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1.
PLoS One ; 15(9): e0239404, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970693

RESUMO

Polynucleotide kinase phosphatase (PNKP) is a DNA repair factor with dual enzymatic functions, i.e., phosphorylation of 5'-end and dephosphorylation of 3'-end, which are prerequisites for DNA ligation and, thus, is involved in multiple DNA repair pathways, i.e., base excision repair, single-strand break repair and double-strand break repair through non-homologous end joining. Mutations in PNKP gene causes inherited diseases, such as microcephaly and seizure (MCSZ) by neural developmental failure and ataxia with oculomotor apraxia 4 (AOA4) and Charcot-Marie-Tooth disease 2B2 (CMT2B2) by neurodegeneration. PNKP consists of the Forkhead-associated (FHA) domain, linker region, phosphatase domain and kinase domain. Although the functional importance of PNKP interaction with XRCC1 and XRCC4 through the FHA domain and that of phosphatase and kinase enzyme activities have been well established, little is known about the function of linker region. In this study, we identified a functional putative nuclear localization signal (NLS) of PNKP located in the linker region, and showed that lysine 138 (K138), arginine 139 (R139) and arginine 141 (R141) residues therein are critically important for nuclear localization. Furthermore, double mutant of K138A and R35A, the latter of which mutates arginine 35, central amino acid of FHA domain, showed additive effect on nuclear localization, indicating that the FHA domain as well as the NLS is important for PNKP nuclear localization. Thus, this study revealed two distinct mechanisms regulating nuclear localization and subnuclear distribution of PNKP. These findings would contribute to deeper understanding of a variety of DNA repair pathway, i.e., base excision repair, single-strand break repair and double-strand break repair.


Assuntos
Núcleo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios Proteicos/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência
2.
PLoS One ; 15(8): e0237999, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822427

RESUMO

Hyper-IgD syndrome (HIDS, OMIM #260920) is a rare autosomal recessive autoinflammatory disorder caused by pathogenic variants in the mevalonate kinase (MVK) gene. HIDS has an incidence of 1:50,000 to 1:5,000, and is thought to be prevalent mainly in northern Europe. Here, we report a case series of HIDS from India, which includes ten patients from six families who presented with a wide spectrum of clinical features such as recurrent fever, oral ulcers, rash, arthritis, recurrent diarrhea, hepatosplenomegaly, and high immunoglobulin levels. Using whole exome sequencing (WES) and/or Sanger capillary sequencing, we identified five distinct genetic variants in the MVK gene from nine patients belonging to six families. The variants were classified as pathogenic or likely pathogenic as per the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for annotation of sequence variants. Over 70% of patients in the present study had two recurrent mutations in MVK gene i.e. a nonsynonymous variant p.V377I, popularly known as the 'Dutch mutation', along with a splicing variant c.226+2delT in a compound heterozygous form. Identity by descent analysis in two patients with the recurrent variants identified a 6.7 MB long haplotype suggesting a founder effect in the South Indian population. Our analysis suggests that a limited number of variants account for the majority of the patients with HIDS in South India. This has implications in clinical diagnosis, as well as in the development of cost-effective approaches for genetic diagnosis and screening. To our best knowledge, this is the first and most comprehensive case series of clinically and genetically characterized patients with HIDS from India.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Deficiência de Mevalonato Quinase/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Adolescente , Pré-Escolar , Feminino , Deleção de Genes , Estudos de Associação Genética , Haplótipos , Heterozigoto , Humanos , Índia , Lactente , Masculino , Deficiência de Mevalonato Quinase/genética , Linhagem , Fosfotransferases (Aceptor do Grupo Álcool)/química , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Sequenciamento Completo do Exoma
3.
Environ Toxicol ; 35(12): 1299-1307, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32652857

RESUMO

Recent evidences show that acylglycerol kinase (AGK) expression is related to the occurrence and development of various human cancers. However, its roles in nasopharyngeal carcinoma (NPC) progression are still unclear. This work aims to explore the roles of AGK in NPC cell stemness. It was shown that AGK expression was higher in NPC tissues compared to the adjacent tissues. Online dataset analysis revealed that AGK expression was negatively correlated with the overall survival of NPC patients. Gain and loss of functional experiments demonstrated that AGK positively regulated the stemness of NPC cells, as evident by the change of the tumor sphere-formation ability, ALDH1 activity and expression of stemness critical regulators. KEGG analysis were performed to determine the potential pathways of AGK involved in NPC cell stemness and showed that the PI3K/Akt pathway exhibited the most correlation with AGK expression. Further mechanistic studies confirmed that AGK promoted the stemness of NPC cells through activating the PI3K/Akt pathway, and thus enhancing ß-catenin accumulation in nucleus. This study demonstrates a novel AGK/PI3K/Akt/ß-catenin axis involving in NPC cell stemness.


Assuntos
Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Adulto , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/genética , Humanos , Masculino , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Proteico
4.
Nucleic Acids Res ; 48(12): 6672-6684, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32504494

RESUMO

Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Apraxias/genética , Apraxias/patologia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Reparo do DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Microcefalia/genética , Microcefalia/patologia , Mutação/genética , Convulsões/genética , Convulsões/patologia
5.
Gene ; 756: 144915, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32580009

RESUMO

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are key enzymes in the process of phosphatidylinositol signaling pathway and have essential functions in growth, development, and biotic and abiotic stresses responses in plants. However, the evolutionary history and patterns of PIP5K genes in plants have not been examined systematically. Here, we use whole-genome sequences from eight plant species of land plants and algae to define the evolutionary relationships between these proteins in plants. 85 PIP5K genes were identified and divided into two subfamilies based on phylogenetic analyses. PIP5K members in subfamily II underwent several duplication events in land plants, resulting in multiple gene copies in angiosperms, while PIP5K members in subfamily I displayed low-copy numbers and lost in eudicots. Furthermore, PIP5K genes within the same subfamily had similar motifs and intron/exon features. Nine duplicated soybean gene pairs, four duplicated Arabidopsis gene pairs and two rice duplicated gene pairs were identified and many of them localized in synteny genomic regions. These duplicate events were formed by Whole-genome duplication (WGD)/segmental duplications. In addition, the ratios of non-synonymous to synonymous substitutions (Ka/Ks) showed that the PIP5K family had undergone purifying selection in higher plants. Expression analysis showed that PIP5K genes had complex and variable expression patterns in different developmental stages. The specificity of these genes is utilized to provide evidence for selective expression in the evolutionary process.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas/enzimologia , Cromossomos de Plantas , Evolução Molecular , Duplicação Gênica , Especiação Genética , Genoma de Planta , Filogenia , Plantas/classificação , Plantas/genética
6.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376619

RESUMO

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The major synthesis pathway of PI(4,5)P2 involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1ß, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared the cellular behavior of Pr55Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1ß is dispensable for Pr55Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P2 pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55Gag PM targeting process.IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired PM localization of Pr55Gag However, cellular factors that control PI(4,5)P2 production required for Pr55Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P2 metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55Gag targeting to the PM.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , HIV-1/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Precursores de Proteínas/metabolismo , Membrana Celular/genética , Membrana Celular/virologia , Endossomos/genética , Endossomos/metabolismo , Endossomos/virologia , HIV-1/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/virologia , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/genética , Proteólise
7.
Nat Commun ; 11(1): 2358, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398649

RESUMO

Sphingosine kinase1 (SphK1) is an acetyl-CoA dependent acetyltransferase which acts on cyclooxygenase2 (COX2) in neurons in a model of Alzheimer's disease (AD). However, the mechanism underlying this activity was unexplored. Here we show that N-acetyl sphingosine (N-AS) is first generated by acetyl-CoA and sphingosine through SphK1. N-AS then acetylates serine 565 (S565) of COX2, and the N-AS-acetylated COX2 induces the production of specialized pro-resolving mediators (SPMs). In a mouse model of AD, microglia show a reduction in N-AS generation, leading to decreased acetyl-S565 COX2 and SPM production. Treatment with N-AS increases acetylated COX2 and N-AS-triggered SPMs in microglia of AD mice, leading to resolution of neuroinflammation, an increase in microglial phagocytosis, and improved memory. Taken together, these results identify a role of N-AS in the dysfunction of microglia in AD.


Assuntos
Doença de Alzheimer/imunologia , Anti-Inflamatórios/farmacologia , Encéfalo/imunologia , Microglia/imunologia , Esfingosina/análogos & derivados , Acetilcoenzima A/metabolismo , Acetilação , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Anti-Inflamatórios/uso terapêutico , Encéfalo/patologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Mutagênese , Neurônios , Fagocitose/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Presenilina-1/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Esfingosina/metabolismo
8.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118714, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32246947

RESUMO

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P). CerK is highly expressed in the brain, and its association with the neuronal function has been reported. Previous reports showed that the activity of CerK is regulated by post-translational modifications including phosphorylation, whereas the cellular fate of CerK protein and its role in neuronal functions have not been clearly elucidated. Therefore, we investigated these issues in PC12 cells. Treatment with nerve growth factor (NGF) for 6 h increased the formation of C1P but not CerK mRNA. Knockdown of CerK and overexpression of HA-tagged CerK down- and up-regulated the formation of C1P, respectively. In PC12-CerK-HA cells, serum withdrawal caused ubiquitination of CerK-HA protein and down-regulated both CerK-HA protein and C1P formation within 6 h, and these down-regulations were abolished by co-treatments with NGF or proteasome inhibitors such as MG132 and clasto-lactacystin. Microscopic analysis showed that treatment with the proteasome inhibitors increased CerK-HA in puncture structures, possibly endosomes and/or vesicles, in cells. Treatment with the lysosome inhibitors reduced serum withdrawal-induced down-regulation of CerK-HA protein but not C1P formation. When knockdown or overexpression of CerK was performed, Ca2+-induced release of [3H] noradrenaline was reduced or enhanced, respectively, but neurite extension was not modified. There was a positive correlation between noradrenaline release and formation of C1P and/or CerK-HA levels in NGF- and clasto-lactacystin-treated cells. These results suggest that levels of CerK were down-regulated by the ubiquitin/proteasome and lysosome pathways and the former pathway-sensitive pool of CerK was suggested to be linked with exocytosis in PC12 cells.


Assuntos
Exocitose/genética , Fator de Crescimento Neural/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Ciclo Celular , Proliferação de Células , Ceramidas , Lisossomos/genética , Lisossomos/metabolismo , Redes e Vias Metabólicas/genética , Fator de Crescimento Neural/metabolismo , Células PC12 , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos
9.
Proc Natl Acad Sci U S A ; 117(17): 9613-9620, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284406

RESUMO

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.


Assuntos
Cloroplastos/microbiologia , Regulação da Expressão Gênica de Plantas/imunologia , Luz , Proteínas NLR/metabolismo , Phytophthora infestans/metabolismo , Proteínas de Plantas/metabolismo , Agrobacterium/metabolismo , Animais , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Inativação Gênica , Microscopia Confocal , Proteínas NLR/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/genética , Plântula , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia , Técnicas do Sistema de Duplo-Híbrido
10.
Artigo em Inglês | MEDLINE | ID: mdl-32112978

RESUMO

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.


Assuntos
Neoplasias Pulmonares/patologia , Metástase Neoplásica/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pseudópodes/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células A549 , Animais , Movimento Celular , Ceramidas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Masculino , Camundongos , Invasividade Neoplásica/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(4): 410-414, 2020 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-32219824

RESUMO

OBJECTIVE: To explore the molecular etiology for a Chinese family with mitochondrial DNA depletion syndrome. METHODS: Genomic DNA was extracted from peripheral blood samples of the patient and her parents.Targeted capture and next-generation sequencing was carried out to detect potential variants. Suspected variant was validated by Sanger sequencing. RESULTS: A novel homozygous frameshift variant c.505_508delTATC was identified in the patient, for which both his mother and father were carriers. CONCLUSION: The frameshift variant c.505_508delTATC probably underlies the mitochondrial DNA depletion syndrome in this patient. The result also enriched the variant spectrum of DGUOK gene.


Assuntos
Grupo com Ancestrais do Continente Asiático , Mutação da Fase de Leitura , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Grupo com Ancestrais do Continente Asiático/genética , DNA Mitocondrial/genética , Feminino , Humanos , Mutação , Síndrome
12.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211893

RESUMO

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.


Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diglicerídeos/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
13.
Sci Rep ; 10(1): 1715, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015447

RESUMO

Palm wine, the most commonly consumed traditional alcoholic beverage in Western Africa, harbours a complex microbiota and metabolites, which plays a crucial role in the overall quality and value of the product. In the present study, a combined metagenomic and metabolomic approach was applied to describe the microbial community structure and metabolites profile of fermented saps from three palm species (Elaeis guineensis, Raphia hookeri, Borassus aethiopum) in Côte d'Ivoire. Lactobacillaceae (47%), Leuconostocaceae (16%) and Acetobacteriaceae (28%) were the most abundant bacteria and Saccharomyces cerevisiae (87%) the predominant yeasts in these beverages. The microbial community structure of Raphia wine was distinctly different from the others. Multivariate analysis based on the metabolites profile clearly separated the three palm wine types. The main differentiating metabolites were putatively identified as gevotroline hydrochloride, sesartemin and methylisocitrate in Elaeis wine; derivative of homoserine, mitoxantrone in Raphia wine; pyrimidine nucleotide sugars (UDP-D-galacturonate) and myo-Inositol derivatives in Borassus wine. The enriched presence of gevotroline (an antipsychotic agent) and mitoxantrone (an anticancer drug) in palm wine supports its therapeutic potential. This work provides a valuable insight into the microbiology and biochemistry of palm wines and a rationale for selecting functional microorganisms for potential biotechnology applications.


Assuntos
Acetobacteraceae/fisiologia , Arecaceae/fisiologia , Genótipo , Lactobacillaceae/fisiologia , Leuconostocaceae/fisiologia , Saccharomyces cerevisiae/fisiologia , Vinho/microbiologia , Biologia Computacional , Costa do Marfim , Fermentação , Metaboloma , Metabolômica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Ribossômico 16S/genética
14.
Anesthesiology ; 132(6): 1503-1515, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32108663

RESUMO

BACKGROUND: Sepsis is the overwhelming inflammatory response to infection, in which nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome plays a crucial role. Shingosine-1-phosphate is reported to evoke NLRP3 inflammasome activation. Sphingosine kinase 1 (SphK1) is the major kinase that catalyzes bioactive lipid shingosine-1-phosphate formation and its role in sepsis remains uncertain. The authors hypothesize that SphK1 elicits NLRP3 inflammasome activation and exacerbates sepsis. METHODS: Peripheral blood mononuclear cells were isolated from septic patients and healthy volunteers to measure messenger RNA (mRNA) expression. In mice, sepsis was induced by cecal ligation and puncture. Bone marrow-derived macrophages were prepared from C57BL/6J wild-type, Casp1, Nlrp3 and SphK1 mice. PF-543 was used as the specific inhibitor of SphK1. Mortality, peripheral perfusion, lung Evan's blue dye index, lung wet/dry ratio, lung injury score, lung myeloperoxidase activity, NLRP3 activation, and function of endothelial adherens junction were measured. RESULTS: SphK1 mRNA expression was higher in cells from septic patients versus healthy volunteers (septic patients vs. healthy volunteers: 50.9 ± 57.0 fold change vs. 1.2 ± 0.1 fold change, P < 0.0001) and was positively correlated with IL-1ß mRNA expression in these cells (r = 0.537, P = 0.012) and negatively correlated with PaO2/FIO2 ratios (r = 0.516, P = 0.017). In mice that had undergone cecal ligation and puncture, the 5-day mortality was 30% in PF-543-treated group and 80% in control group (n = 10 per group, P = 0.028). Compared with controls, PF-543-treated mice demonstrated improved peripheral perfusion and alleviated extravascular Evan's blue dye effusion (control vs. PF-543: 25.5 ± 3.2 ng/g vs. 18.2 ± 1.4 ng/g, P < 0.001), lower lung wet/dry ratio (control vs. PF-543: 8.0 ± 0.2 vs. 7.1 ± 0.4, P < 0.0001), descending lung injury score, and weaker lung myeloperoxidase activity. Inhibition of SphK1 suppressed caspase-1 maturation and interleukin-1ß release through repressing NLRP3 inflammasome activation, and subsequently stabilized vascular endothelial cadherin through suppressing interleukin-1ß-evoked Src-mediated phosphorylation of vascular endothelial cadherin. CONCLUSIONS: SphK1 plays a crucial role in NLRP3 inflammasome activation and contributes to lung injury and mortality in mice polymicrobial sepsis.


Assuntos
Inflamassomos/metabolismo , Lesão Pulmonar/patologia , Macrófagos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sepse/patologia , Animais , Modelos Animais de Doenças , Humanos , Inflamassomos/genética , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sepse/genética , Sepse/metabolismo , Transdução de Sinais/genética
15.
Mol Cell ; 77(6): 1294-1306.e5, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32023483

RESUMO

von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Prognóstico , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Proteínas Repressoras/genética , Células Tumorais Cultivadas , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Development ; 147(5)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32041790

RESUMO

In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development and autophagy. Our CRISPR-generated, fluorescently tagged endogenous Caenorhabditis elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. LET-363/TOR and DAF-15/Raptor are required for development beyond the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs the decision of the animal to progress through development.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Animais , Animais Geneticamente Modificados , Autofagia/fisiologia , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/genética , Estágios do Ciclo de Vida/genética , Longevidade/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Transdução de Sinais
17.
Biochem J ; 477(5): 925-935, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32065229

RESUMO

The interplay of sphingosine 1-phosphate (S1P) synthetic and degradative enzymes as well as S1P exporters creates concentration gradients that are a fundamental to S1P biology. Extracellular S1P levels, such as in blood and lymph, are high relative to cellular S1P. The blood-tissue S1P gradient maintains endothelial integrity while local S1P gradients influence immune cell positioning. Indeed, the importance of S1P gradients was recognized initially when the mechanism of action of an S1P receptor agonist used as a medicine for multiple sclerosis was revealed to be inhibition of T-lymphocytes' recognition of the high S1P in efferent lymph. Furthermore, the increase in erythrocyte S1P in response to hypoxia influences oxygen delivery during high altitude acclimatization. However, understanding of how S1P gradients are maintained is incomplete. For example, S1P is synthesized but is only slowly metabolized by blood yet circulating S1P turns over quickly by an unknown mechanism. Prompted by the counterintuitive observation that blood S1P increases markedly in response to inhibition S1P synthesis (by sphingosine kinase 2 (SphK2)), we studied mice wherein several tissues were made deficient in either SphK2 or S1P degrading enzymes. Our data reveal a mechanism whereby S1P is de-phosphorylated at the hepatocyte surface and the resulting sphingosine is sequestered by SphK phosphorylation and in turn degraded by intracellular S1P lyase. Thus, we identify the liver as the primary site of blood S1P clearance and provide an explanation for the role of SphK2 in this process. Our discovery suggests a general mechanism whereby S1P gradients are shaped.


Assuntos
Hepatócitos/metabolismo , Lisofosfolipídeos/sangue , Taxa de Depuração Metabólica/fisiologia , Esfingosina/análogos & derivados , Animais , Feminino , Humanos , Lisofosfolipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingosina/sangue , Esfingosina/genética
18.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32045479

RESUMO

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de Tempo
19.
Am J Hum Genet ; 106(2): 256-263, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004446

RESUMO

We report an inborn error of metabolism caused by TKFC deficiency in two unrelated families. Rapid trio genome sequencing in family 1 and exome sequencing in family 2 excluded known genetic etiologies, and further variant analysis identified rare homozygous variants in TKFC. TKFC encodes a bifunctional enzyme involved in fructose metabolism through its glyceraldehyde kinase activity and in the generation of riboflavin cyclic 4',5'-phosphate (cyclic FMN) through an FMN lyase domain. The TKFC homozygous variants reported here are located within the FMN lyase domain. Functional assays in yeast support the deleterious effect of these variants on protein function. Shared phenotypes between affected individuals with TKFC deficiency include cataracts and developmental delay, associated with cerebellar hypoplasia in one case. Further complications observed in two affected individuals included liver dysfunction and microcytic anemia, while one had fatal cardiomyopathy with lactic acidosis following a febrile illness. We postulate that deficiency of TKFC causes disruption of endogenous fructose metabolism leading to generation of by-products that can cause cataract. In line with this, an affected individual had mildly elevated urinary galactitol, which has been linked to cataract development in the galactosemias. Further, in light of a previously reported role of TKFC in regulating innate antiviral immunity through suppression of MDA5, we speculate that deficiency of TKFC leads to impaired innate immunity in response to viral illness, which may explain the fatal illness observed in the most severely affected individual.


Assuntos
Catarata/etiologia , Cerebelo/anormalidades , Deficiências do Desenvolvimento/etiologia , Mutação , Malformações do Sistema Nervoso/etiologia , Fósforo-Oxigênio Liases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Alelos , Sequência de Aminoácidos , Catarata/patologia , Cerebelo/patologia , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Feminino , Homozigoto , Humanos , Lactente , Masculino , Malformações do Sistema Nervoso/patologia , Linhagem , Fenótipo , Fosforilação , Homologia de Sequência , Sequenciamento Completo do Exoma
20.
Nat Commun ; 11(1): 867, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054832

RESUMO

Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-ß peptides (Aß) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of Aß42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce Aß42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and α-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of Aß42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Mononucleotídeo de Flavina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Genes Sintéticos , Genoma Fúngico , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Genéticos , Mutação , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Dobramento de Proteína , Proteólise , RNA-Seq , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
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