Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 563
Filter
1.
Molecules ; 29(10)2024 May 15.
Article in English | MEDLINE | ID: mdl-38792190

ABSTRACT

As a conformationally restricted amino acid, hydroxy-l-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened synthetic hydroxy-l-proline derivatives using electrophysiological and radiolabeled uptake methods against amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We have discovered a novel class of alkoxy hydroxy-pyrrolidine carboxylic acids (AHPCs) that act as selective high-affinity inhibitors of the SLC1 family neutral amino acid transporters SLC1A4 and SLC1A5. AHPCs were computationally docked into a homology model and assessed with respect to predicted molecular orientation and functional activity. The series of hydroxyproline analogs identified here represent promising new agents to pharmacologically modulate SLC1A4 and SLC1A5 amino acid exchangers which are implicated in numerous pathophysiological processes such as cancer and neurological diseases.


Subject(s)
Amino Acid Transport System ASC , Minor Histocompatibility Antigens , Amino Acid Transport System ASC/antagonists & inhibitors , Amino Acid Transport System ASC/metabolism , Amino Acid Transport System ASC/chemistry , Minor Histocompatibility Antigens/metabolism , Minor Histocompatibility Antigens/chemistry , Humans , Proline/chemistry , Proline/analogs & derivatives , Animals , Molecular Docking Simulation , Structure-Activity Relationship , HEK293 Cells , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Pyrrolidines/chemical synthesis , Drug Discovery , Amino Acid Transport Systems, Neutral/antagonists & inhibitors , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems, Neutral/genetics
2.
Cells ; 13(7)2024 Apr 06.
Article in English | MEDLINE | ID: mdl-38607085

ABSTRACT

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Humans , Cystine/metabolism , Cystinosis/metabolism , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Cysteamine , Mutation/genetics
3.
Int Immunopharmacol ; 132: 111918, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38537539

ABSTRACT

BACKGROUND: Sideroflexin 1 (SFXN1), a mitochondrial serine transporter implicated in one-carbon metabolism, is a prognostic biomarker in lung adenocarcinoma (LUAD). However, its role in LUAD progression remains elusive. This study aimed to investigate the functional significance of SFXN1 in LUAD and evaluate its potential as a therapeutic target. METHODS: We analyzed SFXN1 expression and its diagnostic and prognostic value in LUAD using the Pan-cancer TCGA dataset. In vitro assays (CCK-8, cell cycle, EDU, wound-healing, and transwell) were employed to assess the role of SFXN1, complemented by in vivo experiments. RNA sequencing elucidated SFXN1-mediated cellular functions and potential mechanisms. Bulk RNA-seq and scRNA-seq data from TCGA and GEO were used to investigate the correlation between SFXN1 and the tumor immune microenvironment. RT-qPCR, Western blot, and IHC assays validated SFXN1 expression and its impact on the immune microenvironment in LUAD. RESULTS: SFXN1 was upregulated in LUAD tissues and associated with poor prognosis. RNA-seq and scRNA-seq analyses revealed increased SFXN1 expression in tumor cells, accompanied by decreased infiltration of NK and cytotoxic T cells. SFXN1 knockdown significantly reduced cell proliferation and migration, and the inhibition of ERK phosphorylation and CCL20 expression may be the molecular mechanism involved. In vivo, targeting SFXN1 decreased Tregs infiltration and inhibited tumor growth. CONCLUSIONS: Our findings suggest that SFXN1 may be a potential therapeutic target for LUAD treatment.


Subject(s)
Adenocarcinoma of Lung , Amino Acid Transport Systems, Neutral , Lung Neoplasms , Lymphocytes, Tumor-Infiltrating , Tumor Microenvironment , Humans , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/immunology , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinogenesis/genetics , Carcinogenesis/immunology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Lung Neoplasms/immunology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lymphocytes, Tumor-Infiltrating/immunology , Prognosis , Tumor Microenvironment/immunology , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism
4.
Am J Physiol Renal Physiol ; 326(6): F981-F987, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38545650

ABSTRACT

Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the CTNS gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate MFSD12 mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of MFSD12 knockout on cystine levels. We showed similar MFSD12 mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC (CTNSΔ57kb) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of mfsd12a translation-blocking morpholino (TB MO) in a ctns-/- zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the mfsd12a TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, MFSD12 mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the ctns-/- zebrafish embryo. In addition, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development warrants further evaluation.NEW & NOTEWORTHY In this study, we show that MFSD12 depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and ctns-/- zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the ctns-/- zebrafish embryos injected with mfsd12a translation-blocking morpholino. Furthermore, a negative effect of the mfsd12a morpholino on the zebrafish development warrants further investigation.


Subject(s)
Cystine , Cystinosis , Disease Models, Animal , Kidney Tubules, Proximal , Zebrafish , Animals , Zebrafish/metabolism , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Cystinosis/metabolism , Cystinosis/genetics , Cystinosis/pathology , Humans , Cystine/metabolism , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Epithelial Cells/metabolism , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , CRISPR-Cas Systems
5.
Int J Mol Sci ; 25(4)2024 Feb 09.
Article in English | MEDLINE | ID: mdl-38396788

ABSTRACT

Innate immune cells, including macrophages, are functionally affected by thyroid hormone (TH). Macrophages can undergo phenotypical alterations, shifting between proinflammatory (M1) and immunomodulatory (M2) profiles. Cellular TH concentrations are, among others, determined by TH transporters. To study the effect of TH and TH transporters on macrophage polarization, specific proinflammatory and immunomodulatory markers were analyzed in bone marrow-derived macrophages (BMDMs) depleted of triiodothyronine (T3) and BMDMs with a knockout (KO) of Mct8 and Mct10 and a double KO (dKO) of Mct10/Mct8. Our findings show that T3 is important for M1 polarization, while a lack of T3 stimulates M2 polarization. Mct8 KO BMDMs are unaffected in their T3 responsiveness, but exhibit slight alterations in M2 polarization, while Mct10 KO BMDMs show reduced T3 responsiveness, but unaltered polarization markers. KO of both the Mct8 and Mct10 transporters decreased T3 availability and, contrary to the T3-depleted BMDMs, showed partially increased M1 markers and unaltered M2 markers. These data suggest a role for TH transporters besides transport of TH in BMDMs. This study highlights the complex role of TH transporters in macrophages and provides a new angle on the interaction between the endocrine and immune systems.


Subject(s)
Macrophages , Symporters , Thyroid Hormones , Animals , Mice , Macrophages/metabolism , Monocarboxylic Acid Transporters/genetics , Symporters/genetics , Thyroid Hormones/metabolism , Thyroid Hormones/pharmacology , Triiodothyronine/pharmacology , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism
6.
Autophagy ; 20(1): 202-204, 2024 01.
Article in English | MEDLINE | ID: mdl-37621073

ABSTRACT

Differentiation and fate decisions are critical for the epithelial cells lining the proximal tubule (PT) of the kidney, but the signals involved remain unknown. Defective cystine mobilization from lysosomes through CTNS (cystinosin, lysosomal cystine transporter), which is mutated in cystinosis, triggers the dedifferentiation and dysfunction of the PT cells, causing kidney disease and severe metabolic complications. Using preclinical models and physiologically relevant cellular systems, along with functional assays and a generative artificial intelligence (AI)-powered engine, we found that cystine storage imparted by CTNS deficiency stimulates Ragulator-RRAG GTPase-dependent recruitment of MTORC1 and its constitutive activation. In turn, this diverts the catabolic trajectories and differentiating states of PT cells toward growth and proliferation, disrupting homeostasis and their specialized functions. Therapeutic MTORC1 inhibition by using low doses of rapamycin corrects lysosome function and differentiation downstream of cystine storage and ameliorates PT dysfunction in preclinical models of cystinosis. These discoveries suggest that cystine may act as a lysosomal fasting signal that tailors MTORC1 signaling to direct fate decisions in the kidney PT epithelium, highlighting novel therapeutic paradigms for cystinosis and other lysosome-related disorders.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Amino Acid Transport Systems, Neutral/metabolism , Artificial Intelligence , Autophagy , Cystine/metabolism , Cystinosis/metabolism , Epithelial Cells/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism
7.
Lab Invest ; 104(1): 100287, 2024 01.
Article in English | MEDLINE | ID: mdl-37949358

ABSTRACT

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis-trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Humans , Cystinosis/genetics , Cystinosis/metabolism , Cystine/genetics , Cystine/metabolism , Proteomics , Biomarkers , Gene Silencing , RNA, Small Interfering/genetics , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism
8.
J Neurol Sci ; 456: 122841, 2024 Jan 15.
Article in English | MEDLINE | ID: mdl-38101161

ABSTRACT

Nephropathic cystinosis is a rare autosomal recessive storage disorder caused by CTNS gene mutations, leading to autophagy-lysosomal pathway impairment and cystine crystals accumulation. Neurologic involvement is highly variable and includes both neurodevelopmental and neurodegenerative disturbances, as well as focal neurologic deficits. By presenting longitudinal data of a 28-year-old patient with a large infratentorial lesion, we summarized the pathology, clinical and imaging features of neurological involvement in cystinosis patients. Brain damage in form of cystinosis-related cerebral lesions occurs in advanced disease phases and is characterized by the accumulation of cystine crystals, subsequent inflammation with vasculitis-like features, necrosis, and calcification. Epilepsy is a frequent comorbidity in affected individuals. Steroids might play a role in the symptomatic treatment of "stroke-like" episodes due to edematous-inflammatory lesions, but probably do not change the overall prognosis. Lifelong compliance to depleting therapy with cysteamine still represents the main therapeutic option. However, consequences of CTNS gene defects are not restricted to cystine accumulation. New evidence of four-repeat (4R-) Tau immunoreactivity suggests concurrent progressive neurodegeneration in cystinosis patients, highlighting the need of innovative therapeutic strategies, and shedding light on the crosstalk between proteinopathies and autophagy-lysosomal system defects. Eventually, emerging easily accessible biomarkers such as serum neurofilament light chains (NfL) might detect subclinical neurologic involvement in cystinosis patients.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Humans , Adult , Cystinosis/complications , Cystinosis/genetics , Cystinosis/drug therapy , Cystine/metabolism , Cystine/therapeutic use , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems, Neutral/therapeutic use , Cysteamine/therapeutic use , Inflammation/drug therapy , Brain/diagnostic imaging , Brain/metabolism
9.
Int J Mol Sci ; 24(23)2023 Nov 30.
Article in English | MEDLINE | ID: mdl-38069326

ABSTRACT

Cystinosis is an autosomal recessive disease resulting from mutations in ctns, which encodes for cystinosin, a proton-coupled cystine transporter that exports cystine from lysosomes. The major clinical form, infantile cystinosis, is associated with renal failure due to the malfunctioning of the renal proximal tubule (RPT). To examine the hypothesis that the malfunctioning of the cystinotic RPT arises from defective differentiation, human-induced pluripotent stem cells (hiPSCs) were generated from human dermal fibroblasts from an individual with infantile cystinosis, as well as a normal individual. The results indicate that both the cystinotic and normal hiPSCs are pluripotent and can form embryoid bodies (EBs) with the three primordial germ layers. When the normal hiPSCs were subjected to a differentiation regime that induces RPT formation, organoids containing tubules with lumens emerged that expressed distinctive RPT proteins, including villin, the Na+/H+ Exchanger (NHE) isoform 3 (NHE3), and the NHE Regulatory Factor 1 (NHERF1). The formation of tubules with lumens was less pronounced in organoids derived from cystinotic hiPSCs, although the organoids expressed villin, NHE3, and NHERF1. These observations can be attributed to an impairment in differentiation and/or by other defects which cause cystinotic RPTs to have an increased propensity to undergo apoptosis or other types of programmed cell death.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Induced Pluripotent Stem Cells , Humans , Cystinosis/genetics , Cystine/metabolism , Induced Pluripotent Stem Cells/metabolism , Sodium-Hydrogen Exchanger 3/genetics , Mutation , Amino Acid Transport Systems, Neutral/metabolism , Lysosomes/metabolism
10.
Pharmacol Rev ; 76(1): 142-193, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37940347

ABSTRACT

The neutral amino acid transporter subfamily that consists of six members, consecutively SLC6A15-SLC620, also called orphan transporters, represents membrane, sodium-dependent symporter proteins that belong to the family of solute carrier 6 (SLC6). Primarily, they mediate the transport of neutral amino acids from the extracellular milieu toward cell or storage vesicles utilizing an electric membrane potential as the driving force. Orphan transporters are widely distributed throughout the body, covering many systems; for instance, the central nervous, renal, or intestinal system, supplying cells into molecules used in biochemical, signaling, and building pathways afterward. They are responsible for intestinal absorption and renal reabsorption of amino acids. In the central nervous system, orphan transporters constitute a significant medium for the provision of neurotransmitter precursors. Diseases related with aforementioned transporters highlight their significance; SLC6A19 mutations are associated with metabolic Hartnup disorder, whereas altered expression of SLC6A15 has been associated with a depression/stress-related disorders. Mutations of SLC6A18-SLCA20 cause iminoglycinuria and/or hyperglycinuria. SLC6A18-SLC6A20 to reach the cellular membrane require an ancillary unit ACE2 that is a molecular target for the spike protein of the SARS-CoV-2 virus. SLC6A19 has been proposed as a molecular target for the treatment of metabolic disorders resembling gastric surgery bypass. Inhibition of SLC6A15 appears to have a promising outcome in the treatment of psychiatric disorders. SLC6A19 and SLC6A20 have been suggested as potential targets in the treatment of COVID-19. In this review, we gathered recent advances on orphan transporters, their structure, functions, related disorders, and diseases, and in particular their relevance as therapeutic targets. SIGNIFICANCE STATEMENT: The following review systematizes current knowledge about the SLC6A15-SLCA20 neutral amino acid transporter subfamily and their therapeutic relevance in the treatment of different diseases.


Subject(s)
Amino Acid Transport Systems, Neutral , Amino Acids, Neutral , COVID-19 , Humans , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Kidney/metabolism , Amino Acids/metabolism , Amino Acids, Neutral/metabolism , COVID-19/metabolism , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism
11.
Sci Rep ; 13(1): 20961, 2023 11 28.
Article in English | MEDLINE | ID: mdl-38016974

ABSTRACT

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns-/- larvae, and restoration of the zebrafish pronephros function.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Animals , Cystinosis/genetics , Cystinosis/therapy , Cystine/metabolism , Zebrafish/genetics , Zebrafish/metabolism , RNA, Messenger/genetics , RNA, Messenger/therapeutic use , Models, Theoretical , Dietary Supplements , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism
12.
Am J Physiol Cell Physiol ; 325(2): C550-C562, 2023 08 01.
Article in English | MEDLINE | ID: mdl-37458433

ABSTRACT

SLC38A5/SNAT5 is a system N transporter that can mediate net inward or outward transmembrane fluxes of neutral amino acids coupled with Na+ (symport) and H+ (antiport). Its preferential substrates are not only amino acids with side chains containing amide (glutamine and asparagine) or imidazole (histidine) groups, but also serine, glycine, and alanine are transported by the carrier. Expressed in the pancreas, intestinal tract, brain, liver, bone marrow, and placenta, it is regulated at mRNA and protein levels by mTORC1 and WNT/ß-catenin pathways, and it is sensitive to pH, nutritional stress, inflammation, and hypoxia. SNAT5 expression has been found to be altered in pathological conditions such as chronic inflammatory diseases, gestational complications, chronic metabolic acidosis, and malnutrition. Growing experimental evidence shows that SNAT5 is overexpressed in several types of cancer cells. Moreover, recently published results indicate that SNAT5 expression in stromal cells can support the metabolic exchanges occurring in the tumor microenvironment of asparagine-auxotroph tumors. We review the functional role of the SNAT5 transporter in pathophysiology and propose that, due to its peculiar operational and regulatory features, SNAT5 may play important pro-cancer roles when expressed either in neoplastic or in stromal cells of glutamine-auxotroph tumors.NEW & NOTEWORTHY The transporter SLC38A5/SNAT5 provides net influx or efflux of glutamine, asparagine, and serine. These amino acids are of particular metabolic relevance in several conditions. Changes in transporter expression or activity have been described in selected types of human cancers, where SNAT5 can mediate amino acid exchanges between tumor and stromal cells, thus providing a potential therapeutic target. This is the first review that recapitulates the characteristics and roles of the transporter in physiology and pathology.


Subject(s)
Amino Acid Transport Systems, Neutral , Neoplasms , Pregnancy , Female , Humans , Glutamine , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Asparagine , Tumor Microenvironment , Amino Acid Transport Systems , Amino Acids , Serine , Neoplasms/genetics
13.
Int J Mol Sci ; 24(12)2023 Jun 16.
Article in English | MEDLINE | ID: mdl-37373388

ABSTRACT

d-amino acids have recently been found to be present in the extracellular milieu at millimolar levels and are therefore assumed to play a physiological function. However, the pathway (or potential pathways) by which these d-amino acids are secreted remains unknown. Recently, Escherichia coli has been found to possess one or more energy-dependent d-alanine export systems. To gain insight into these systems, we developed a novel screening system in which cells expressing a putative d-alanine exporter could support the growth of d-alanine auxotrophs in the presence of l-alanyl-l-alanine. In the initial screening, five d-alanine exporter candidates, AlaE, YmcD, YciC, YraM, and YidH, were identified. Transport assays of radiolabeled d-alanine in cells expressing these candidates indicated that YciC and AlaE resulted in lower intracellular levels of d-alanine. Further detailed transport assays of AlaE in intact cells showed that it exports d-alanine in an expression-dependent manner. In addition, the growth constraints on cells in the presence of 90 mM d-alanine were mitigated by the overexpression of AlaE, implying that AlaE could export free d-alanine in addition to l-alanine under conditions in which intracellular d/l-alanine levels are raised. This study also shows, for the first time, that YciC could function as a d-alanine exporter in intact cells.


Subject(s)
Amino Acid Transport Systems, Neutral , Escherichia coli Proteins , Escherichia coli , Alanine/metabolism , Escherichia coli Proteins/metabolism , Amino Acids/metabolism , Biological Transport , Amino Acid Transport Systems, Neutral/metabolism
14.
Med Sci (Paris) ; 39(3): 253-261, 2023 Mar.
Article in French | MEDLINE | ID: mdl-36943122

ABSTRACT

Cystinosis is an autosomal recessive metabolic disease characterized by lysosomal accumulation of cystine in all the cells of the body. Infantile cystinosis begins in infancy by a renal Fanconi syndrome and eventually leads to multi-organ failure, including the kidney, eye, thyroid, muscle, and pancreas, eventually causing premature death in early adulthood. The current treatment is the drug cysteamine that only delays the progression of the disease. We identified the gene involved, CTNS, and showed that the encoded protein, cystinosin, is a proton-driven cystine transporter. We generated a mouse model of cystinosis, the Ctns-/- mice, that recapitulates the main disease complications. The goal was next to develop a gene therapy approach for cystinosis. We used bone marrow stem cells as a vehicle to bring the healthy CTNS gene to tissues, and we showed that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation led to abundant tissue integration of bone marrow-derived cells, significant decrease of tissue cystine accumulation and long-term kidney, eye and thyroid preservation. We then developed an autologous transplantation approach of HSPCs modified ex vivo using a lentiviral vector to introduce a functional CTNS cDNA, and showed its efficacy in Ctns-/- mice. We conducted the pharmacology/toxicology studies, developed the manufacturing process using human CD34+ cells, and design the clinical trial. We received Food and Drug Administration (FDA)-clearance to start a phase 1/2 clinical trial for cystinosis in December 2018. Six patients have been treated so far. In this review, we describe the path to go from the gene to a gene therapy approach for cystinosis.


Title: Cystinose - De la découverte du gène aux premiers essais de thérapie génique. Abstract: La cystinose est une maladie métabolique autosomique récessive caractérisée par une accumulation lysosomale de cystine dans toutes les cellules de l'organisme. La cystinose infantile débute dans la petite enfance par un syndrome de Fanconi et aboutit à une détérioration progressive de la fonction de la plupart des organes, y compris les reins, les yeux, la thyroïde, les muscles et le pancréas, et finit par entraîner une mort prématurée. Le traitement par la cystéamine ne permet que de retarder la progression de la maladie. Afin de développer une approche de thérapie génique pour la cystinose, un modèle murin qui présente les principales complications de la maladie a été développé grâce à l'identification du gène CTNS, dont le produit, la cystinosine, est un co-transporteur de cystine-protons. Cette revue décrit les étapes allant de la découverte du gène à la thérapie génique pour la cystinose, qui a permis de traiter six patients jusqu'à présent.


Subject(s)
Amino Acid Transport Systems, Neutral , Cystinosis , Adult , Animals , Humans , Mice , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems, Neutral/therapeutic use , Cysteamine/therapeutic use , Cysteamine/adverse effects , Cystine/genetics , Cystine/metabolism , Cystine/therapeutic use , Cystinosis/genetics , Cystinosis/therapy , Cystinosis/complications , Genetic Therapy/adverse effects , Kidney , Clinical Trials as Topic
15.
Elife ; 122023 02 01.
Article in English | MEDLINE | ID: mdl-36722887

ABSTRACT

Hyperosmolarity of the renal medulla is essential for urine concentration and water homeostasis. However, how renal medullary collecting duct (MCD) cells survive and function under harsh hyperosmotic stress remains unclear. Using RNA-Seq, we identified SLC38A2 as a novel osmoresponsive neutral amino acid transporter in MCD cells. Hyperosmotic stress-induced cell death in MCD cells occurred mainly via ferroptosis, and it was significantly attenuated by SLC38A2 overexpression but worsened by Slc38a2-gene deletion or silencing. Mechanistic studies revealed that the osmoprotective effect of SLC38A2 is dependent on the activation of mTORC1. Moreover, an in vivo study demonstrated that Slc38a2-knockout mice exhibited significantly increased medullary ferroptosis following water restriction. Collectively, these findings reveal that Slc38a2 is an important osmoresponsive gene in the renal medulla and provide novel insights into the critical role of SLC38A2 in protecting MCD cells from hyperosmolarity-induced ferroptosis via the mTORC1 signalling pathway.


Subject(s)
Amino Acid Transport Systems, Neutral , Ferroptosis , Animals , Mice , Amino Acid Transport Systems, Neutral/metabolism , Kidney/metabolism , Kidney Medulla/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism
16.
Respir Res ; 24(1): 33, 2023 Jan 27.
Article in English | MEDLINE | ID: mdl-36707853

ABSTRACT

Pulmonary inflammation involves complex changes of the immune cells, in which macrophages play important roles and their function might be influenced by metabolism. Slc38a6 acts as a carrier of nutrient for macrophages (Mφ) to exert the function. In this study, pneumonia patient blood was found up-regulated SLC38A6 expression, which correlated with monocytes number and white blood cell number. The similar result was also shown in LPS induced sepsis mice. To reveal the key role of Slc38a6, we used systemic and conditional knock-out mice. Either systemic or LyzCRE specific knock-out could alleviate the severity of sepsis mice, reduce the proinflammatory cytokine TNF-α and IL-1ß expression in serum and decrease the monocytes number in bronchial alveolar lavage and peritoneal lavage via flow cytometry. In order to reveal the signal of up-regulated Slc38a6, the Tlr4 signal inhibitor TAK242 and TLR4 knock-out mice were used. By blocking Tlr4 signal in macrophages via TAK242, the expression of Slc38a6 was down-regulated synchronously, and the same results were also found in Tlr4 knock-out macrophages. However, in the overexpressed Slc38a6 macrophages, blocking Tlr4 signal via TAK242, 20% of the mRNA expression of IL-1ß still could be expressed, indicating that up-regulated Slc38a6 participates in IL-1ß expression process. Collectively, it is the first time showed that an amino acid transporter SLC38A6 up-regulated in monocytes/macrophages promotes activation in pulmonary inflammation. SLC38A6 might be a promising target molecule for pulmonary inflammation treatment.


Subject(s)
Pneumonia , Toll-Like Receptor 4 , Animals , Mice , Inflammation/chemically induced , Inflammation/genetics , Inflammation/metabolism , Interleukin-1beta/metabolism , Lipopolysaccharides/toxicity , Macrophages/metabolism , Mice, Knockout , Pneumonia/chemically induced , Pneumonia/genetics , Pneumonia/metabolism , Signal Transduction/physiology , Nerve Tissue Proteins/metabolism , Amino Acid Transport Systems, Neutral/metabolism
17.
Dis Model Mech ; 16(5)2023 05 01.
Article in English | MEDLINE | ID: mdl-36374036

ABSTRACT

Nicotinamide adenine dinucleotide (NAD) is a key metabolite synthesised from vitamin B3 or tryptophan. Disruption of genes encoding NAD synthesis enzymes reduces NAD levels and causes congenital NAD deficiency disorder (CNDD), characterised by multiple congenital malformations. SLC6A19 (encoding B0AT1, a neutral amino acid transporter), represents the main transporter for free tryptophan in the intestine and kidney. Here, we tested whether Slc6a19 heterozygosity in mice limits the tryptophan available for NAD synthesis during pregnancy and causes adverse pregnancy outcomes. Pregnant Slc6a19+/- mice were fed diets depleted of vitamin B3, so that tryptophan was the source of NAD during gestation. This perturbed the NAD metabolome in pregnant Slc6a19+/- females, resulting in reduced NAD levels and increased rates of embryo loss. Surviving embryos were small and exhibited specific combinations of CNDD-associated malformations. Our results show that genes not directly involved in NAD synthesis can affect NAD metabolism and cause CNDD. They also suggest that human female carriers of a SLC6A19 loss-of-function allele might be susceptible to adverse pregnancy outcomes unless sufficient NAD precursor amounts are available during gestation. This article has an associated First Person interview with the first author of the paper.


Subject(s)
Amino Acid Transport Systems, Neutral , Congenital Abnormalities , NAD , Animals , Female , Mice , Pregnancy , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Heterozygote , Kidney/metabolism , NAD/deficiency , Niacinamide , Tryptophan/genetics , Tryptophan/metabolism , Congenital Abnormalities/genetics
18.
J Cell Biol ; 221(10)2022 10 03.
Article in English | MEDLINE | ID: mdl-36099524

ABSTRACT

Mutant huntingtin, which causes Huntington's disease (HD), is ubiquitously expressed but induces preferential loss of striatal neurons by unclear mechanisms. Rab11 dysfunction mediates homeostatic disturbance of HD neurons. Here, we report that Rab11 dysfunction also underscores the striatal vulnerability in HD. We profiled the proteome of Rab11-positive endosomes of HD-vulnerable striatal cells to look for protein(s) linking Rab11 dysfunction to striatal vulnerability in HD and found XK, which triggers the selective death of striatal neurons in McLeod syndrome. XK was trafficked together with Rab11 and was diminished on the surface of immortalized HD striatal cells and striatal neurons in HD mouse brains. We found that XK participated in transporting manganese, an essential trace metal depleted in HD brains. Introducing dominantly active Rab11 into HD striatal cells improved XK dynamics and increased manganese accumulation in an XK-dependent manner. Our study suggests that impaired Rab11-based recycling of XK onto cell surfaces for importing manganese is a driver of striatal dysfunction in Huntington's disease.


Subject(s)
Amino Acid Transport Systems, Neutral , Corpus Striatum , Huntington Disease , Amino Acid Transport Systems, Neutral/metabolism , Animals , Corpus Striatum/metabolism , Corpus Striatum/physiopathology , Endosomes/metabolism , Huntington Disease/genetics , Huntington Disease/metabolism , Manganese/metabolism , Mice , Neurons/metabolism , Proteome , rab GTP-Binding Proteins/metabolism
19.
J Cell Biol ; 221(10)2022 10 03.
Article in English | MEDLINE | ID: mdl-36112418

ABSTRACT

Chhetri and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202112073) show that Rab11-mediated endosomal recycling regulates cell surface expression of McLeod syndrome protein XK. Mutant huntingtin interferes with the recycling of XK to the cell surface and significantly reduces manganese transport across cell membrane.


Subject(s)
Amino Acid Transport Systems, Neutral , Endosomes , Huntington Disease , Manganese , Neuroacanthocytosis , Amino Acid Transport Systems, Neutral/metabolism , Cell Membrane/metabolism , Endosomes/metabolism , Humans , Huntington Disease/complications , Manganese/metabolism , Neuroacanthocytosis/complications , rab GTP-Binding Proteins/metabolism
20.
Biomed Pharmacother ; 155: 113726, 2022 Nov.
Article in English | MEDLINE | ID: mdl-36166962

ABSTRACT

D-serine has attracted increasing attention for its possible role in depression. L-4-Fluorophenylglycine (L-4FPG), an inhibitor of the neutral amino acid transporter ASCT1/2, has been shown to regulate extracellular D-serine levels. The present study aimed to explore the potential antidepressant effects of L-4FPG. First, the acute effects of L-4FPG on the forced swimming test, elevated plus maze test, and novelty-suppressed feeding test were examined. L-4FPG showed antidepressant-like effects, which could be reversed by rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist. The phosphorylation levels of mTOR and GluR1 in the hippocampus were also increased after L-4FPG treatment. Next, the therapeutic effects of L-4FPG were examined in a chronic social defeat stress (CSDS) model of depression. L-4FPG ameliorated depression-like behaviors in mice subjected to CSDS. Furthermore, treatment with L-4FPG prior to each social defeat stress session not only decreased defensive behaviors but also prevented CSDS-induced social avoidance and anxiety-like and depression-like behaviors. These findings suggest that L-4FPG may be useful not only in alleviating depression but also in protecting against chronic stress-related psychiatric disorders.


Subject(s)
Amino Acid Transport Systems, Neutral , Depression , Mice , Animals , 6-Cyano-7-nitroquinoxaline-2,3-dione/metabolism , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Depression/drug therapy , Depression/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/pharmacology , Stress, Psychological/drug therapy , Stress, Psychological/metabolism , Mice, Inbred C57BL , Antidepressive Agents/pharmacology , TOR Serine-Threonine Kinases/metabolism , Hippocampus , Serine/metabolism , Serine/pharmacology , Sirolimus/pharmacology , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems, Neutral/pharmacology , Mammals
SELECTION OF CITATIONS
SEARCH DETAIL
...