RESUMO
Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1ß treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis.
Assuntos
Infecções por Escherichia coli , Mucosa Intestinal , Animais , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Camundongos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Células CACO-2 , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Escherichia coli , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Creatina/metabolismoRESUMO
X-linked creatine transporter deficiency is caused by hemizygous or heterozygous pathogenic variants in SLC6A8 that cause neuropsychiatric symptoms because of impaired uptake of creatine into tissues throughout the body. Small cohorts have suggested that supplementation of creatine, arginine, and glycine can stop disease progression in males, but only six cases of supplementation in females have been published. Here, we present a female with a de-novo pathogenic SLC6A8 variant who had ongoing weight loss, mild intellectual disability, and neuropsychiatric symptoms. Magnetic resonance spectroscopy of the brain showed reduced creatine on all acquired spectra. The patient was started on creatine-monohydrate, l -arginine, and l -glycine supplementation, and she had significant symptomatic improvement within the following 3 weeks. After 8 months of supplementation, magnetic resonance spectroscopy showed improved creatine concentrations with normalizing semiquantitative ratios with other brain metabolites. Current data supports clinicians trialing creatine, arginine, and glycine supplements for female patients with creatine transporter deficiency.
Assuntos
Arginina , Creatina , Suplementos Nutricionais , Glicina , Deficiência Intelectual Ligada ao Cromossomo X , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores , Humanos , Feminino , Creatina/metabolismo , Creatina/deficiência , Glicina/metabolismo , Arginina/metabolismo , Arginina/uso terapêutico , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Encéfalo/metabolismo , Adulto , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Espectroscopia de Ressonância Magnética , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/tratamento farmacológico , Encefalopatias Metabólicas Congênitas , Proteínas de Membrana TransportadorasRESUMO
INTRODUCTION: Lung cancer is a major cause of cancer-related death worldwide and effective therapies, besides surgery, are available only for a small proportion of patients. Since cellular respiration is known to be broadly altered in malignant tumors, the cellular processes of respiration can be a potential therapeutic target. One important element of cellular respiration is creatine and its transport by the creatine transporter SLC6A8. Here we describe the expression of SLC6A8 at the RNA and protein level, epigenetic modifications as well as survival analysis in NSCLC tissues and matched controls. MATERIALS AND METHODS: We analyzed epigenetic modifications of the SLC68A gene in 32 patients, of which 18 were additionally analyzed by transcriptome analysis. The expression of SLC6A8 at the protein level was assessed by immunohistochemistry using an independent cohort and correlated with clinicopathological data including survival. Kaplan-Meier analysis was performed to analyze the possible effects of the transcriptional levels of SLC6A8 in another separate cohort (n=1925). RESULTS: SLC6A8 loci are epigenetically modified in NSCLC compared with tumor-free controls. SLC6A8 is upregulated in NSCLC at the RNA and protein level. High mRNA expression of SLC6A8 was associated with an overall poor prognosis in lung adenocarcinoma patients and displayed the strongest adverse prognostic effect in male smokers with adenocarcinomas. Results of transcriptome analysis were partially confirmed at the protein level. CONCLUSIONS: Our results suggest an important role of creatine and its transport via SLC6A8 in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Epigênese Genética , Neoplasias Pulmonares , Regulação para Cima , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Regulação Neoplásica da Expressão Gênica , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Prognóstico , Estimativa de Kaplan-Meier , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Adulto , Proteínas de Membrana TransportadorasRESUMO
The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.
Assuntos
Sódio , Simportadores , Sódio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Simportadores/metabolismo , Sítios de Ligação , NeurotransmissoresRESUMO
Cerebral creatine deficiency syndromes (CCDS) are neurodevelopmental disorders caused by a decrease in creatine levels in the central nervous system (CNS) due to functional mutations in creatine synthetic enzymes or creatine transporter (CRT/SLC6A8). Although SLC6A8 mutations have been reported to be the most frequent cause of CCDS, sufficient treatment for patients with CCDS harboring SLC6A8 mutations has not yet been achieved. This study aimed to elucidate the molecular mechanism of SLC6A8 dysfunction caused by the c. 1699T > C missense mutation, which is thought to induce dysfunction through an unidentified mechanism. A study on SLC6A8-expressing oocytes showed that the c.1699T > C mutation decreased creatine uptake compared to that in wild-type (WT) oocytes. In addition, a kinetics study of creatine uptake revealed that the c.1699T > C mutation reduced the maximum uptake rate but not Michaelis-Menten constant. In contrast, the c.1699T > C mutation did not attenuate SLC6A8 protein levels or alter its cellular localization. Based on the SLC6A8 structure in the AlphaFold protein structure database, it is possible that the c.1699T > C mutation alters the interaction between the S567 and Y143 residues of SLC6A8, leading to decreased creatine transport function. These findings contribute to the understanding of the pathology of CCDS and to the development of strategies for CCDS treatment.