RESUMEN
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.
Asunto(s)
Cistina , Cistinosis , Modelos Animales de Enfermedad , Túbulos Renales Proximales , Pez Cebra , Animales , Pez Cebra/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Cistinosis/metabolismo , Cistinosis/genética , Cistinosis/patología , Humanos , Cistina/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Células Epiteliales/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas CRISPR-CasRESUMEN
Cystinosis is a rare autosomal recessive disease with an incidence 1 per 100,000-200,000 live births. It is caused by pathogenic variants of the cystinosin (CTNS) gene that lead to impaired cystine transport from lysosomes to cystosol, resulting in cystine accumulation in lysosomes and subsequent cellular dysfunction. The initial manifestation, cystine accumulation in proximal tubular cells (PTCs), causes renal Fanconi syndrome, which presents with proximal renal tubular acidosis and generalized dysfunction of the proximal tubule, including the presence of polyuria, glycosuria, phosphaturia, aminoaciduria, tubular proteinuria, growth retardation, and rickets. Eventually, glomerular involvement, glomerular proteinuria, focal segmental glomerulosclerosis (FSGS), and progression to kidney failure occur. Although the kidneys are the first organs affected, and play a key role in morbidity and mortality, extrarenal multiorgan involvement can occur in patients with cystinosis, which is seen not only in adults but in early ages in untreated patients, patients with insufficient treatment, and in those that don't comply with treatment. The treatment of cystinosis consists of supportive treatment for Fanconi syndrome, and specific lifelong cystine-depleting therapy using oral cysteamine. There is strong evidence that as early as possible, initiation and ongoing appropriate therapy with cysteamine are essential for delaying the progression to kidney failure, end-organ damage, and extrarenal involvement. The present review aimed to evaluate the extra renal complications of cystinosis.
Asunto(s)
Cistinosis , Síndrome de Fanconi , Humanos , Cistinosis/complicaciones , Cistinosis/genética , Síndrome de Fanconi/etiología , Síndrome de Fanconi/complicaciones , Cisteamina/uso terapéutico , Depletores de Cistina/uso terapéutico , Sistemas de Transporte de Aminoácidos Neutros/genéticaRESUMEN
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.
Asunto(s)
Macrófagos , Simportadores , Hormonas Tiroideas , Animales , Ratones , Macrófagos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Simportadores/genética , Hormonas Tiroideas/metabolismo , Hormonas Tiroideas/farmacología , Triyodotironina/farmacología , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismoRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Cistinosis , Humanos , Cistina/metabolismo , Cistinosis/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cisteamina , Mutación/genéticaRESUMEN
The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Microscopía por Crioelectrón , Animales , Humanos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Ratones , Sitio Alostérico , Células HEK293 , Sitios de Unión , Conformación ProteicaRESUMEN
Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Microscopía por Crioelectrón , Prolina , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Prolina/metabolismo , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , COVID-19/virología , COVID-19/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Modelos MolecularesRESUMEN
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.
Asunto(s)
Adenocarcinoma del Pulmón , Sistemas de Transporte de Aminoácidos Neutros , Neoplasias Pulmonares , Linfocitos Infiltrantes de Tumor , Microambiente Tumoral , Humanos , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinogénesis/genética , Carcinogénesis/inmunología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/inmunología , Pronóstico , Microambiente Tumoral/inmunología , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismoRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Cistinosis , Humanos , Adulto , Cistinosis/complicaciones , Cistinosis/genética , Cistinosis/tratamiento farmacológico , Cistina/metabolismo , Cistina/uso terapéutico , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/uso terapéutico , Cisteamina/uso terapéutico , Inflamación/tratamiento farmacológico , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismoRESUMEN
Sepsis is a systemic inflammatory response syndrome resulting from the invasion of the human body by bacteria and other pathogenic microorganisms. One of its most prevalent complications is acute lung injury, which places a significant medical burden on numerous countries and regions due to its high morbidity and mortality rates. MicroRNA (miRNA) plays a critical role in the body's inflammatory response and immune regulation. Recent studies have focused on miR-21-5p in the context of acute lung injury, but its role appears to vary in different models of this condition. In the LPS-induced acute injury model of A549 cells, there is differential expression, but the specific mechanism remains unclear. Therefore, our aim is to investigate the changes in the expression of miR-21-5p and SLC16A10 in a type II alveolar epithelial cell injury model induced by LPS and explore the therapeutic effects of their targeted regulation. A549 cells were directly stimulated with 10 µg/ml of LPS to construct a model of LPS-induced cell injury. Cells were collected at different time points and the expression of interleukin 1 beta (IL-1ß), tumor necrosis factor-α (TNF-α) and miR-21-5p were measured by RT-qPCR and western blot. Then miR-21-5p mimic transfection was used to up-regulate the expression of miR-21-5p in A549 cells and the expression of IL-1ß and TNF-α in each group of cells was measured by RT-qPCR and western blot. The miRDB, TargetScan, miRWalk, Starbase, Tarbase and miR Tarbase databases were used to predict the miR-21-5p target genes and simultaneously, the DisGeNet database was used to search the sepsis-related gene groups. The intersection of the two groups was taken as the core gene. Luciferase reporter assay further verified SLC16A10 as the core gene with miR-21-5p. The expression of miR-21-5p and SLC16A10 were regulated by transfection or inhibitors in A549 cells with or without LPS stimulation. And then the expression of IL-1ß and TNF-α in A549 cells was tested by RT-qPCR and western blot in different groups, clarifying the role of miR-21-5p-SLC16A10 axis in LPS-induced inflammatory injury in A549 cells. (1) IL-1ß and TNF-α mRNA and protein expression significantly increased at 6, 12, and 24 h after LPS stimulation as well as the miR-21-5p expression compared with the control group (P < 0.05). (2) After overexpression of miR-21-5p in A549 cells, the expression of IL-1ß and TNF-α was significantly reduced after LPS stimulation, suggesting that miR-21-5p has a protection against LPS-induced injury. (3) The core gene set, comprising 51 target genes of miR-21-5p intersecting with the 1448 sepsis-related genes, was identified. This set includes SLC16A10, TNPO1, STAT3, PIK3R1, and FASLG. Following a literature review, SLC16A10 was selected as the ultimate target gene. Dual luciferase assay results confirmed that SLC16A10 is indeed a target gene of miR-21-5p. (4) Knocking down SLC16A10 expression by siRNA significantly reduced the expression of IL-1ß and TNF-α in A549 cells after LPS treatment (P < 0.05). (5) miR-21-5p inhibitor increased the expression levels of IL-1ß and TNF-α in A549 cells after LPS stimulation (P < 0.05). In comparison to cells solely transfected with miR-21-5p inhibitor, co-transfection of miR-21-5p inhibitor and si-SLC6A10 significantly reduced the expression of IL-1ß and TNF-α (P < 0.05). MiR-21-5p plays a protective role in LPS-induced acute inflammatory injury of A549 cells. By targeting SLC16A10, it effectively mitigates the inflammatory response in A549 cells induced by LPS. Furthermore, SLC16A10 holds promise as a potential target for the treatment of acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda , Células Epiteliales Alveolares , Sistemas de Transporte de Aminoácidos Neutros , MicroARNs , Humanos , Células A549 , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/efectos de los fármacos , Regulación de la Expresión Génica , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Lipopolisacáridos/toxicidad , MicroARNs/genética , MicroARNs/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismoRESUMEN
Neurological abnormalities associated with spiculated, "acanthocytic" red cells in blood have been described as neuroacanthocytosis. This is a heterogeneous group of conditions that can be clearly subdivided on the basis of recent genetic findings. The McLeod Syndrome, one of the core neuroacanthocytosis syndromes, is a rare X-linked disorder caused by mutations of the XK gene, an X-chromosomal gene of unknown function characterized by haemopoietic abnormalities and late-onset neurological and muscular defects. We report two Chilean brothers with the McLeod phenotype who showed important psychiatric features. The diagnosis may be elusive if the presence of acanthocytosis is not properly studied. We describe a method which allowed the diagnosis that unmasked acanthocytosis. Otherwise the condition could have remained undiagnosed as it had been for decades in this family. This syndrome must be considered when assessing a familial movement disorder, specially affecting males with relevant psychiatric features. A reliable test for acanthocytosis assessment is available.