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1.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068417

RESUMO

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.


Assuntos
Ataxia/patologia , Canais de Cálcio/genética , Mutação , Adulto , Sequência de Aminoácidos , Ataxia/congênito , Ataxia/etiologia , Ataxia/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Criança , Feminino , Humanos , Masculino , Neuroimagem , Fenótipo , Conformação Proteica , Homologia de Sequência , Relação Estrutura-Atividade , Adulto Jovem
2.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946947

RESUMO

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM+ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM+ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206+ and peritubular MHC II+ macrophages, with higher levels in CD206+ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2+ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM+ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206+MHC II+ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2+ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM+ mice. The changes in testis are distinct from the described alterations in other organs of AROM+, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM+ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM+ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.


Assuntos
Canais de Cálcio/fisiologia , Macrófagos/metabolismo , Orquite/metabolismo , Canais de Cátion TRPV/fisiologia , Testículo/metabolismo , Glândulas Suprarrenais/metabolismo , Fatores Etários , Animais , Aromatase/genética , Encéfalo/metabolismo , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Modelos Animais de Doenças , Genótipo , Infertilidade Masculina/metabolismo , Lectinas Tipo C/análise , Masculino , Lectinas de Ligação a Manose/análise , Camundongos , Camundongos Transgênicos , NADPH Oxidase 2/biossíntese , NADPH Oxidase 2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Superfície Celular/análise , Espermatogênese , Canais de Cátion TRPV/biossíntese , Canais de Cátion TRPV/genética , Fator de Necrose Tumoral alfa/biossíntese
3.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806823

RESUMO

Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca2+, which acts as a secondary messenger. The maintenance of adequate Ca2+ concentrations is possible thanks to proper concentrations of other ions, such as K+ and Na+, among others, that modulate plasma membrane potential and the intracellular pH. Like in every cell, ion homeostasis in spermatozoa is ensured by a vast spectrum of ion channels supported by the work of ion pumps and transporters. To achieve success in fertilization, sperm ion channels have to be sensitive to various external and internal factors. This sensitivity is provided by specific channel structures. In addition, novel sperm-specific channels or isoforms have been found with compositions that increase the chance of fertilization. Notably, the most significant sperm ion channel is the cation channel of sperm (CatSper), which is a sperm-specific Ca2+ channel required for the hyperactivation of sperm motility. The role of other ion channels in the spermatozoa, such as voltage-gated Ca2+ channels (VGCCs), Ca2+-activated Cl-channels (CaCCs), SLO K+ channels or voltage-gated H+ channels (VGHCs), is to ensure the activation and modulation of CatSper. As the activation of sperm motility differs among metazoa, different ion channels may participate; however, knowledge regarding these channels is still scarce. In the present review, the roles and structures of the most important known ion channels are described in regard to regulation of sperm motility in animals.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Motilidade Espermática , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Canais de Cloreto/química , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Humanos , Canais Iônicos/genética , Masculino , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Sódio/química , Canais de Sódio/genética , Canais de Sódio/metabolismo , Relação Estrutura-Atividade
4.
Int J Mol Sci ; 22(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33799975

RESUMO

Migraine is a common neurological disease that affects about 11% of the adult population. The disease is divided into two main clinical subtypes: migraine with aura and migraine without aura. According to the neurovascular theory of migraine, the activation of the trigeminovascular system (TGVS) and the release of numerous neuropeptides, including calcitonin gene-related peptide (CGRP) are involved in headache pathogenesis. TGVS can be activated by cortical spreading depression (CSD), a phenomenon responsible for the aura. The mechanism of CSD, stemming in part from aberrant interactions between neurons and glia have been studied in models of familial hemiplegic migraine (FHM), a rare monogenic form of migraine with aura. The present review focuses on those interactions, especially as seen in FHM type 1, a variant of the disease caused by a mutation in CACNA1A, which encodes the α1A subunit of the P/Q-type voltage-gated calcium channel.


Assuntos
Canais de Cálcio/metabolismo , Transtornos de Enxaqueca/etiologia , Neuroglia/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/fisiopatologia , Mutação , Neuroglia/metabolismo
5.
Nat Genet ; 53(3): 313-321, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33664507

RESUMO

Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.


Assuntos
Variação Genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Locos de Características Quantitativas , Síndrome de Bardet-Biedl/genética , Canais de Cálcio/genética , Linhagem Celular , Ataxia Cerebelar/genética , Metilação de DNA , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Doenças Raras/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
6.
Nucleic Acids Res ; 49(6): 3168-3184, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33684213

RESUMO

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes.


Assuntos
Processamento Alternativo , Esclerose Amiotrófica Lateral/genética , Astrócitos/metabolismo , Íntrons , Animais , Astrócitos/efeitos dos fármacos , Canais de Cálcio/genética , Núcleo Celular/genética , Células Cultivadas , Citocinas/farmacologia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Camundongos , Mutação , Superóxido Dismutase-1/genética , Translocação Genética , Proteína com Valosina/genética
7.
Toxicol Appl Pharmacol ; 419: 115513, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33785354

RESUMO

The early characterization of ligands at the dopamine and serotonin transporters, DAT and SERT, respectively, is important for drug discovery, forensic sciences, and drug abuse research. 4-Methyl amphetamine (4-MA) is a good example of an abused drug whose overdose can be fatal. It is a potent substrate at DAT and SERT where its simplest secondary amine (N-methyl 4-MA) retains substrate activity at them. In contrast, N-n-butyl 4-MA is very weak, therefore it was categorized as inactive at these transporters. Here, N-octyl 4-MA and other related compounds were synthesized, and their activities were evaluated at DAT and SERT. To expedite this endeavor, cells expressing DAT or SERT were co-transfected with a voltage-gated Ca2+ channel and, the genetically-encoded Ca2+ sensor, GCaMP6s. Control compounds and the newly synthesized molecules were tested on these cells using an automated multi-well fluorescence plate reader; substrates and inhibitors were identified successfully at DAT and SERT. N-Octyl 4-MA and three bivalent compounds were inhibitors at these transporters. These findings were validated by measuring Ca2+-mobilization using quantitative fluorescence microscopy. The bivalent molecules were the most potent of the series and were further characterized in an uptake-inhibition assay. Compared to cocaine, they showed comparable potency inhibiting uptake at DAT and higher potency at SERT. These observations support a previous hypothesis that amphetamine-related (and, here, N-extended alkyl and) bivalent arylalkylamine molecules are active at monoamine transporters, showing potent activity as reuptake inhibitors, and implicate the involvement of a distant auxiliary binding feature to account for their actions at DAT and SERT.


Assuntos
Técnicas Biossensoriais , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Inibidores da Captação de Dopamina/toxicidade , Proteínas de Fluorescência Verde/metabolismo , Metanfetamina/toxicidade , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Inibidores de Captação de Serotonina/toxicidade , Canais de Cálcio/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Metanfetamina/análogos & derivados , Metanfetamina/síntese química , Microscopia de Fluorescência , Estrutura Molecular , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(2): 127-130, 2021 Feb 10.
Artigo em Chinês | MEDLINE | ID: mdl-33565063

RESUMO

OBJECTIVE: To analyze the clinical phenotype and genetic characterization of a child with early infantile epileptic encephalopathy. METHODS: The proband was subjected to history taking and was diagnosed based on his clinical manifestation, magnetic resonance imaging (MRI) and whole exome sequencing (WES). Sanger sequencing was carried out to determine the origin of pathogenic variant. RESULTS: The proband unconsciously tilts his head to one side with squint, which revealed an abnormal discharge. MRI indicated suspicious abnormal signal shadow in the left posterior frontal cortex in addition with inflammation signs in the right maxillary sinus and ethmoid sinus. WES revealed that the proband has carried a heterozygous c.5789G>A variant in the CACNAIA gene. The result of Sanger sequencing was in keeping with that of WES. Neither of his parents has carried the same variant. CONCLUSION: The heterozygous c.5789G>A variant of the CACNAIA gene probably underlay the early infantile epileptic encephalopathy 42 in the proband, which has a de novo origin.


Assuntos
Canais de Cálcio/genética , Testes Genéticos , Espasmos Infantis/genética , Heterozigoto , Humanos , Lactente , Mutação , Espasmos Infantis/diagnóstico , Sequenciamento Completo do Exoma
9.
Nat Commun ; 12(1): 907, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568645

RESUMO

Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are two distinct autoimmune diseases that manifest with chronic synovial inflammation. Here, we show that CD4+ T cells from patients with RA and PsA have increased expression of the pore-forming calcium channel component ORAI3, thereby increasing the activity of the arachidonic acid-regulated calcium-selective (ARC) channel and making T cells sensitive to arachidonic acid. A similar increase does not occur in T cells from patients with systemic lupus erythematosus. Increased ORAI3 transcription in RA and PsA T cells is caused by reduced IKAROS expression, a transcriptional repressor of the ORAI3 promoter. Stimulation of the ARC channel with arachidonic acid induces not only a calcium influx, but also the phosphorylation of components of the T cell receptor signaling cascade. In a human synovium chimeric mouse model, silencing ORAI3 expression in adoptively transferred T cells from patients with RA attenuates tissue inflammation, while adoptive transfer of T cells from healthy individuals with reduced expression of IKAROS induces synovitis. We propose that increased ARC activity due to reduced IKAROS expression makes T cells more responsive and contributes to chronic inflammation in RA and PsA.


Assuntos
Ácido Araquidônico/imunologia , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Membrana Sinovial/imunologia , Idoso , Artrite Psoriásica/genética , Artrite Psoriásica/imunologia , Artrite Reumatoide/genética , Cálcio/imunologia , Canais de Cálcio/genética , Canais de Cálcio/imunologia , Sinalização do Cálcio , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
10.
Mol Biol Rep ; 48(2): 1217-1223, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33523372

RESUMO

Cardiac complications are leading causes of death in diabetic patients. Imbalance of Ca2+ homeostasis is a hallmark of cardiac dysfunction in diabetes, while TRPV channels are non-selective for cations and are permeable to Ca2+. Our aim was to evaluate the expression levels of TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6 genes and proteins in cardiac tissue at 3 days and 4, 8, and 12 weeks after induction of diabetes. Sprague-Dawley rats were assigned to control and DM groups. DM was induced by intraperitoneal injection of streptozotocin (60 mg/kg). The expression levels of TRPV genes were analyzed by the quantitative reverse transcription polymerase chain reaction, and TRPV proteins were determined by western blotting. Compared to controls, the expression levels of TRPV2, TRPV3, and TRPV6 in diabetic myocardium did not change, while TRPV1 decreased at 4, 8, and 12 weeks, TRPV4 was upregulated at 3 days and 4, 8, and 12 weeks, TRPV5 mRNA increased at 8 and 12 weeks, and TRPV5 protein increased at 4, 8, and 12 weeks. Our findings showed that TRPV1, TRPV4, and TRPV5 are associated with the diabetic heart.


Assuntos
Complicações do Diabetes/genética , Diabetes Mellitus Experimental/genética , Cardiomiopatias Diabéticas/genética , Miocárdio/metabolismo , Animais , Canais de Cálcio/genética , Complicações do Diabetes/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Homeostase/genética , Humanos , Miocárdio/patologia , Ratos , Canais de Cátion TRPV/genética
11.
Toxicology ; 453: 152726, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33617915

RESUMO

Cadmium (Cd) is a ubiquitous environmental and occupational pollutant that is considered as a high-risk factor for neurodegenerative diseases. However, the mechanism underlying Cd-induced neurotoxicity has not been fully elucidated. Abnormal mitochondrial distribution and excessive mitochondrial fission are increasingly implicated in various neurological pathologies. Herein, by exposing primary cortical neurons to Cd (10 and 100 µM) for various times (0, 6, 12, and 24 h), we observed that the rapid motility of the mitochondria in neurons progressively slowed. Many more mitochondria were transported and distributed to the somas of Cd-treated neurons. Coupled with abnormal mitochondrial distribution, Cd exposure triggered excessive mitochondrial fragmentation, followed by mitochondrial membrane potential loss and neuronal damage. However, BAPTA-AM, a chelator of cytosolic calcium ([Ca2+]c), significantly attenuated Cd-induced abnormal mitochondrial distribution and excessive mitochondrial fission, which protected against Cd-induced mitochondrial damage and neuronal toxicity. In contrast to the increase in [Ca2+]c, Cd exposure had no effect on the level of mitochondrial calcium ([Ca2+]m). Inhibiting [Ca2+]m uptake, either by ruthenium 360 (Ru360) or by knock-out of mitochondrial calcium uniporter (MCU), failed to alleviate Cd-induced mitochondrial damage and neuronal toxicity. Additionally, in MCU knock-out neurons, BAPTA-AM effectively prevented Cd-induced abnormal mitochondrial distribution and excessive mitochondrial fission. Taken together, Cd exposure disrupts mitochondrial distribution and activates excessive mitochondrial fission by elevating [Ca2+]c independent of MCU-mediated mitochondrial calcium uptake, thereby leading to neurotoxicity. Chelating overloaded [Ca2+]c is a promising strategy to prevent the neurotoxicity of Cd.


Assuntos
Cádmio/toxicidade , Canais de Cálcio/deficiência , Cálcio/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Animais Recém-Nascidos , Canais de Cálcio/genética , Células Cultivadas , Citosol/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
12.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430180

RESUMO

Cell competition (CC) is a feature that allows tumor cells to outcompete and eliminate adjacent cells that are deemed less fit. Studies of CC, first described in Drosophila melanogaster, reveal a diversity of underlying mechanisms. In this review, we will discuss three recent studies that expand our understanding of the molecular features governing CC. In particular, we will focus on a molecular fitness fingerprint, oncogenic pathways, and the importance of cell junction stability. A fitness fingerprint, mediated by flower (hFWE) protein isoforms, dictates that cells expressing the flower-win isoforms will outcompete adjacent flower-loss-expressing cells. The impact of the flower protein isoforms is seen in cancer progression and may have diagnostic potential. The yes-associated protein (YAP) and TAZ transcription factors, central mediators of the oncogenic Hippo pathway, elevate peritumoral fitness thereby protecting against tumor progression and provide a suppressive barrier. Similarly, COL17A1 is a key component in hemidesmosome stability, and its expression in epidermal stem cells contributes to fitness competition and aging characteristics. The contributions of these pathways to disease development and progression will help define how CC is hijacked to favor cancer growth. Understanding these features will also help frame the diagnostic and therapeutic possibilities that may place CC in the crosshairs of cancer therapeutics.


Assuntos
Autoantígenos/genética , Canais de Cálcio/genética , Proteínas de Drosophila/genética , Aptidão Genética/genética , Colágenos não Fibrilares/genética , Animais , Drosophila melanogaster/genética , Variação Genética/genética , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Transativadores/genética
13.
PLoS Genet ; 17(1): e1009236, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465068

RESUMO

The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity.


Assuntos
Canais de Cálcio/genética , Estudo de Associação Genômica Ampla , Cor de Cabelo/genética , Animais , Fibroblastos/metabolismo , Mutação com Ganho de Função/genética , Genoma Humano/genética , Humanos , Lisossomos/genética , Camundongos , Camundongos Knockout , NADP/genética , Pigmentação/genética , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética
14.
Nat Microbiol ; 6(3): 366-379, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33462436

RESUMO

Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes-an intracellular bacterial pathogen-suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca2+ (mtCa2+) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa2+ uptake through the mtCa2+ uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa2+ signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host-microbial interactions.


Assuntos
Sinalização do Cálcio , Listeria monocytogenes/fisiologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Mitocôndrias/metabolismo , Fagocitose , Acetilcoenzima A/metabolismo , Acetilação , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , NADPH Oxidases/metabolismo
15.
Arch Biochem Biophys ; 698: 108724, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33309615

RESUMO

Abdominal aortic aneurysm (AAA) is a fatal vascular disease with insidious symptoms. However, the mechanism behind its development remains unclear. The transient receptor potential vanilloid (TRPV) family has crucial protective effects against cardiovascular diseases, but the role of TRPV5 in AAA has yet to be reported. In this study, ApoE-/- mice were intraperitoneally injected with AAV-GFP or AAV-TRPV5. After 30 days, mice were further administered with angiotensin II (Ang II, 1.44 mg/kg/day) by using osmotic pumps to induce the AAA model or Saline for 28 days, (i.e., Saline + AAV-GFP, Saline + AAV-TRPV5, Ang II + AAV-GFP and Ang II + AAV-TRPV5 groups were established). Compared with the control group, the incidence of AAA and the maximal diameter of the abdominal aorta markedly decreased in Ang II + AAV-TRPV5, which was detected by vascular ultrasound at 28 day. Meanwhile, less collagen and elastin degradation were observed in the Ang II + AAV-TRPV5 group by using Masson and Elastin stains. Moreover, more α-SMA and less MMP2 was observed in the abdominal aortas collected at 28 day by immunohistochemistry. In vitro, primary mouse vascular smooth muscle cells (VSMCs) were treated with Ang II (1 µM) to induce phenotype switch. Sh-TRPV5 and AdTRPV5 were used to transfect VSMCs. PCR and Western blotting were used to access the expression of contractile marker, including α-SMA and SM-22α. The results showed that the mRNA and protein level of α-SMA and SM-22α were decreased under the stimulation of Ang II, but could be attenuated by TRPV5 overexpression. The cell scratch assay demonstrated that the migration ability of VSMCs was increased in Ang II treated group and could be ameliorated by TRPV5 overexpression. Above all, VSMCs transformed from the contractile into secretory phenotype under Ang II stimuli, but could be rescued by TRPV5 overexpression. Furthermore, TRPV5 overexpression suppressed the increased expression of KLF4 induced by Ang II treatment in VSMCs. The data demonstrated that TRPV5 could inhibit AAA formation and play a critical role in the VSMC phenotype switch by downregulating KLF4, suggesting TRPV5 as a new strategy for treating AAA.


Assuntos
Aneurisma da Aorta Abdominal/tratamento farmacológico , Canais de Cálcio/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Canais de Cátion TRPV/farmacologia , Angiotensina II , Animais , Aorta/citologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/patologia , Canais de Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Dependovirus/genética , Regulação para Baixo , Técnicas de Transferência de Genes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Canais de Cátion TRPV/genética , Regulação para Cima
17.
Eur J Paediatr Neurol ; 30: 144-154, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33349592

RESUMO

BACKGROUND: CACNA1A-related disorders present with persistent progressive and non-progressive cerebellar ataxia and paroxysmal events: epileptic seizures and non-epileptic attacks. These phenotypes overlap and co-exist in the majority of patients. OBJECTIVE: To describe phenotypes in infantile onset CACNA1A-related disorder and to explore intra-familial variations and genotype-phenotype correlations. MATERIAL AND METHODS: This study was a multicenter international collaboration. A retrospective chart review of CACNA1A patients was performed. Clinical, radiological, and genetic data were collected and analyzed in 47 patients with infantile-onset disorder. RESULTS: Paroxysmal non-epileptic events (PNEE) were observed in 68% of infants, with paroxysmal tonic upward gaze (PTU) noticed in 47% of infants. Congenital cerebellar ataxia (CCA) was diagnosed in 51% of patients including four patients with developmental delay and only one neurological sign. PNEEs were found in 63% of patients at follow-up, with episodic ataxia (EA) in 40% of the sample. Cerebellar ataxia was found in 58% of the patients at follow-up. Four patients had epilepsy in infancy and nine in childhood. Seven infants had febrile convulsions, three of which developed epilepsy later; all three patients had CCA. Cognitive difficulties were demonstrated in 70% of the children. Cerebellar atrophy was found in only one infant but was depicted in 64% of MRIs after age two. CONCLUSIONS: Nearly all of the infants had CCA, PNEE or both. Cognitive difficulties were frequent and appeared to be associated with CCA. Epilepsy was more frequent after age two. Febrile convulsions in association with CCA may indicate risk of epilepsy in later childhood. Brain MRI was normal in infancy. There were no genotype-phenotype correlations found.


Assuntos
Canais de Cálcio/genética , Ataxia Cerebelar/genética , Transtornos Cognitivos/genética , Distonia/genética , Epilepsia/genética , Criança , Feminino , Humanos , Lactente , Masculino , Fenótipo , Estudos Retrospectivos
18.
Ann Clin Lab Sci ; 50(6): 775-780, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33334793

RESUMO

OBJECTIVE: To investigate the relationship between Transient Receptor Potential Vanilloid 6 (TRPV6) and ST-elevation acute myocardial infarction (STEMI) patients. METHODS: This observational research included a total of 221 patients with STEMI admitted during January 2017~August 2019. Additionally, 50 cases of non-ST-elevation acute myocardial infarction (NSTEMI) patients and 50 healthy individuals were enrolled as the control. Serum levels of TRPV6 were detected by ELISA method. The relationship between TRPV6, clinical characteristics, laboratory indices of CK-MB, TnI, NT-pro-B-type natriuretic peptide (NT-pro-BNP), C-reactive protein (CRP), and the left ventricular ejection fraction (LVEF%) was analyzed by statistical methods. K-M curve was performed for survival time. RESULTS: Serum levels of TRPV6 were remarkably lower in STEMI and NSTEMI patients compared with the healthy control. Levels of NT-pro-BNP and CK-MB were significantly higher and serum levels of TRPV6 were dramatically lower in deceased STEMI patients in comparison with the surviving patients. The levels of TRPV6 were negatively correlated with CK-MB and NT-pro-BNP. Meanwhile, TRPV6 was negatively expressed in tissues of STEMI patients and positively expressed in normal tissues. Patients with lower TRPV6 levels had remarkably lower LVEF ratio, higher GRACE scores, higher CK-MB and NT-pro-BNP levels, as well as higher ratios of cardiovascular death, malignant arrhythmia, cumulative MACE, and shorter survival time than patients with higher TRPV6. CONCLUSION: The lower expression of TRPV6 was associated with poor clinical outcomes and prognosis of STEMI patients.


Assuntos
Canais de Cálcio/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Canais de Cátion TRPV/metabolismo , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Canais de Cálcio/sangue , Canais de Cálcio/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Fragmentos de Peptídeos/metabolismo , Prognóstico , Curva ROC , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/genética , Volume Sistólico/fisiologia , Canais de Cátion TRPV/sangue , Canais de Cátion TRPV/genética , Função Ventricular Esquerda/fisiologia
19.
Nat Commun ; 11(1): 4031, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788582

RESUMO

Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies.


Assuntos
Canais de Cálcio/genética , Evolução Molecular , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/química , Quitridiomicetos/genética , Proteínas Fúngicas/química , Células HeLa , Humanos , Funções Verossimilhança , Filogenia , Especificidade da Espécie
20.
Neuron ; 107(4): 667-683.e9, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32616470

RESUMO

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.


Assuntos
Canais de Cálcio/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Exocitose/fisiologia , Camundongos Knockout , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo
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