal glycoproteins. We have previously reported that galectin-2 (Gal-2) forms a complex with cationic amino acid transporter 3 (CAT3) in pancreatic β cells, although the biological significance of the molecular interaction between Gal-2 and CAT3 has not been elucidated. In this study, we demonstrated that the structure of N-glycan of CAT3 was either tetra- or tri-antennary branch structure carrying β-galactosides, which works as galectin-ligands. Indeed, CAT3 bound to Gal-2 using β-galactoside epitope. Moreover, the disruption of the glycan-mediated bindings between galectins and CAT3 significantly reduced cell surface expression levels of CAT3. The reduced cell surface residency of CAT3 attenuated the cellular arginine uptake activities and subsequently reduced nitric oxide production, and thus impaired the arginine-stimulated insulin secretion of pancreatic β cells. These results indicate that galectin-lattice stabilizes CAT3 by preventing endocytosis to sustain the arginine-stimulated insulin secretion of pancreatic β cells. This provides a novel cell biological insight into the endocrinological mechanism of nutrition metabolism and homeostasis., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Nitrogen (N) is essential for plant growth and crop productivity. Organic N is a major form of remobilized N in plants' response to N limitation. It is necessary to understand the regulatory role of N limitation adaption (NLA) in organic N remobilization for this adaptive response. Transcriptional and proteomic analyses were integrated to investigate differential responses of wild-type (WT) and nla mutant plants to N limitation and to identify the core organic N transporters targeted by NLA. Under N limitation, the nla mutant presented an early senescence with faster chlorophyll loss and less anthocyanin accumulation than the WT, and more N was transported out of the aging leaves in the form of amino acids. High-throughput transcriptomic and proteomic analyses revealed that N limitation repressed genes involved in photosynthesis and protein synthesis, and promoted proteolysis; these changes were higher in the nla mutant than in the WT. Both transcriptional and proteomic profiling demonstrated that LHT1, responsible for amino acid remobilization, were only significantly upregulated in the nla mutant under N limitation. These findings indicate that NLA might target LHT1 and regulate organic N remobilization, thereby improving our understanding of the regulatory role of NLA on N remobilization under N limitation.

A direct inhibiting effect of NO on the function of CAT-1 and -2A has been postulated to occur via nitrosylation of cysteine residues in the transporters. Neither the NO donor SNAP nor a mixture of SIN-1 and Spermine NONOate, that generates the strong nitrosating agent N 2 O 3 , reduced CAT-mediated L-arginine transport. Direct nitros(yl)ation does either not occur in CATs or does not affect their transport function. A regulatory effect of NO or nitrosating agents on CAT-mediated transport under physiological conditions seems, therefore, unlikely.

The C9orf72 protein is required for normal lysosome function. In support of such functions, C9orf72 forms a heterotrimeric complex with SMCR8 and WDR41 that is recruited to lysosomes when amino acids are scarce. These properties raise questions about the identity of the lysosomal binding partner of the C9orf72 complex and the amino acid-sensing mechanism that regulates C9orf72 complex abundance on lysosomes. We now demonstrate that an interaction with the lysosomal cationic amino acid transporter PQLC2 mediates C9orf72 complex recruitment to lysosomes. This is achieved through an interaction between PQLC2 and WDR41. The interaction between PQLC2 and the C9orf72 complex is negatively regulated by arginine, lysine, and histidine, the amino acids that PQLC2 transports across the membrane of lysosomes. These results define a new role for PQLC2 in the regulated recruitment of the C9orf72 complex to lysosomes and reveal a novel mechanism that allows cells to sense and respond to changes in the availability of cationic amino acids within lysosomes., (© 2019 Amick et al.)

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein-rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L-mediated arginine transport is mainly accounted for by y+LAT1 in monocyte-derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco-2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco-2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Porcine epidemic diarrhea virus (PEDV) infection can induce intestinal dysfunction, resulting in severe diarrhea and even death, but the mode of action underlying these viral effects remains unclear. This study determined the effects of PEDV infection on intestinal absorption and the expression of genes for nutrient transporters via biochemical tests and microarray analysis. Sixteen 7-day-old healthy piglets fed a milk replacer were randomly allocated to one of two groups. After 5-day adaption, piglets (n = 8/group) were orally administrated with either sterile saline or PEDV (the strain from Yunnan province) at 10 4.5 TCID 50 (50% tissue culture infectious dose) per pig. All pigs were orally infused D-xylose (0.1 g/kg BW) on day 5 post PEDV or saline administration. One hour later, jugular vein blood samples as well as intestinal samples were collected for further analysis. In comparison with the control group, PEDV infection increased diarrhea incidence, blood diamine oxidase activity, and iFABP level, while reducing growth and plasma D-xylose concentration in piglets. Moreover, PEDV infection altered plasma and jejunal amino acid profiles, and decreased the expression of aquaporins and amino acid transporters (L-type amino acid transporter 1, sodium-independent amino acid transporter, B(° ,+ )-type amino acid transport protein, sodium-dependent neutral amino acid transporter 1, sodium-dependent glutamate/aspartate transporter 3, and peptide transporter (1), lipid transport and metabolism-related genes (lipoprotein lipase, apolipoprotein A1, apolipoprotein A4, apolipoprotein C2, solute carrier family 27 member 2, solute carrier family 27 member 4, fatty acid synthase, and long-chain acyl-CoA synthetase (3), and glucose transport genes (glucose transporter-2 and insulin receptor) in the jejunum. However, PEDV administration increased mRNA levels for phosphoenolpyruvate carboxykinase 1, argininosuccinate synthase 1, sodium/glucose co-transporter-1, and cystic fibrosis transmembrane conductance regulator in the jejunum. Collectively, these comprehensive results indicate that PEDV infection induces intestinal injury and inhibits the expression of genes encoding for nutrient transporters.

Background: Growth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. However, it is currently unknown whether there is a direct link between these external growth signals and intracellular amino acid levels. In primary prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1), and we therefore investigated whether LAT3 is regulated by growth factor signalling., Methods: To investigate how PI3K/Akt signalling regulates leucine transport, prostate cancer cells were treated with different PI3K/Akt inhibitors, or stable knock down of LAT3 by shRNA, followed by analysis of leucine uptake, western blotting, immunofluorescent staining and proximity ligation assay., Results: Inhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines, while growth factor addition significantly increased leucine uptake. These effects appeared to be mediated by LAT3 transport, as LAT3 knockdown blocked leucine uptake, and was not rescued by growth factor activation or further inhibited by signalling pathway inhibition. We further demonstrated that EGF significantly increased LAT3 protein levels when Akt was phosphorylated, and that Akt and LAT3 co-localised on the plasma membrane in EGF-activated LNCaP cells. These effects were likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation., Conclusion: Growth factor-activated PI3K/Akt signalling pathway regulates leucine transport through LAT3 in prostate cancer cell lines. These data support a direct link between growth factor and amino acid uptake, providing a mechanism by which the cells rapidly coordinate amino acid uptake for cell growth.

Background: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity., Results: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter., Conclusions: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.

The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae , we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsE A14 , a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30× higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1 , when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsE A14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability., (Copyright © 2019 by the Genetics Society of America.)

The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication., (Published by Oxford University Press on behalf of Nucleic Acids Research 2019.)

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection., Competing Interests: The authors have declared that no competing interests exist.

The pathology of acute lung injury (ALI) involves inducible nitric oxide (NO) synthase (iNOS)-derived NO-induced apoptosis of pulmonary endothelial cells. In vitro, iNOS-derived NO production has been shown to depend on the uptake of l-arginine by the cationic amino acid transporters (CAT). To test the hypothesis that mice deficient in CAT-2 ( slc7a2 -/- on a C57BL/6 background) would be protected from hyperoxia-induced ALI, mice ( slc7a2 -/- or wild-type) were placed in >95% oxygen (hyperoxia) or 21% oxygen (control) for 60 h. In wild-type mice exposed to hyperoxia, the exhaled nitric oxide (exNO) was twofold greater than in wild-type mice exposed to normoxia ( P < 0.005), whereas in slc7a2 -/- mice there was no significant difference between exNO in animals exposed to hyperoxia or normoxia ( P = 0.95). Hyperoxia-exposed wild-type mice had greater ( P < 0.05) lung resistance and a lower ( P < 0.05) lung compliance than did hyperoxia-exposed slc7a2 -/- mice. The lung wet-to-dry weight ratio was greater ( P < 0.005) in the hyperoxia-exposed wild-type mice than in hyperoxia-exposed slc7a2 -/- mice. Neutrophil infiltration was lower ( P < 0.05) in the hyperoxia-exposed slc7a2 -/- mice than in the hyperoxia-exposed wild-type mice as measured by myeloperoxidase activity. The protein concentration in bronchoalveolar lavage fluid was lower ( P < 0.001) in the hyperoxia-exposed slc7a2 -/- mice than in similarly exposed wild-type mice. The percent of TUNEL-positive cells in the lung following hyperoxia exposure was significantly lower ( P < 0.001) in the slc7a2 -/- mice than in the wild-type mice. These results are consistent with our hypothesis that lack of CAT-2 protects mice from acute lung injury.

Tumor cells overexpress amino acid transporters to meet the increased demand for amino acids. PQ loop repeat-containing (PQLC)2 is a cationic amino acid transporter that might be involved in cancer progression. Here, we show that upregulation of PQLC2 is critical to gastric cancer (GC) development in vitro and in vivo. Both PQLC2 mRNA and protein were overexpressed in GC tissues, especially of the diffuse type. Overexpression of PQLC2 promoted cell growth, anchorage independence, and tumor formation in nude mice. This was due to activation of MEK/ERK1/2 and PI3K/AKT signaling. Conversely, PQLC2 knockdown caused growth arrest and cell death of cancer cells and suppressed tumor growth in a mouse xenograft model. These results suggest that targeting PQLC2 is an effective strategy for GC treatment., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)