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2.
Int J Mol Sci ; 25(4)2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38396788

RESUMO

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.


Assuntos
Macrófagos , Simportadores , Hormônios Tireóideos , Animais , Camundongos , Macrófagos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Simportadores/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologia , Tri-Iodotironina/farmacologia , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo
3.
BMC Ophthalmol ; 24(1): 48, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38291379

RESUMO

BACKGROUND: The polymer-based facile and effective drug carrier approach was developed to treat superficial fungal infected retinopathy infections. METHODS: Here, biotin-glutathione (B-GHS) functionalized with chitosan grafted proline (CS-g-P) moieties were fabricated with the loading of fluconazole (FLZ) for the treatment of retinopathy. FT-IR and XRD techniques were used to characterize chemical structural and phase changes of the prepared carriers The SEM results show that the sphere morphology with interconnection particle nature. RESULTS: The particle diameter was found as ~ 6.5 and ~ 8.6 nm for CS-g-P/B-GHS and FLZ-loaded CS-g-P/B-GHS carriers, respectively. The negative surface charge was found as the values of CS-g-P/B-GHS and FLZ-loaded CS-g-P/B-GHS, such as -20.7 mV and - 32.2 mV, from zeta potential analysis. The in-vitro FLZ releases from the CS-g-P/B-GHS were investigated at pH 7.4 (PBS) as the tear fluid environment, and it was observed at 85.02% of FLZ release in 8 h reaction time. The sustained release was observed, leading to the necessity for prolonged therapeutic effects. The antifungal effect of the carrier was studied by the minimum inhibitory concentration (MIC) and the percentage inhibition of viable fungal count against Candida albicans, and it observed 81.02% of the zone of inhibition by the FLZ carrier. CONCLUSION: FLZ-loaded CS-g-P/B-GHS carrier could inhibit the biofilm formation in a concentration-dependent inhibition. Hence, A novel FLZ/B-GHS-CS-g-P carrier is a hopeful approach for effectively treating superficial fungal contaminations of the retina region.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Antifúngicos , Quitosana , Fluconazol , Retinite , Humanos , Antifúngicos/farmacologia , Biotina , Fluconazol/administração & dosagem , Doenças Retinianas , Retinite/tratamento farmacológico , Espectroscopia de Infravermelho com Transformada de Fourier , Micoses/tratamento farmacológico
4.
Breast Cancer ; 31(1): 96-104, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37914960

RESUMO

BACKGROUND: Solute carrier family 38 member 5 (SLC38A5), as an amino acid transporter, play a vital role in cellular biological processes. In this study, we analyzed the function of SLC38A5 and its potential mechanism in breast cancer (BC) progression. METHODS: The expression of SLC38A5 in cancer and adjacent-normal tissues was analyzed by qRT-PCR and Western blot, and its correlation with patient prognosis was analyzed. The immunohistochemical staining of cancer tissues and adjacent-normal tissues was performed on SLC38A5-positive specimens. BC mice were successfully applied to examine the role of SLC38A5 on tumor proliferation using the CCK-8 assay. In BC cells and mouse tumor tissues, SLC38A5 and PCNA expression were determined by Western blotting. RESULTS: The study found that SLC38A5 was highly expressed in BC patients and associated with a poor survival. SLC38A5 silencing inhibited BC cell viability and glutamine uptake. In addition, SLC38A5 overexpression promoted BC cell viability via the glutamine metabolism. SLC38A5 inhibited cisplatin chemosensitivity in BC cells. Importantly, SLC38A5 silencing inhibited tumor growth in vivo. CONCLUSION: Our findings suggest that SLC38A5 enhances BC cell viability by glutamine metabolism, inhibits the chemical sensitivity of cisplatin in BC cells, and promotes tumor growth, emphasizing the clinical relevance of SLC38A5 in BC management as a novel potential therapeutic target.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Glutamina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Sistemas de Transporte de Aminoácidos Neutros/uso terapêutico
5.
Clin Genet ; 105(3): 323-328, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38009794

RESUMO

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Humanos , Cistinose/genética , Splicing de RNA/genética , Éxons/genética , Sequências Reguladoras de Ácido Nucleico , RNA , Processamento Alternativo , Sítios de Splice de RNA , Sistemas de Transporte de Aminoácidos Neutros/genética
6.
Lab Invest ; 104(1): 100287, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37949358

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Humanos , Cistinose/genética , Cistinose/metabolismo , Cistina/genética , Cistina/metabolismo , Proteômica , Biomarcadores , Inativação Gênica , RNA Interferente Pequeno/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo
7.
Autophagy ; 20(1): 202-204, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37621073

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Inteligência Artificial , Autofagia , Cistina/metabolismo , Cistinose/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
8.
Neurochem Res ; 49(1): 170-183, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37684384

RESUMO

The glutamatergic hypothesis of schizophrenia suggests a correlation between NMDA receptor hypofunction and negative psychotic symptoms. It has been observed that the expression of the proline transporter (PROT) in the central nervous system (CNS) is associated with glutamatergic neurotransmission, as L-proline has the capacity to activate and modulate AMPA and NMDA receptors. In this study, we aimed to investigate whether inhibition of proline transporters could enhance glutamatergic neurotransmission and potentially exhibit antipsychotic effects in an experimental schizophrenia model. Using molecular dynamics analysis in silico, we validated an innovative PROT inhibitor, LQFM215. We quantified the cytotoxicity of LQFM215 in the Lund human mesencephalic cell line (LUHMES). Subsequently, we employed the ketamine-induced psychosis model to evaluate the antipsychotic potential of the inhibitor, employing behavioral tests including open-field, three-chamber interaction, and prepulse inhibition (PPI). Our results demonstrate that LQFM215, at pharmacologically active concentrations, exhibited negligible neurotoxicity when astrocytes were co-cultured with neurons. In the ketamine-induced psychosis model, LQFM215 effectively reduced hyperlocomotion and enhanced social interaction in a three-chamber social approach task across all administered doses. Moreover, the compound successfully prevented the ketamine-induced disruption of sensorimotor gating in the PPI test at all tested doses. Overall, these findings suggest that PROT inhibition could serve as a potential therapeutic target for managing symptoms of schizophrenia model.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Antipsicóticos , Ketamina , Esquizofrenia , Humanos , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Ketamina/farmacologia , Ketamina/uso terapêutico , Sistemas de Transporte de Aminoácidos Neutros/uso terapêutico , Receptores de N-Metil-D-Aspartato
9.
J Neurol Sci ; 456: 122841, 2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38101161

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Humanos , Adulto , Cistinose/complicações , Cistinose/genética , Cistinose/tratamento farmacológico , Cistina/metabolismo , Cistina/uso terapêutico , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/uso terapêutico , Cisteamina/uso terapêutico , Inflamação/tratamento farmacológico , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo
10.
Int J Mol Sci ; 24(23)2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38069326

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Células-Tronco Pluripotentes Induzidas , Humanos , Cistinose/genética , Cistina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Mutação , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Lisossomos/metabolismo
11.
Orphanet J Rare Dis ; 18(1): 389, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38087330

RESUMO

BACKGROUND: Cystinosis, a rare lysosomal storage disease caused by mutations in the CTNS gene, is characterized by cystine crystallization and accumulation within multiple tissues, including kidney and brain. Its impact on neural function appears mild relative to its effects on other organs during early disease, but since therapeutic advances have led to substantially increased life expectancy, neurological implications are of increasing interest, necessitating deeper understanding of the impact of cystinosis on neurocognitive function. Behavioral difficulties have been reported in cystinosis in the visual domain. Very little is known, however, about how the brains of people living with cystinosis process visual information. This is especially interesting given that cystine accumulation in the cornea and posterior ocular structures is a hallmark of cystinosis. METHODS: Here, high-density scalp electrophysiology was recorded to visual stimuli (during a Go/No-Go task) to investigate visual processing in individuals with cystinosis, compared to age-matched controls. Analyses focused on early stages of cortical visual processing. RESULTS: The groups differed in their initial cortical response, with individuals with cystinosis exhibiting a significantly larger visual evoked potential (VEP) in the 130-150 ms time window. The groups also differed in the associations between neural responses and verbal abilities: While controls with higher IQ scores presented larger neural responses, that relationship was not observed in cystinosis. CONCLUSIONS: The enlarged VEP in cystinosis could be the result of cortical hyperexcitability and/or differences in attentional engagement and explain, at least partially, the visual and visual-spatial difficulties described in this population.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Oftalmopatias , Criança , Adulto , Humanos , Cistinose/genética , Cistinose/tratamento farmacológico , Cistina/genética , Cistina/metabolismo , Cistina/uso terapêutico , Potenciais Evocados Visuais , Mutação/genética , Percepção Visual , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/uso terapêutico
12.
Pharmacol Rev ; 76(1): 142-193, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37940347

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Aminoácidos Neutros , COVID-19 , Humanos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Rim/metabolismo , Aminoácidos/metabolismo , Aminoácidos Neutros/metabolismo , COVID-19/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
13.
Sci Rep ; 13(1): 20961, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-38016974

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Animais , Cistinose/genética , Cistinose/terapia , Cistina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/uso terapêutico , Modelos Teóricos , Suplementos Nutricionais , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo
14.
Cells ; 12(20)2023 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-37887353

RESUMO

Pancreatic cancer is characterized by a poor prognosis, with its five-year survival rate lower than that of any other cancer type. Gemcitabine, a standard treatment for pancreatic cancer, often has poor outcomes for patients as a result of chemoresistance. Therefore, novel therapeutic targets must be identified to overcome gemcitabine resistance. Here, we found that SLC38A5, a glutamine transporter, is more highly overexpressed in gemcitabine-resistant patients than in gemcitabine-sensitive patients. Furthermore, the deletion of SLC38A5 decreased the proliferation and migration of gemcitabine-resistant PDAC cells. We also found that the inhibition of SLC38A5 triggered the ferroptosis signaling pathway via RNA sequencing. Also, silencing SLC38A5 induced mitochondrial dysfunction and reduced glutamine uptake and glutathione (GSH) levels, and downregulated the expressions of GSH-related genes NRF2 and GPX4. The blockade of glutamine uptake negatively modulated the mTOR-SREBP1-SCD1 signaling pathway. Therefore, suppression of SLC38A5 triggers ferroptosis via two pathways that regulate lipid ROS levels. Similarly, we observed that knockdown of SLC38A5 restored gemcitabine sensitivity by hindering tumor growth and metastasis in the orthotopic mouse model. Altogether, our results demonstrate that SLC38A5 could be a novel target to overcome gemcitabine resistance in PDAC therapy.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Ferroptose , Neoplasias Pancreáticas , Animais , Camundongos , Humanos , Gencitabina , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Glutamina , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas
15.
Invest Ophthalmol Vis Sci ; 64(13): 32, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37862028

RESUMO

Purpose: We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation. Methods: The FHONDA mouse model was generated with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). Results: From numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin content. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labeling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. Conclusions: Slc38a8 mutant mice recapitulate the phenotype of patients with FHONDA concerning their normal pigmentation and their abnormal visual system, in the latter being a hallmark of all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.


Assuntos
Albinismo , Sistemas de Transporte de Aminoácidos Neutros , Anormalidades do Olho , Adulto , Humanos , Camundongos , Animais , Melaninas , Camundongos Endogâmicos C57BL , Pigmentação , Sistemas de Transporte de Aminoácidos Neutros/genética
16.
Sci Rep ; 13(1): 16863, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37803043

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) cells have a great demand for nutrients in the form of sugars, amino acids, and lipids. Particularly, amino acids are critical for cancer growth and, as intermediates, connect glucose, lipid and nucleotide metabolism. PDAC cells meet these requirements by upregulating selective amino acid transporters. Here we show that SLC38A5 (SN2/SNAT5), a neutral amino acid transporter is highly upregulated and functional in PDAC cells. Using CRISPR/Cas9-mediated knockout of SLC38A5, we show its tumor promoting role in an in vitro cell line model as well as in a subcutaneous xenograft mouse model. Using metabolomics and RNA sequencing, we show significant reduction in many amino acid substrates of SLC38A5 as well as OXPHOS inactivation in response to SLC38A5 deletion. Experimental validation demonstrates inhibition of mTORC1, glycolysis and mitochondrial respiration in KO cells, suggesting a serious metabolic crisis associated with SLC38A5 deletion. Since many SLC38A5 substrates are activators of mTORC1 as well as TCA cycle intermediates/precursors, we speculate amino acid insufficiency as a possible link between SLC38A5 deletion and inactivation of mTORC1, glycolysis and mitochondrial respiration, and the underlying mechanism for PDAC attenuation. Overall, we show that SLC38A5 promotes PDAC, thereby identifying a novel, hitherto unknown, therapeutic target for PDAC.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Carcinógenos , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Sistemas de Transporte de Aminoácidos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Pancreáticas
17.
Sci Rep ; 13(1): 12728, 2023 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-37543674

RESUMO

Colon adenocarcinoma (COAD), one of the common clinical cancers, exhibits high morbidity and mortality, and its pathogenesis and treatment are still underdeveloped. Numerous studies have demonstrated the involvement of bile acids in tumour development, while the potential role of their metabolism in the tumor microenvironment (TME) has not been explored. A collection of 481 genes related to bile acid metabolism were obtained, and The Cancer Genome Atlas-based COAD risk model was developed using the least absolute shrinkage selection operator (LASSO) regression analysis. The Gene Expression Omnibus dataset was used to validate the results. The predictive performance of the model was verified using column line plots, principal component analysis and receiver operating characteristic curves. Then, we analysed the differences between the high- and low-risk groups from training set based on clinical characteristics, immune cell infiltration, immune-related functions, chemotherapeutic drug sensitivity and immunotherapy efficacy. Additionally, we constructed a protein-protein interaction network to screen for target genes, which were further investigated in terms of differential immune cell distribution. A total of 234 bile acids-related differentially expressed genes were obtained between normal and tumour colon tissues. Among them, 111 genes were upregulated and 123 genes were down-regulated in the tumour samples. Relying on the LASSO logistic regression algorithm, we constructed a model of bile acid risk score, comprising 12 genes: CPT2, SLCO1A2, CD36, ACOX1, CDKN2A, HADH, GABRD, LEP, TIMP1, MAT1A, SLC6A15 and PPARGC1A. This model was validated in the GEO-COAD set. Age and risk score were observed to be independent prognostic factors in patients with COAD. Genes related to bile acid metabolism in COAD were closely related to bile secretion, intestinal transport, steroid and fatty acid metabolism. Furthermore, the high-risk group was more sensitive to Oxaliplatin than the low-risk group. Finally, the three target genes screened were closely associated with immune cells. We identified a set of 12 genes (CPT2, SLCO1A2, CD36, ACOX1, CDKN2A, HADH, GABRD, LEP, TIMP1, MAT1A, SLC6A15, and PPARGC1A) associated with bile acid metabolism and developed a bile acid risk score model using LASSO regression analysis. The model demonstrated good predictive performance and was validated using an independent dataset. Our findings revealed that the bile acid risk score were independent prognostic factors in COAD patients.


Assuntos
Adenocarcinoma , Sistemas de Transporte de Aminoácidos Neutros , Neoplasias do Colo , Humanos , Prognóstico , Adenocarcinoma/genética , Neoplasias do Colo/genética , Genes p16 , Proteínas Inibidoras de Quinase Dependente de Ciclina , Ácidos e Sais Biliares , Microambiente Tumoral/genética , Proteínas do Tecido Nervoso
18.
Cell Rep ; 42(8): 112910, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37531255

RESUMO

Amino acid (aa) metabolism is closely correlated with the pathogenesis of psoriasis; however, details on aa transportation during this process are barely known. Here, we find that SLC38A5, a sodium-dependent neutral aa transporter that counter-transports protons, is markedly upregulated in the psoriatic skin of both human patients and mouse models. SLC38A5 deficiency significantly ameliorates the pathogenesis of psoriasis, indicating a pathogenic role of SLC38A5. Surprisingly, SLC38A5 is almost exclusively expressed in dendritic cells (DCs) when analyzing the psoriatic lesion and mainly locates on the lysosome. Mechanistically, SLC38A5 potentiates lysosomal acidification, which dictates the cleavage and activation of TLR7 with ensuing production of pro-inflammatory cytokines such as interleukin-23 (IL-23) and IL-1ß from DCs and eventually aggravates psoriatic inflammation. In summary, this work uncovers an auxiliary mechanism in driving lysosomal acidification, provides inspiring insights for DC biology and psoriasis etiology, and reveals SLC38A5 as a promising therapeutic target for treating psoriasis.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Psoríase , Animais , Camundongos , Humanos , Células Dendríticas/metabolismo , Pele/patologia , Psoríase/patologia , Inflamação/patologia , Modelos Animais de Doenças , Lisossomos/patologia , Concentração de Íons de Hidrogênio
19.
Nat Commun ; 14(1): 3994, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37452023

RESUMO

Differentiation is critical for cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in preclinical models of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Humanos , Cistina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Rim/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética
20.
Am J Physiol Cell Physiol ; 325(2): C550-C562, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37458433

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Neoplasias , Gravidez , Feminino , Humanos , Glutamina , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Asparagina , Microambiente Tumoral , Sistemas de Transporte de Aminoácidos , Aminoácidos , Serina , Neoplasias/genética
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