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1.
Viruses ; 13(6)2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205489

RESUMO

The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC-EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies.


Assuntos
Adenoviridae/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Metapneumovirus/fisiologia , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral , Células A549 , Linhagem Celular , Quimiocinas/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Células HEK293 , Humanos , Quinolinas/farmacologia
2.
Int J Mol Sci ; 22(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062838

RESUMO

BACKGROUND: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to ß-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. METHODS: Cellular arrhythmia, ion currents, and Ca2+-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. RESULTS: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in ICaL seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in INaL and Ca2+-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. CONCLUSION: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.


Assuntos
Arritmias Cardíacas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/patologia , Cálcio/metabolismo , AMP Cíclico/genética , GMP Cíclico/genética , Regulação da Expressão Gênica/genética , Coração/fisiopatologia , Humanos , Isoproterenol/toxicidade , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo
3.
Cancer Med ; 10(10): 3346-3357, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33932127

RESUMO

BACKGROUND: Chronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression. METHODS: R-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC. RESULTS: We identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo. CONCLUSIONS: PSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.


Assuntos
Álcoois/efeitos adversos , Carcinoma Hepatocelular/genética , Epigênese Genética/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias Hepáticas/genética , NF-kappa B/genética , Transcrição Genética/genética , Fator de Necrose Tumoral alfa/genética , Consumo de Bebidas Alcoólicas/genética , Animais , Caderinas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Gravidez , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Fator de Transcrição RelA/genética
4.
FASEB J ; 35(6): e21627, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33948992

RESUMO

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.


Assuntos
Derme/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Junções Íntimas/fisiologia , Proteína rhoB de Ligação ao GTP/metabolismo , Permeabilidade Capilar , Derme/citologia , Derme/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Proteína rhoB de Ligação ao GTP/genética
5.
Int J Mol Sci ; 22(7)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917494

RESUMO

Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Circadianas Period/metabolismo , Proteólise , Caseína Quinase Idelta/genética , Caseína Quinase Idelta/metabolismo , Relógios Circadianos , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Proteínas Circadianas Period/genética , Fosforilação
6.
Nat Commun ; 12(1): 2198, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850160

RESUMO

Cancer is initiated by somatic mutations in oncogenes or tumor suppressor genes. However, additional alterations provide selective advantages to the tumor cells to resist treatment and develop metastases. Their identification is of paramount importance. Reduced expression of EFA6B (Exchange Factor for ARF6, B) is associated with breast cancer of poor prognosis. Here, we report that loss of EFA6B triggers a transcriptional reprogramming of the cell-to-ECM interaction machinery and unleashes CDC42-dependent collective invasion in collagen. In xenograft experiments, MCF10 DCIS.com cells, a DCIS-to-IDC transition model, invades faster when knocked-out for EFA6B. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knock-out cells. Thus, we reveal an EFA6B-regulated molecular mechanism that controls the invasive potential of mammary cells; this finding opens up avenues for the treatment of invasive breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Nus , Transcriptoma , Proteína cdc42 de Ligação ao GTP
7.
Transl Psychiatry ; 11(1): 234, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888678

RESUMO

In this study we tested the hypothesis that pharmacological modulation of glutamatergic neurotransmission could rescue behavioral deficits exhibited by mice carrying a specific mutation in the Iqsec2 gene. The IQSEC2 protein plays a key role in glutamatergic synapses and mutations in the IQSEC2 gene are a frequent cause of neurodevelopmental disorders. We have recently reported on the molecular pathophysiology of one such mutation A350V and demonstrated that this mutation downregulates AMPA type glutamatergic receptors (AMPAR) in A350V mice. Here we sought to identify behavioral deficits in A350V mice and hypothesized that we could rescue these deficits by PF-4778574, a positive AMPAR modulator. Using a battery of social behavioral tasks, we found that A350V Iqsec2 mice exhibit specific deficits in sex preference and emotional state preference behaviors as well as in vocalizations when encountering a female mouse. The social discrimination deficits, but not the impaired vocalization, were rescued with a single dose of PF-4778574. We conclude that social behavior deficits associated with the A350V Iqsec2 mutation may be rescued by enhancing AMPAR mediated synaptic transmission.


Assuntos
Receptores de AMPA , Comportamento Social , Animais , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Transmissão Sináptica
8.
Nat Commun ; 12(1): 1623, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712589

RESUMO

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Metástase Neoplásica , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Fatores de Ribosilação do ADP/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Xenoenxertos , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Medicine (Baltimore) ; 100(9): e24633, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33655927

RESUMO

RATIONALE: Familial exudative vitreoretinopathy (FEVR) is an inherited disorder, which is mostly reported to be associated with the mutation of genes involved in the Wnt signaling pathway related to ß-catenin. To the best of our knowledge, the involvement of Adams-Oliver syndrome (AOS) genes in FEVR patients have not been reported before. PATIENT CONCERNS: Two patients with FEVR presented with microcephaly. One of them showed slight scarring of the scalp vertex which is a typical manifestation of AOS. The whole exon sequencing confirmed the diagnosis of AOS with 2 AOS-gene mutations at DOCK6 and ARHGAP31. Further clinical examination revealed that their parents with the same mutations showed FEVR-like vascular anomalies. DIAGNOSIS: Both patients were diagnosed with AOS through whole exon sequencing, and they presented with some FEVR-like retinopathy including retinal detachment. INTERVENTIONS: Both patients received vitrectomy for tractional retinal detachment with proliferative vitreoretinopathy. During the follow-up, 1 patient received additional laser photocoagulation for tractional retinal detachment. OUTCOMES: The 2 patients remained stable in the latest follow up after the treatment. LESSONS: Microcephaly could be associated with some form of retinopathy. We proposed that mutation of DOCK6 and ARHGAP31 genes could be the possible cause of FEVR associated with microcephaly. Our study suggested that these genes may be candidate genes of FEVR.


Assuntos
Displasia Ectodérmica/genética , Vitreorretinopatias Exsudativas Familiares/genética , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Deformidades Congênitas dos Membros/genética , Microcefalia/genética , Fosfoproteínas/genética , Dermatoses do Couro Cabeludo/congênito , Oftalmopatias Hereditárias/genética , Feminino , Humanos , Lactente , Masculino , Mutação/genética , Descolamento Retiniano/genética , Dermatoses do Couro Cabeludo/genética
10.
Sci Transl Med ; 13(586)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33762435

RESUMO

Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which have the worst prognosis and distant metastasis-free survival among breast cancer subtypes. Now, no targeted therapies are available for patients with BLBC due to the lack of reliable and effective molecular targets. Here, we performed the BLBC tissue microarray-based immunohistochemical analysis and showed that Faciogenital Dysplasia 5 (FGD5) abundance is associated with poor prognosis in BLBCs. FGD5 deletion decreased the proliferation, invasion, and tumorsphere formation capacity of BLBC cells. Furthermore, genetic inhibition of Fgd5 in mouse mammary epithelial cells attenuated BLBC initiation and progression by reducing the self-renewal ability of tumor-initiating cells. In addition, FGD5 abundance was positively correlated with the abundance of epidermal growth factor receptor (EGFR) in BLBCs. FGD5 ablation decreased EGFR abundance by reducing EGFR stability in TNBC cells in 2D and 3D culture conditions. Mechanistically, FGD5 binds to EGFR and interferes with basal EGFR ubiquitination and degradation induced by the E3 ligase ITCH. Impaired EGFR degradation caused BLBC cell proliferation and promoted invasive properties and self-renewal. To verify the role of the FGD5-EGFR interaction in the regulation of EGFR stability, we screened a cell-penetrating α-helical peptide PER3 binding with FGD5 to disrupt the interaction. Treatment of BLBC patient-derived xenograft-bearing mice with the peptide PER3 disrupting the FGD5-EGFR interaction either with or without chemotherapy reduced BLBC progression. Our study identified FGD5 as a positive modulator of tumor-initiating cells and suggests a potential therapeutic option for the BLBC subtype of breast cancer.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Células-Tronco Neoplásicas , Neoplasias de Mama Triplo Negativas , Animais , Receptores ErbB , Feminino , Humanos , Camundongos , Neoplasias de Mama Triplo Negativas/genética
11.
Transl Psychiatry ; 11(1): 181, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753721

RESUMO

IQSEC2 is an X-linked gene that is associated with autism spectrum disorder (ASD), intellectual disability, and epilepsy. IQSEC2 is a postsynaptic density protein, localized on excitatory synapses as part of the NMDA receptor complex and is suggested to play a role in AMPA receptor trafficking and mediation of long-term depression. Here, we present brain-wide structural volumetric and functional connectivity characterization in a novel mouse model with a missense mutation in the IQ domain of IQSEC2 (A350V). Using high-resolution structural and functional MRI, we show that animals with the A350V mutation display increased whole-brain volume which was further found to be specific to the cerebral cortex and hippocampus. Moreover, using a data-driven approach we identify putative alterations in structure-function relations of the frontal, auditory, and visual networks in A350V mice. Examination of these alterations revealed an increase in functional connectivity between the anterior cingulate cortex and the dorsomedial striatum. We also show that corticostriatal functional connectivity is correlated with individual variability in social behavior only in A350V mice, as assessed using the three-chamber social preference test. Our results at the systems-level bridge the impact of previously reported changes in AMPA receptor trafficking to network-level disruption and impaired social behavior. Further, the A350V mouse model recapitulates similarly reported brain-wide changes in other ASD mouse models, with substantially different cellular-level pathologies that nonetheless result in similar brain-wide alterations, suggesting that novel therapeutic approaches in ASD that result in systems-level rescue will be relevant to IQSEC2 mutations.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Deficiência Intelectual , Animais , Transtorno do Espectro Autista/diagnóstico por imagem , Transtorno do Espectro Autista/genética , Transtorno Autístico/diagnóstico por imagem , Transtorno Autístico/genética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/genética , Imageamento por Ressonância Magnética , Camundongos , Proteínas do Tecido Nervoso
12.
Medicine (Baltimore) ; 100(3): e23967, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33545980

RESUMO

ABSTRACT: Trisomy 9p is one of the most common chromosomal partial trisomies in newborns. However, reports on prenatal 9p microduplications are rare in the clinic. This study aimed to examine the genotype-phenotype correlation and assess the clinical significance of 9p24.3 microduplication encompassing the DOCK8 gene. Eight pregnant women underwent amniocentesis for cytogenetic and genetic testing for various indications for prenatal diagnosis from January 2019 to January 2020. Chromosomal karyotypic analysis was performed on G-band metaphases that were prepared from cultured amniotic fluid cells. Chromosomal microarray analysis was carried out to detect chromosomal copy number variations. We also performed a literature review on clinical data on similar 9p24.3 microduplications to determine the genotype-phenotype correlation. We detected 123-248-kb microduplications in the region of 9p24.3 (chr9: 208454-469022), involving part of or the entire DOCK8 gene. The indications for prenatal diagnosis mainly focused on the risk of maternal serum screening for trisomy 21/18, advanced maternal age, and increased nuchal translucency. No evident structural abnormalities were observed for all fetuses, except for case 5 who presented with increased nuchal translucency in prenatal ultrasound findings. Follow-up of postnatal health was performed and showed no apparent abnormalities for cases 1 to 6 after birth. The parents of case 7 chose to terminate the pregnancy while the parents of case 8 chose to continue the pregnancy. We propose that 9p24.3 microduplications that encompass part of or the entire DOCK8 gene are variants that might be benign. However, further large-scale studies are necessary to evaluate the clinical pathogenicity. For prenatal cases with 9p24.3 microduplication, postnatal health and growth should be followed up and assessed regularly from childhood to adulthood.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Diagnóstico Pré-Natal/métodos , Adulto , Líquido Amniótico , Cromossomos Humanos Par 9/fisiologia , Variações do Número de Cópias de DNA , Feminino , Fatores de Troca do Nucleotídeo Guanina/análise , Humanos , Gravidez , Trissomia/fisiopatologia
13.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557244

RESUMO

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1's function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1's functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1's NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.


Assuntos
Suscetibilidade a Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Nível de Saúde , Chaperonas Moleculares/metabolismo , Animais , Biomarcadores , Gerenciamento Clínico , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Estudos de Associação Genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Fenótipo , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/etiologia , Degenerações Espinocerebelares/metabolismo , Degenerações Espinocerebelares/terapia , Relação Estrutura-Atividade , Resposta a Proteínas não Dobradas
14.
Nat Commun ; 12(1): 879, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563986

RESUMO

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages. Here we show that macrophages infected with S. Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates. The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1. The changes in host metabolism promote intracellular replication of S. Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors. Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S. Typhimurium replication or virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Macrófagos/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Glucose/metabolismo , Ácidos Glicéricos/metabolismo , Glicólise , Fatores de Troca do Nucleotídeo Guanina/genética , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Salmonella typhimurium/metabolismo , Serina/biossíntese , Transdução de Sinais , Sistemas de Secreção Tipo III/genética , Virulência
15.
J Neuroimmunol ; 353: 577507, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548618

RESUMO

Interferon-ß (IFN-ß) is among the first drugs used for reducing the symptoms of multiple sclerosis (MS). Many studies show that the genetic predisposition of patients might modulate their response to IFN-ß treatment. In this study GAPVD1 gene expression and the genotyping of rs2291858 variant were analysed in 100 responder and 100 non-responder patients with MS treated using IFN-ß. Moreover, rs2291858 genotyping was performed for 200 patients with MS and 200 healthy controls. GAPVD1 expression was significantly increased in the responder patients than in non-responders and the distribution of rs2291858 polymorphism was significantly different between them. The GAPVD1 expression level in AA genotype of the responder group was higher than that in other genotypes of these two groups. The results show that the GAPVD1 expression level and rs2291858 genotype probably affect the response to IFN- ß in patients with MS.


Assuntos
Resistência a Medicamentos/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores Imunológicos/uso terapêutico , Interferon beta/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/genética , Adulto , Feminino , Genótipo , Humanos , Masculino , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Polimorfismo de Nucleotídeo Único
16.
PLoS Pathog ; 17(2): e1009294, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33539420

RESUMO

Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs that can decoy other RNAs to inhibit their functions. Kaposi's sarcoma (KS), caused by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), is a highly angiogenic and invasive vascular tumor of endothelial origin commonly found in AIDS patients. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) induces cell invasion, angiogenesis and cellular transformation; however, the role of circRNAs is largely unknown in the context of KSHV vIRF1. Herein, transcriptome analysis identified 22 differentially expressed cellular circRNAs regulated by vIRF1 in an endothelial cell line. Among them, circARFGEF1 was the highest upregulated circRNA. Mechanistically, vIRF1 induced circARFGEF1 transcription by binding to transcription factor lymphoid enhancer binding factor 1 (Lef1). Importantly, upregulation of circARFGEF1 was required for vIRF1-induced cell motility, proliferation and in vivo angiogenesis. circARFGEF1 functioned as a competing endogenous RNAs (ceRNAs) by binding to and inducing degradation of miR-125a-3p. Mass spectrometry analysis demonstrated that glutaredoxin 3 (GLRX3) was a direct target of miR-125a-3p. Knockdown of GLRX3 impaired cell motility, proliferation and angiogenesis induced by vIRF1. Taken together, vIRF1 transcriptionally activates circARFGEF1, potentially by binding to Lef1, to promote cell oncogenic phenotypes via inhibiting miR-125a-3p and inducing GLRX3. These findings define a novel mechanism responsible for vIRF1-induced oncogenesis and establish the scientific basis for targeting these molecules for treating KSHV-associated cancers.


Assuntos
Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Herpesvirus Humano 8/fisiologia , Fatores Reguladores de Interferon/metabolismo , Neovascularização Patológica/patologia , RNA Circular/genética , Sarcoma de Kaposi/patologia , Proteínas Virais/metabolismo , Proteínas de Transporte/genética , Movimento Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Fatores Reguladores de Interferon/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/virologia , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Proteínas Virais/genética
17.
Mol Cell ; 81(6): 1276-1291.e9, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33539787

RESUMO

Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.


Assuntos
Neoplasias Encefálicas , Carcinogênese , Proteínas de Ciclo Celular , Glioblastoma , Fatores de Troca do Nucleotídeo Guanina , Mitose/efeitos da radiação , Proteínas de Neoplasias , Proteínas Nucleares , Proteína-Arginina N-Metiltransferases , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/efeitos da radiação , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Mitose/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
18.
PLoS One ; 16(1): e0245584, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471859

RESUMO

The COVID19 pandemic caused by SARS-CoV-2 virus has severely affected most countries of the world including Bangladesh. We conducted comparative analysis of publicly available whole-genome sequences of 64 SARS-CoV-2 isolates in Bangladesh and 371 isolates from another 27 countries to predict possible transmission routes of COVID19 to Bangladesh and genomic variations among the viruses. Phylogenetic analysis indicated that the pathogen was imported in Bangladesh from multiple countries. The viruses found in the southern district of Chattogram were closely related to strains from Saudi Arabia whereas those in Dhaka were similar to that of United Kingdom and France. The 64 SARS-CoV-2 sequences from Bangladesh belonged to three clusters. Compared to the ancestral SARS-CoV-2 sequence reported from China, the isolates in Bangladesh had a total of 180 mutations in the coding region of the genome, and 110 of these were missense. Among these, 99 missense mutations (90%) were predicted to destabilize protein structures. Remarkably, a mutation that leads to an I300F change in the nsp2 protein and a mutation leading to D614G change in the spike protein were prevalent in SARS-CoV-2 genomic sequences, and might have influenced the epidemiological properties of the virus in Bangladesh.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Sequenciamento Completo do Genoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Bangladesh , Simulação por Computador , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Filogenia , SARS-CoV-2/classificação , Glicoproteína da Espícula de Coronavírus/genética
19.
Nat Commun ; 12(1): 460, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469029

RESUMO

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação/genética , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Guanosina Trifosfato/metabolismo , Humanos , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Fagocitose , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/isolamento & purificação , Proteínas rab1 de Ligação ao GTP/ultraestrutura
20.
Int J Mol Sci ; 22(1)2021 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-33401608

RESUMO

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor-coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis-Golgi compartment.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator 1 de Ribosilação do ADP/genética , Complexo I de Proteína do Envoltório/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
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