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1.
Hum Mol Genet ; 2024 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-39271101

RESUMEN

Craniofacial anomalies encompassing the orofacial cleft are associated with > 30% of systemic congenital malformations. Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF) is a rare genetic disorder attributed to variants in the actin beta (ACTB) or actin gamma genes that are correlated with a range of craniofacial abnormalities, including cleft lip and/or palate. The underlying pathological mechanism of BWCFF remains elusive, and it is necessary to investigate the etiology of orofacial clefts in patients with BWCFF. In this study, we identified a missense variant (c.1043C > T: p.S348L) in the ACTB gene of a patient with BWCFF and concomitant cleft lip and palate. Furthermore, we performed functional assessments of this variant using various disease models such as the MDCK cell line and Xenopus laevis. These models revealed a compromised capacity of mutated ACTB to localize to the epithelial junction, consequently affecting the behavior of epithelial cells. Additionally, we discovered that the mutated ACTB exhibited an impaired ability to bind PROFILIN1, a critical factor in actin polymerization. This defective ability may contribute to the molecular etiology of aberrant epithelial cell adhesion and migration, resulting in orofacial cleft formation in BWCFF.

2.
J Hum Genet ; 2024 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-39164360

RESUMEN

Hydrops fetalis, characterized by abnormal fluid accumulation in fetuses, presents a significant risk of stillbirth and neonatal mortality. Although the etiology of nonimmune hydrops fetalis (NIHF) is multifaceted, recent studies have highlighted genetic factors as crucial determinants. This study focused on a family with three consecutive stillbirths, each with pronounced hydrops fetalis. Using whole-exome sequencing (WES), we identified compound heterozygous variants of the SCN4A gene encoding the voltage-gated sodium channel of the skeletal muscle (hNav1.4), c.2429T>A p.L810Q and c.4556T>C p.F1519S, in all three deceased infants. A functional analysis conducted using the whole-cell patch-clamp technique revealed loss-of-function defects in both variant channels, with F1519S exhibiting a complete loss of ionic current and L810Q showing a reduced channel opening. These findings support the pathogenicity of SCN4A variants in NIHF and underscore the significance of functional studies in elucidating genotype-phenotype correlations. Furthermore, our study emphasizes the diagnostic value of WES in cases of NIHF in where standard genetic testing fails to identify causative variants.

3.
Clin Genet ; 105(2): 159-172, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-37899590

RESUMEN

The investigation of environmental effects on clinical measurements using individual samples is challenging because their genetic and environmental factors are different. However, using monozygotic twins (MZ) makes it possible to investigate the influence of environmental factors as they have the same genetic factors within pairs because the difference in the clinical traits within the MZ mostly reflect the influence of environmental factors. We hypothesized that the within-pair differences in the traits that are strongly affected by genetic factors become larger after genetic risk score (GRS) correction. Using 278 Japanese MZ pairs, we compared the change in within-pair differences in each of the 45 normalized clinical measurements before and after GRS correction, and we also attempted to correct for the effects of genetic factors to identify Cytosine-phosphodiester-Guanine (CpG) sites in DNA sequences with epigenetic effects that are regulated by genetic factors. Five traits were classified into the high heritability group, which was strongly affected by genetic factors. CpG sites could be classified into three groups: regulated only by environmental factors, regulated by environmental factors masked by genetic factors, and regulated only by genetic factors. Our method has the potential to identify trait-related methylation sites that have not yet been discovered.


Asunto(s)
Metilación de ADN , Epigénesis Genética , Humanos , Islas de CpG/genética , Metilación de ADN/genética , Puntuación de Riesgo Genético , Japón , Laboratorios Clínicos , Gemelos Monocigóticos/genética
4.
J Med Genet ; 60(7): 722-731, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-36543533

RESUMEN

BACKGROUND: Fabry disease is a rare, multisystemic disorder caused by GLA gene variants that lead to alpha galactosidase A deficiency, resulting in accumulation of glycosphingolipids and cellular dysfunction. Fabry-associated clinical events (FACEs) cause significant morbidity and mortality, yet the long-term effect of Fabry therapies on FACE incidence remains unclear. METHODS: This posthoc analysis evaluated incidence of FACEs (as a composite outcome and separately for renal, cardiac and cerebrovascular events) in 97 enzyme replacement therapy (ERT)-naïve and ERT-experienced adults with Fabry disease and amenable GLA variants who were treated with migalastat for up to 8.6 years (median: 5 years) in Phase III clinical trials of migalastat. Associations between baseline characteristics and incidence of FACEs were also evaluated. RESULTS: During long-term migalastat treatment, 17 patients (17.5%) experienced 22 FACEs and there were no deaths. The incidence rate of FACEs was 48.3 events per 1000 patient-years overall. Numerically higher incidence rates were observed in men versus women, patients aged >40 years versus younger patients, ERT-naïve versus ERT-experienced patients and men with the classic phenotype versus men and women with all other phenotypes. There was no statistically significant difference in time to first FACE when analysed by patient sex, phenotype, prior treatment status or age. Lower baseline estimated glomerular filtration rate (eGFR) was associated with an increased risk of FACEs across patient populations. CONCLUSIONS: The overall incidence of FACEs for patients during long-term treatment with migalastat compared favourably with historic reports involving ERT. Lower baseline eGFR was a significant predictor of FACEs.


Asunto(s)
Enfermedad de Fabry , Humanos , Femenino , Enfermedad de Fabry/complicaciones , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/genética , alfa-Galactosidasa/genética , alfa-Galactosidasa/uso terapéutico , Riñón , 1-Desoxinojirimicina/uso terapéutico , Terapia de Reemplazo Enzimático
5.
Twin Res Hum Genet ; : 1-7, 2024 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-39463157

RESUMEN

Twins lag behind singletons in their early psychomotor development, but little is known about how chorionicity affects this difference. We compared early psychomotor development in singletons, monochorionic (MC) twins and dichorionic (DC) twins. Our longitudinal data from the Japan Environment and Children's Study (JECS; see Appendix) included 98,042 singletons, 577 MC twins and 1051 DC twins representing the general Japanese population. Chorionicity was evaluated by ultrasound images and complemented by postnatal pathological examinations. Five domains of psychomotor development were evaluated at 6 time points from 6 months to 3 years of age using the Ages and Stages Questionnaires (ASQ-3). The data were analyzed using linear regression models. Twins lagged behind singletons in all areas of psychomotor development during infancy. This gap decreased over time but was still noticeable at 3 years of age. More than half of this difference was attributed to twins having lower birth weight and being born earlier in gestation. MC twins showed slightly delayed development compared to DC twins, but this difference was minor compared to the overall gap between twins and singletons. Twins delay singletons in their early psychomotor development, and this delay is not specific to MC twinning.

6.
Biochem Biophys Res Commun ; 662: 58-65, 2023 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-37099811

RESUMEN

The neurotransmitter serotonin (5-HT) is transported back into serotonergic neurons by the serotonin transporter (SERT). SERT is a main target of antidepressants, and much effort has therefore focused on finding relationships between SERT and depression. However, it is not fully understood how SERT is regulated at the cellular level. Here, we report post-translational regulation of SERT by S-palmitoylation, in which palmitate is covalently attached to cysteine residues of proteins. Using AD293 cells (a human embryonic kidney 293-derived cell line with improved cell adherence) transiently transfected with FLAG-tagged human SERT, we observed S-palmitoylation of immature SERT containing high-mannose type N-glycans or no N-glycan, which is presumed to be localized in the early secretory pathway, such as the endoplasmic reticulum. Mutational analysis by alanine substitutions shows that S-palmitoylation of immature SERT occurs at least at Cys-147 and Cys-155, juxtamembrane cysteine residues within the first intracellular loop. Furthermore, mutation of Cys-147 reduced cellular uptake of a fluorescent SERT substrate that mimics 5-HT without decreasing SERT on the cell surface. On the other hand, combined mutation of Cys-147 and Cys-155 inhibited SERT surface expression and reduced the uptake of the 5-HT mimic. Thus, S-palmitoylation of Cys-147 and Cys-155 is important for both the cell surface expression and 5-HT uptake capacity of SERT. Given the importance of S-palmitoylation in brain homeostasis, further investigation of SERT S-palmitoylation could provide new insights into the treatment of depression.


Asunto(s)
Proteínas de Transporte de Serotonina en la Membrana Plasmática , Serotonina , Humanos , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Lipoilación , Cisteína/metabolismo , Membrana Celular/metabolismo
7.
Glycoconj J ; 40(2): 191-198, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36787035

RESUMEN

Changes in protein glycosylation are clinically used as biomarkers. In the present study, we employed a twin cohort to investigate the contributions of genetic and environmental factors to glycan modifications of glycoproteins. Mac-2 binding protein (Mac-2 bp), haptoglobin (Hp), and their glycosylated forms are liver fibrosis and cancer biomarkers. Sera from 107 twin pairs without clinical information were used as a training cohort for the Mac-2 bp and Mac-2 bp glycosylation isomer (M2BPGi) assay. As a validation cohort, 22 twin pairs were enrolled in the study. For each twin pair, one twin was diagnosed with liver or pancreatic disease. For the training cohort, the correlation ratios of serum Mac-2 bp and M2BPGi levels in twin sera with random sequences were 0.30 and 0.018, respectively. The correlation ratios between twin pairs in the validation cohort for serum Mac-2 bp and M2BPGi levels were 0.75 and 0.35, respectively. In contrast, correlation ratios of serum Hp and fucosylated haptoglobin (Fuc-Hp) levels between twin sera with liver and pancreatic disease were 0.49 and 0.16, respectively. Although serum protein levels of glycoproteins are susceptible to genetic factors, characteristic glycan changes of these glycoproteins are more susceptible to environmental factors, including liver and pancreatic disease.


Asunto(s)
Haptoglobinas , Glicoproteínas de Membrana , Humanos , Haptoglobinas/metabolismo , Glicoproteínas/metabolismo , Biomarcadores , Cirrosis Hepática/genética , Glicosilación , Antígenos de Neoplasias/metabolismo
8.
Am J Med Genet A ; 191(7): 1984-1989, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37141439

RESUMEN

Craniofacial defects are one of the most frequent phenotypes in syndromic diseases. More than 30% of syndromic diseases are associated with craniofacial defects, which are important for the precise diagnosis of systemic diseases. Special AT-rich sequence-binding protein 2 (SATB2)-associated syndrome (SAS) is a rare syndromic disease associated with a wide variety of phenotypes, including intellectual disability and craniofacial defects. Among them, dental anomalies are the most frequently observed phenotype and thus becomes an important diagnostic criterion for SAS. In this report, we demonstrate three Japanese cases of genetically diagnosed SAS with detailed craniofacial phenotypes. The cases showed multiple dental problems, which have been previously reported to be linked to SAS, including abnormal crown morphologies and pulp stones. One case showed a characteristic enamel pearl at the root furcation. These phenotypes add new insights for differentiating SAS from other disorders.


Asunto(s)
Discapacidad Intelectual , Proteínas de Unión a la Región de Fijación a la Matriz , Humanos , Pueblos del Este de Asia , Síndrome , Fenotipo , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Factores de Transcripción/genética
9.
J Inherit Metab Dis ; 46(4): 618-633, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37114839

RESUMEN

BACKGROUND: Glycogen storage disease type Ia (GSDIa) is caused by biallelic pathogenic variants in the glucose-6-phosphatase gene (G6PC) and mainly characterized by hypoglycemia, hepatomegaly, and renal insufficiency. Although its symptoms are reportedly mild in patients carrying the G6PC c.648G>T variant, the predominant variant in Japanese patients, details remain unclear. Therefore, we examined continuous glucose monitoring (CGM) data and daily nutritional intake to clarify their associations in Japanese patients with GSDIa with G6PC c.648G>T. METHODS: This cross-sectional study enrolled 32 patients across 10 hospitals. CGM was performed for 14 days, and nutritional intake was recorded using electronic diaries. Patients were divided according to genotype (homozygous/compound heterozygous) and age. The durations of biochemical hypoglycemia and corresponding nutritional intake were analyzed. Multiple regression analysis was performed to identify factors associated with the duration of biochemical hypoglycemia. RESULTS: Data were analyzed for 30 patients. The mean daily duration of hypoglycemia (<4.0 mmol/L) in the homozygous group increased with age (2-11 years [N = 8]: 79.8 min; 12-18 years [5]: 84.8 min; ≥19 years [10]: 131.5 min). No severe hypoglycemic symptoms were recorded in the patients' diaries. The mean frequency of snack intake was approximately three times greater in patients aged 2-11 years (7.1 times/day) than in those aged 12-18 years (1.9 times/day) or ≥19 years (2.2 times/day). Total cholesterol and lactate were independently associated with the duration of biochemical hypoglycemia. CONCLUSION: Although nutritional therapy prevents severe hypoglycemia in patients with GSDIa with G6PC c.648G>T, patients often experience asymptomatic hypoglycemia.


Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo I , Hipoglucemia , Humanos , Glucemia , Estudios Transversales , Automonitorización de la Glucosa Sanguínea , Enfermedad del Almacenamiento de Glucógeno Tipo I/complicaciones , Glucosa-6-Fosfatasa/genética , Hipoglucemia/complicaciones
10.
J Pharmacol Sci ; 153(1): 55-67, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37524455

RESUMEN

Microglial removal of dying cells plays a beneficial role in maintaining homeostasis in the CNS, whereas under some pathological conditions, inflammatory microglia can cause excessive clearance, leading to neuronal death. However, the mechanisms underlying dying cell removal by inflammatory microglia remain poorly understood. In this study, we performed live imaging to examine the purinergic regulation of dying cell removal by inflammatory activated microglia. Lipopolysaccharide (LPS) stimulation induces rapid death of primary rat microglia, and the surviving microglia actively remove dying cells. The nonselective P2 receptor antagonist, suramin, inhibited dying cell removal to the same degree as that of the selective P2Y2 antagonist, AR-C118925. This inhibition was more potent in LPS-stimulated microglia than in non-stimulated ones. LPS stimulation elicited distribution of the P2Y2 receptor on the leading edge of the plasma membrane and then induced drastic upregulation of P2Y2 receptor mRNA expression in microglia. LPS stimulation caused upregulation of the dying cell-sensing inflammatory Axl phagocytic receptor, which was suppressed by blocking the P2Y2 receptor and its downstream signaling effector, proline-rich tyrosine kinase (Pyk2). Together, these results indicate that inflammatory stimuli may activate the P2Y2 receptor, thereby mediating dying cell removal, at least partially, through upregulating phagocytic Axl in microglia.


Asunto(s)
Lipopolisacáridos , Microglía , Ratas , Animales , Microglía/metabolismo , Lipopolisacáridos/farmacología , Transducción de Señal , Proteínas Tirosina Quinasas/metabolismo , Apoptosis
11.
Mol Cell Neurosci ; 118: 103691, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34871769

RESUMEN

During neuronal development, immature neurons extend neurites and subsequently polarize to form an axon and dendrites. We have previously reported that G protein-coupled receptor 3 (GPR3) levels increase during neuronal development, and that GPR3 has functions in neurite outgrowth and neuronal differentiation in cerebellar granular neurons. Moreover, GPR3 is transported and concentrated at the tips of neurite, thereby contributing to the local activation of protein kinase A (PKA). However, the signaling pathways for GPR3-mediated neurite outgrowth and its subsequent effects on neuronal polarization have not yet been elucidated. We therefore analyzed the signaling pathways related to GPR3-mediated neurite outgrowth, and also focused on the possible roles of GPR3 in axon polarization. We demonstrated that, in cerebellar granular neurons, GPR3-mediated neurite outgrowth was mediated by multiple signaling pathways, including those of PKA, extracellular signal-regulated kinases (ERKs), and most strongly phosphatidylinositol 3-kinase (PI3K). In addition, the GPR3-mediated activation of neurite outgrowth was associated with G protein-coupled receptor kinase 2 (GRK2)-mediated signaling and phosphorylation of the C-terminus serine/threonine residues of GPR3, which affected downstream protein kinase B (Akt) signaling. We further demonstrated that GPR3 was transiently increased early in the development of rodent hippocampal neurons. It was subsequently concentrated at the tip of the longest neurite, and was thus associated with accelerated polarity formation in a PI3K-dependent manner in rat hippocampal neurons. In addition, GPR3 knockout in mouse hippocampal neurons led to delayed neuronal polarity formation, thereby affecting the dephosphorylation of collapsing response mediator protein 2 (CRMP2), which is downstream of the PI3K signaling pathway. Taken together, these findings suggest that the intrinsic expression of GPR3 in differentiated neurons constitutively activates PI3K-mediated signaling pathway predominantly, thus accelerating neurite outgrowth and further augmenting polarity formation in primary cultured neurons.


Asunto(s)
Neuronas , Fosfatidilinositol 3-Quinasas , Receptores Acoplados a Proteínas G , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Neuritas/metabolismo , Proyección Neuronal , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal
12.
Neurobiol Dis ; 172: 105811, 2022 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-35809764

RESUMEN

Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice.


Asunto(s)
Glaucoma , Traumatismos del Nervio Óptico , Animales , Axones/patología , Glaucoma/metabolismo , Ratones , Ratones Noqueados , Compresión Nerviosa , Regeneración Nerviosa/fisiología , Nervio Óptico , Traumatismos del Nervio Óptico/patología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Ganglionares de la Retina/metabolismo , Zimosan/metabolismo , Zimosan/farmacología
13.
J Hum Genet ; 67(10): 595-599, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35760954

RESUMEN

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant SCA caused by variants of the PRKCG encoding protein kinase C gamma (PKCγ). Although the toxic gain-of-function mechanism is the main cause of SCA14, its molecular pathophysiology remains unclear. To elucidate the molecular pathogenesis of SCA14, we analyzed two families with the variants in PRKCG. Clinical symptoms and neurological findings of two Japanese families were evaluated by neurologists. Exome sequencing was performed using the BGI platform. GFP-tagged PRKCGs harboring the identified variants were transfected into the HeLa cells, and aggregation of PKCγ was analyzed using confocal laser microscopy. Solubility of PKCγ was evaluated by assessing the proportion of insoluble fraction present in1% Triton-X. Patients in family 1 presented with only cerebellar atrophy without ataxia; however, patients in family 2 exhibited cerebellar ataxia, dystonia, and more severe cerebellar atrophy than those in family 1. Exome sequencing identified two novel missense variants of PRKCG:c.171 G > C,p.W57C (family 1), and c.400 T > C,p.C134R (family 2). Both the mutant PKCγ aggregated in the cytoplasm. Although the solubility of PKCγ of the C134R variant was lower than that of the wild-type, PKCγ of W57C retained its solubility. In conclusion, we identified two novel variants of PRKCG. The difference in severity between the two families may be due to the difference in solubility changes observed between the two variants. Decreased solubility of the PKCγ may play an important role in the pathogenesis of SCA14.


Asunto(s)
Ataxia Cerebelosa , Atrofia , Células HeLa , Humanos , Proteína Quinasa C , Ataxias Espinocerebelosas
14.
J Pharmacol Sci ; 148(3): 307-314, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-35177210

RESUMEN

G protein-coupled receptor 3 (GPR3) constitutively activates Gαs proteins without any ligands and is predominantly expressed in neurons. Since the expression and physiological role of GPR3 in immune cells is still unknown, we examined the possible role of GPR3 in T lymphocytes. The expression of GPR3 was upregulated 2 h after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation and was sustained in Jurkat cells, a human T lymphocyte cell line. In addition, the expression of nuclear receptor 4 group A member 2 (NR4A2) was highly modulated by GPR3 expression. Additionally, GPR3 expression was linked with the transcriptional promoter activity of NR4A in Jurkat cells. In mouse CD4+ T cells, transient GPR3 expression was induced immediately after the antigen receptor stimulation. However, the expression of NR4A2 was not modulated in CD4+ T cells from GPR3-knockout mice after stimulation, and the population of Treg cells in thymocytes and splenocytes was not affected by GPR3 knockout. By contrast, spontaneous effector activation in both CD4+ T cells and CD8+ T cells was observed in GPR3-knockout mice. In summary, GPR3 is immediately induced by T cell stimulation and play an important role in the suppression of effector T cell activation.


Asunto(s)
Activación de Linfocitos/genética , Receptores Acoplados a Proteínas G/fisiología , Linfocitos T/inmunología , Animales , Línea Celular , Cromograninas/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Expresión Génica , Ratones Noqueados , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Linfocitos T/metabolismo
15.
J Pharmacol Sci ; 148(1): 187-195, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34924125

RESUMEN

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.


Asunto(s)
Antiinflamatorios no Esteroideos , Flurbiprofeno/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Chaperonas Moleculares , Mutación , Pliegue de Proteína , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Chaperón BiP del Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Glicosilación , Ubiquitina-Proteína Ligasas
16.
Mol Ther ; 29(2): 671-679, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33038326

RESUMEN

Pabinafusp alfa (JR-141) is a novel enzyme drug that crosses the blood-brain barrier by transcytosis via transferrin receptors. In order to establish its efficacy and safety, a multicenter, single-arm, open-label phase 2/3 clinical trial was conducted in 28 Japanese patients with mucopolysaccharidosis II (MPS-II, Hunter syndrome) by intravenous administrations of 2.0 mg/kg of pabinafusp alfa for 52 weeks. The primary efficacy endpoint was changes in heparan sulfate (HS) concentrations in the cerebrospinal fluid (CSF). Secondary endpoints included assessments of neurocognitive development for central efficacy, and changes in plasma HS and dermatan sulfate (DS) concentrations for peripheral efficacy. HS concentrations in the CSF significantly decreased from baseline to week 52 (p < 0.001), suggesting continuous inhibition of substrate accumulations in the CNS, i.e., hitherto unaddressed progressive neurodegeneration. Evaluations of neurocognitive developments showed positive changes in 21 of the 28 patients. Serum HS and DS concentrations, liver and spleen volumes, and other assessments suggested the peripheral efficacy of pabinafusp alfa was comparable to that of idursulfase. Drug-related adverse events were mild or moderate in severity, transient, and manageable. The results establish delivery across the BBB of pabinafusp alfa as an effective therapeutic for treating both the CNS and peripheral symptoms of patients with MPS-II.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Iduronato Sulfatasa/administración & dosificación , Mucopolisacaridosis II/tratamiento farmacológico , Receptores de Transferrina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Quimioterapia Combinada , Humanos , Mucopolisacaridosis II/diagnóstico , Resultado del Tratamiento
17.
Medicina (Kaunas) ; 58(12)2022 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-36557058

RESUMEN

Background and Objectives: Our aim was to assess genetic and environmental effects on surface morphological parameters for quantifying anterior cingulate cortex (ACC) changes in middle- to advanced-age East Asians using twin analysis. Materials and Methods: Normal twins over 39 years old comprising 37 monozygotic pairs and 17 dizygotic pairs underwent 3-dimensional (3D) T1-weighted imaging of the brain at 3T. Freesurfer-derived ACC parameters including thickness, standard deviation of thickness (STDthickness), volume, surface area, and sulcal morphological parameters (folding, mean, and Gaussian curvatures) were calculated from 3D T1-weighted volume images. Twin analysis with a model involving phenotype variance components of additive genetic effects (A), common environmental effects (C), and unique environmental effects (E) was performed to assess the magnitude of each genetic and environmental influence on parameters. Results: Most parameters fit best with an AE model. Both thickness (A: left 0.73/right 0.71) and surface area (A: left 0.63/right 0.71) were highly heritable. STDthickness was low to moderately heritable (A: left 0.48/right 0.29). Volume was moderately heritable (A: left 0.37). Folding was low to moderately heritable (A: left 0.44/right 0.28). Mean curvature (A: left 0.37/right 0.65) and Gaussian curvature (A: right 0.79) were moderately to highly heritable. Right volume and left Gaussian curvature fit best with a CE model, indicating a relatively weak contribution of genetic factors to these parameters. Conclusions: When assessing ACC changes in middle- to advanced-age East Asians, one must keep in mind that thickness and surface area appear to be strongly affected by genetic factors, whereas sulcal morphological parameters tend to involve environmental factors.


Asunto(s)
Encéfalo , Giro del Cíngulo , Giro del Cíngulo/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Asia Oriental , Biomarcadores , Gemelos Dicigóticos/genética , Gemelos Monocigóticos/genética
18.
Curr Issues Mol Biol ; 43(1): 389-404, 2021 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-34205365

RESUMEN

Fabry disease is an X-linked disorder of α-galactosidase A (GLA) deficiency. Our previous interim analysis (1 July 2014 to 31 December 2015) revealed plasma globotriaosylsphingosine as a promising primary screening biomarker for Fabry disease probands. Herein, we report the final results, including patients enrolled from 1 January to 31 December 2016 for evaluating the potential of plasma globotriaosylsphingosine and GLA activity as a combined screening marker. We screened 5691 patients (3439 males) referred from 237 Japanese specialty clinics based on clinical findings suggestive of Fabry disease using plasma globotriaosylsphingosine and GLA activity as primary screening markers, and GLA variant status as a secondary screening marker. Of the 14 males who tested positive in the globotriaosylsphingosine screen (≥2.0 ng/mL), 11 with low GLA activity (<4.0 nmol/h/mL) displayed GLA variants (four classic, seven late-onset) and one with normal GLA activity and no pathogenic variant displayed lamellar bodies in affected organs, indicating late-onset biopsy-proven Fabry disease. Of the 19 females who tested positive in the globotriaosylsphingosine screen, eight with low GLA activity displayed GLA variants (six classic, two late-onset) and five with normal GLA activity displayed a GLA variant (one classic) and no pathogenic variant (four late-onset biopsy-proven). The combination of plasma globotriaosylsphingosine and GLA activity can be a primary screening biomarker for classic, late-onset, and late-onset biopsy-proven Fabry disease probands.


Asunto(s)
Biomarcadores/sangre , Enfermedad de Fabry/sangre , Glucolípidos/sangre , Tamizaje Masivo/métodos , Esfingolípidos/sangre , alfa-Galactosidasa/sangre , Adolescente , Adulto , Anciano , Pueblo Asiatico , Niño , Estudios de Cohortes , Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/etnología , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , alfa-Galactosidasa/metabolismo
19.
Biochem Biophys Res Commun ; 534: 583-589, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33243462

RESUMEN

To elucidate the regulation of serotonin transporter (SERT) function via its membrane trafficking, we investigated the involvement of the ubiquitin E3 ligase HRD1 (HMG-CoA reductase degradation protein), which participates in endoplasmic reticulum (ER)-associated degradation (ERAD), in the functional regulation of SERT. Cells transiently expressing wild-type SERT or a SERT C-terminal deletion mutant (SERTΔCT), a SERT protein predicted to be misfolded, were used for experiments. Studies using HRD1-overexpressing or HRD1-knockdown cells demonstrated that HRD1 is involved in SERT proteolysis. Overexpression of HRD1 promoted SERT ubiquitination, the effect of which was augmented by treatment with the proteasome inhibitor MG132. Immunoprecipitation studies revealed that HRD1 interacts with SERT in the presence of MG132. In addition, HRD1 was intracellularly colocalized with SERT, especially with aggregates of SERTΔCT in the ER. HRD1 also affected SERT uptake activity in accordance with the expression levels of the SERT protein. These results suggest that HRD1 contributes to the membrane trafficking and functional regulation of SERT through its involvement in ERAD-mediated SERT degradation.


Asunto(s)
Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Degradación Asociada con el Retículo Endoplásmico , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Leupeptinas/farmacología , Inhibidores de Proteasoma/farmacología , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacos
20.
Biochem Biophys Res Commun ; 567: 42-48, 2021 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-34139556

RESUMEN

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Espectroscopía de Resonancia por Spin del Electrón , Histonas/química , Humanos , Metilación , Modelos Moleculares , Dominios Proteicos
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