RESUMO
BACKGROUND: HCFC1 encodes transcriptional co-regulator HCF-1, which undergoes an unusual proteolytic maturation at a centrally located proteolysis domain. HCFC1 variants were associated with X-linked cobalamin metabolism disorders and mental retardation-3. This study aimed to explore the role of HCFC1 variants in common epilepsy and the mechanism underlying phenotype heterogeneity. METHODS: Whole-exome sequencing was performed in a cohort of 313 patients with idiopathic partial (focal) epilepsy. Functional studies determined the effects of the variants on the proteolytic maturation of HCF-1, cell proliferation and MMACHC expression. The role of HCFC1 variants in partial epilepsy was validated in another cohort from multiple centers. RESULTS: We identified seven hemizygous HCFC1 variants in 11 cases and confirmed the finding in the validation cohort with additional 13 cases and six more hemizygous variants. All patients showed partial epilepsies with favorable outcome. None of them had cobalamin disorders. Functional studies demonstrated that the variants in the proteolysis domain impaired the maturation by disrupting the cleavage process with loss of inhibition of cell growth but did not affect MMACHC expression that was associated with cobalamin disorder. The degree of functional impairment was correlated with the severity of phenotype. Further analysis demonstrated that variants within the proteolysis domain were associated with common and mild partial epilepsy, whereas those in the kelch domain were associated with cobalamin disorder featured by severe and even fatal epileptic encephalopathy, and those in the basic and acidic domains were associated with mainly intellectual disability. CONCLUSION: HCFC1 is potentially a candidate gene for common partial epilepsy with distinct underlying mechanism of proteolysis dysfunction. The HCF-1 domains played distinct functional roles and were associated with different clinical phenotypes, suggesting a sub-molecular effect. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype and pathogenic mechanism, implied a clinical significance in early diagnosis and management.
Assuntos
Epilepsias Parciais , Epilepsia , Humanos , Proteólise , Epilepsia/genética , Vitamina B 12/genética , Vitamina B 12/metabolismo , Regulação da Expressão Gênica , Epilepsias Parciais/genética , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Potential health risks for humans from exposure to acrylamide (AA) and its epoxide metabolite glycidamide (GA) have garnered much attention lately because substantial amounts of AA are present in a variety of fried and baked starchy foods. AA is tumorigenic in rodents, and a large number of in vitro and in vivo studies indicate that AA is genotoxic. A recent cancer bioassay on AA demonstrated that the lung was one of the target organs for tumor induction in mice; however, the mutagenicity of AA in this tissue is unclear. Therefore, to investigate whether or not gene mutation is involved in the etiology of AA- or GA-induced mouse lung carcinogenicity, we screened for cII mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19-111 mg/kg bw/days). Both doses of AA and GA produced significant increases in cII MFs, with the high doses producing responses 2.7-5.6-fold higher than the corresponding controls (P ≤ 0.05; control MFs = 17.2 ± 2.2 and 15.8 ± 3.5 × 10(-6) in males and females, respectively). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P ≤ 0.01). The predominant types of mutations in the lung cII gene from AA- and GA-treated mice were A:T â T:A, and G:C â C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted. Environ. Mol. Mutagen. 56:446-456, 2015. © 2015 Wiley Periodicals, Inc.
Assuntos
Acrilamida/toxicidade , Compostos de Epóxi/toxicidade , Neoplasias Pulmonares/genética , Mutagênicos/toxicidade , Mutação , Fatores de Transcrição/genética , Proteínas Virais/genética , Acrilamida/metabolismo , Administração Oral , Animais , DNA/genética , Relação Dose-Resposta a Droga , Compostos de Epóxi/metabolismo , Feminino , Neoplasias Pulmonares/induzido quimicamente , Masculino , Camundongos Endogâmicos , Mutagênicos/metabolismo , Análise de Sequência de DNA , Caracteres SexuaisRESUMO
Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quimiotaxia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Pseudópodes/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas de Ancoragem à Quinase A/química , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo , Estrutura Terciária de Proteína , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs) in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Enzimas/genética , Enzimas/metabolismo , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Transcriptoma , Redes Reguladoras de Genes , Humanos , Fígado/enzimologia , Medicina de Precisão , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Lymphocyte activation results in profound changes in the abundance of mRNA transcripts many of which are downregulated. The Wiskott-Aldrich syndrome (WAS) protein (WASP) family is critical for productive T-cell receptor signaling and actin reorganization. The WASP signal pathway includes the WAS/WAS-like (WASL) interacting protein family 2 (WIPF2) gene also known as WIRE/WICH. We show that both human WIPF2 and mouse Wipf2 are mice, alternatively spliced within the 3' untranslated region (3'UTR) resulting in two major transcripts of approximately 4.5 and 6 kb in size. Following T-cell activation, the level of human WIPF2 and mouse Wipf2 mRNA rapidly declines. In mice, this decline is accompanied by a marked reduction in WIPF2 protein levels. Transgenic expression of a 240-bp fragment derived from a highly conserved terminal 3'UTR found within the 6-kb transcript blocks Wipf2 downregulation. These effects may be mediated by competitive inhibition of microinhibitory RNA (miRNA) regulation since the 6-kb-derived transgene and the 4.5-kb transcript share functional binding sites for miRNA146a. Blocking Wipf2 gene and protein repression resulted in improved T-cell responses to antigen immunization in vivo as well as in vitro cytotoxic T-cell killing. Collectively, these data suggest that early downregulation of this immunologically relevant gene controls the intensity of selective lymphocyte functions.
Assuntos
Regiões 3' não Traduzidas , RNA Mensageiro/genética , Proteína da Síndrome de Wiskott-Aldrich/antagonistas & inibidores , Proteína da Síndrome de Wiskott-Aldrich/genética , Actinas/genética , Actinas/imunologia , Animais , Antígenos/imunologia , Sítios de Ligação , Regulação para Baixo , Feminino , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , RNA Mensageiro/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinação/métodos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Humans at all ages are continually exposed to triclosan (TCS), a widely used antimicrobial agent that can be found in many daily hygiene products, such as toothpastes and shampoos; however, the toxicological and biological effects of TCS in the human body after long-term and low-concentration exposure are far from being well understood. In the current study, we investigated the effects of TCS on the differentiation of human mesenchymal stem cells (hMSCs) by measuring the cytotoxicity, morphological changes, lipid accumulation, and the expression of adipocyte differentiation biomarkers during 21-day adipogenesis. Significant cytotoxicity was observed in un-induced hMSCs treated with high-concentration TCS (≥ 5.0 µM TCS), but not with low-concentration treatments (≤ 2.5 µM TCS). TCS inhibited adipocyte differentiation of hMSCs in a concentration-dependent manner in the 0.156 to 2.5 µM range as indicated by morphological changes with Oil Red O staining, which is an index of lipid accumulation. The inhibitory effect was confirmed by a decrease in gene expression of specific adipocyte differentiation biomarkers including adipocyte protein 2, lipoprotein lipase, and adiponectin. Our study demonstrates that TCS inhibits adipocyte differentiation of hMSCs under concentrations that are not cytotoxic and in the range observed in human blood.
Assuntos
Adipogenia/efeitos dos fármacos , Anti-Infecciosos Locais/toxicidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Triclosan/toxicidade , Adiponectina/genética , Adiponectina/metabolismo , Compostos Azo/química , Relação Dose-Resposta a Droga , Humanos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human sex differences in the gene expression of drug metabolizing enzymes and transporters (DMETs) introduce differences in drug absorption, distribution, metabolism and excretion, possibly affecting drug efficacy and adverse reactions. However, existing studies aimed at identifying dimorphic expression differences of DMET genes are limited by sample size and the number of genes profiled. Focusing on a list of 374 DMET genes, we analyzed a previously published gene expression data set consisting of human male (n=234) and female (n=193) liver samples, and identified 77 genes showing differential expression due to sex. To delineate the biological functionalities and regulatory mechanisms for the differentially expressed DMET genes, we conducted a co-expression network analysis. Moreover, clinical implications of sex differences in the expression of human hepatic DMETs are discussed. This study may contribute to the realization of personalized medicine by better understanding the inter-individual differences between males and females in drug/xenobiotic responses and human disease susceptibilities.
RESUMO
The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely through its ability to scaffold multiple signaling mediators such as PKC, PKA, cyclins, calmodulin, and Src. Although SSeCKS and PKCα bind phosphatidylserine, we demonstrate that phosphatidylserine-independent binding of PKC by SSeCKS is facilitated by two homologous SSeCKS motifs, EG(I/V)(T/S)XWXSFK(K/R)(M/L)VTP(K/R)K(K/R)X(K/R)XXXEXXXE(E/D) (amino acids 592-620 and 741-769). SSeCKS binding to PKCα decreased kinase activity and was dependent on the two PKC-binding motifs. SSeCKS scaffolding of PKC was increased in confluent cell cultures, correlating with significantly increased SSeCKS protein levels and decreased PKCα activity, suggesting a role for SSeCKS in suppressing PKC activation during contact inhibition. SSeCKS-null mouse embryo fibroblasts displayed increased relative basal and phorbol ester (phorbol 12-myristate 13-acetate)-induced PKC activity but were defective in phorbol 12-myristate 13-acetate-induced actin cytoskeletal reorganization and cell shape change; these responses could be rescued by the forced expression of full-length SSeCKS but not by an SSeCKS variant deleted of its PKC-binding domains. Finally, the PKC binding sites in SSeCKS were required to restore cell rounding and/or decreased apoptosis in phorbol ester-treated LNCaP, LNCaP-C4-2, and MAT-LyLu prostate cancer cells. Thus, PKC-mediated remodeling of the actin cytoskeleton is likely regulated by the ability of SSeCKS to control PKC signaling and activity through a direct scaffolding function.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas de Ciclo Celular/fisiologia , Citoesqueleto/metabolismo , Ésteres de Forbol/farmacologia , Proteína Quinase C/química , Proteínas de Ancoragem à Quinase A/química , Animais , Apoptose , Sítios de Ligação , Proteínas de Ciclo Celular/química , Sobrevivência Celular , Fibroblastos/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Isoformas de ProteínasRESUMO
Cell signaling pathways induce Sp1 phosphorylation, which allows for the upregulation of Sp1-dependent genes that control cell growth, cell-cycle progression, survival and tumorigenesis. Sp1 activity is under constitutive repression through the sumoylation of Lysine-16, and Lysine-16 dependent N-terminal cleavage relieves this repression. The present investigation probes further into the mechanisms of Sp1 processing, desumoylation, and degradation to reveal that phosphorylation is the major driving force behind these coupled activities. The first 7 amino acid residues of Sp1 enhance the accessibility of Lysine-16 to the homologous modifiers SUMO-1 and ubiquitin; and Serine-7 specifically enhances ubiquitinylation. Our data show that Serine-59 regulates Sp1 proteolytic processing, and thereby provides a mechanism for the upregulation of Sp1-dependent transcription by CyclinA/cdk2 phosphorylation of Serine-59. Sp1 activators, forskolin and PMA, enhance Sp1 processing in MCFE cells through distinct signaling pathways. PKC, ERK, and ERBB2 kinase inhibitors suppress PMA induction of Sp1 and the specific isozyme PKCalpha enhances Sp1 cleavage. Sp1 contains several NFkappaB2-like proteolytic processing components including a functional phosphorylation-dependent beta-TrCP binding motif. From these data, we propose a model by which cell-cycle and mitotic kinases induce Sp1 proteolytic processing resulting in a desumoylated, derepressed and unstable Sp1 product.