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1.
Anticancer Res ; 40(7): 3645-3649, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620603

RESUMO

Despite the establishment of the traditional prognostic factors for breast cancer, patients belonging to the same histological and molecular subgroup often present quite different outcomes. Recently, the introduction of gene expression profiling, assessed by RT-qPCR and microarray DNA analysis, offered a view of the whole cell gene activity and the ability to identify new transcripts that are associated with outcome. This review aimed to gather all recent trials about new breast cancer prognostic factors, focusing on the most promising one, the FGD3 gene, and to discuss the real feasibility of a molecular approach in everyday clinical practice. In conclusion, all literature concerning this subject indicated that expression of the FGD3 gene is a strong marker of good prognosis in breast cancer patients and that immunohistochemistry represents an efficient, inexpensive, reproducible evaluation method, affordable also by small Institutions.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Biomarcadores Tumorais/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Prognóstico
2.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651375

RESUMO

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Calorimetria , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica , Fosforilação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
3.
Gene ; 755: 144886, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32534055

RESUMO

Non-small cell lung cancer (NSCLC) is a common lung cancer with high mortality worldwide. Cisplatin (DDP) resistance is a huge limitation for NSCLC therapy. FGD5 antisense RNA 1 (FGD5-AS1) was recognized as a significant cancer cell regulator. However, the molecular mechanism of FGD5-AS1 in cisplatin resistance of NSCLC cells is poorly understood. FGD5-AS1 and WEE1 expression were up-regulated in DDP-resistant tumors and cells compared with DDP-sensitive ones. Interestingly, down-regulation of FGD5-AS1 or WEE1 inhibited cell proliferation, migration, invasion, autophagy and stimulated cell apoptosis in NSCLC DDP-resistant cells. What's more, restoration of WEE1 abrogated FGD5-AS1 silencing-induced suppression on cell proliferation, migration, invasion, autophagy and promotion on cell apoptosis in NSCLC DDP-resistant cells. Next, we discovered that FGD5-AS1 was able to enhance WEE1 expression by interacting with miR-140-5p. Furthermore, FGD5-AS1 silencing restrained tumor growth of cisplatin-resistant mice. Overexpression of FGD5-AS1 accelerated cell proliferation, migration, invasion and autophagy by enhancing cisplatin resistance against NSCLC cells through miR-140-5p/WEE1 axis, presenting promising biomarkers for the diagnosis of DDP-resistant NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Cisplatino/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Antissenso/metabolismo , Células A549 , Adulto , Animais , Apoptose/genética , Autofagia/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/genética , RNA Antissenso/biossíntese , RNA Antissenso/genética , RNA Longo não Codificante/genética
4.
Life Sci ; 256: 117998, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32585241

RESUMO

AIMS: Accumulating evidence elucidates the biological significance of long non-coding RNA (lncRNAs) in tumorigenesis and development. FGD5 antisense RNA 1 (FGD5-AS1) was previously revealed as an oncogene in several types of malignancies. However, the roles of FGD5-AS1 in glioblastoma (GBM) and its potential molecular mechanisms remain unclear. MATERIALS AND METHODS: The expression of FGD5-AS1, miR-129-5p, and heterogeneous nuclear ribonucleoprotein K (HNRNPK) mRNA were measured by qRT-PCR. Cell proliferation, invasion and apoptosis were determined by MTT, colony formation, transwell and flow cytometry assays. The protein levels of Ki-67, HNRNPK and Wnt signaling-associated genes were examined by western blot assay. The possible action mechanism of FGD5-AS1 was detected by bioinformatic tools, luciferase reporter, RIP and TOP/FOP Flash reporter assays. A nude mouse xenograft model was built to analyze the function of FGD5-AS1 in vivo. KEY FINDINGS: FGD5-AS1 expression was increased in GBM tumor tissues and cells. Knockdown of FGD5-AS1 inhibited cell proliferation and invasion in vitro, and slowed tumor growth in vivo. Mechanistically, FGD5-AS1 served as a sponge of miR-129-5p to relieve its suppression on HNRNPK. Moreover, down-regulation of HNRNPK repressed cell proliferation and invasion, while enhanced apoptosis. Additionally, si-FGD5-AS1-mediated suppression of cell proliferation and invasion was obviously reversed by the decrease of miR-129-5p or restoration of HNRNPK. Furthermore, FGD5-AS1 promoted cell growth and invasion by stimulating Wnt/ß-catenin signaling via regulation of miR-129-5p/HNRNPK. SIGNIFICANCE: FGD5-AS1 promoted GBM progression at least partly by regulating miR-129-5p/HNRNPK to activate Wnt/ß-catenin signaling, suggesting the potential of FGD5-AS1 as a candidate target to improve GBM therapy.


Assuntos
Glioblastoma/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , MicroRNAs/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Longo não Codificante/genética , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Genet ; 992020.
Artigo em Inglês | MEDLINE | ID: mdl-32529990

RESUMO

IQSEC2 is an X-linked gene highly expressed at the excitatory synapses where it plays a crucial role in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking and synaptic plasticity. To date, several males and females with severe to profound intellectual disability have been reported harbouring frameshift and nonsense variants in this gene, whereas a milder phenotype has been recognized in females carrying missense pathogenic variants. Here, we report two novel IQSEC2 variants in four females with psychiatric features and otherwise variable cognitive impairment. A female (case 1) with severe verbal language learning disorder and a psychotic episode (precipitated by exposure to anti-contraceptive pill) harboured a de novo pathogenic frameshift variant (c.1170dupG,p.Gln391Alafs*5), whereas the female proband of family 2, displaying severe psychomotor regression and complex psychiatric features carried a missense variant of uncertain significance (c.770G[A,p.Ser257Asn) that was maternally inherited. Skewed X-inactivation was noted in the carrier mother. The maternal aunt, affected by schizophrenia, was found to bear the same IQSEC2 variant. We discuss the variable clinical presentation of IQSEC2 spectrum disorders and the challenging genotype-phenotype correlation, including the possible role of environmental factors as triggers for decompensation. Our report highlights how psychiatric features may be the main clinical presentation in subtle IQSEC2 phenotype, suggesting that the prevalence of IQSEC2 mutations in patients with psychiatric disorders may be underestimated.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Mutação/genética , Feminino , Genes Ligados ao Cromossomo X , Estudos de Associação Genética , Humanos , Sequenciamento Completo do Exoma , Inativação do Cromossomo X
6.
PLoS Pathog ; 16(5): e1008503, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365138

RESUMO

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.


Assuntos
Aderência Bacteriana , Mucosa Intestinal/microbiologia , Infecções por Salmonella , Salmonella typhimurium , Sistemas de Secreção Tipo I/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cães , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Sistemas de Secreção Tipo I/genética
7.
PLoS Genet ; 16(5): e1008255, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392211

RESUMO

mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Animais , Células Cultivadas , Regulação para Baixo/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética
8.
PLoS Genet ; 16(4): e1008721, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339198

RESUMO

Current estimates suggest 50% of glaucoma blindness worldwide is caused by primary angle-closure glaucoma (PACG) but the causative gene is not known. We used genetic linkage and whole genome sequencing to identify Spermatogenesis Associated Protein 13, SPATA13 (NM_001166271; NP_001159743, SPATA13 isoform I), also known as ASEF2 (Adenomatous polyposis coli-stimulated guanine nucleotide exchange factor 2), as the causal gene for PACG in a large seven-generation white British family showing variable expression and incomplete penetrance. The 9 bp deletion, c.1432_1440del; p.478_480del was present in all affected individuals with angle-closure disease. We show ubiquitous expression of this transcript in cell lines derived from human tissues and in iris, retina, retinal pigment and ciliary epithelia, cornea and lens. We also identified eight additional mutations in SPATA13 in a cohort of 189 unrelated PACS/PAC/PACG samples. This gene encodes a 1277 residue protein which localises to the nucleus with partial co-localisation with nuclear speckles. In cells undergoing mitosis SPATA13 isoform I becomes part of the kinetochore complex co-localising with two kinetochore markers, polo like kinase 1 (PLK-1) and centrosome-associated protein E (CENP-E). The 9 bp deletion reported in this study increases the RAC1-dependent guanine nucleotide exchange factors (GEF) activity. The increase in GEF activity was also observed in three other variants identified in this study. Taken together, our data suggest that SPATA13 is involved in the regulation of mitosis and the mutations dysregulate GEF activity affecting homeostasis in tissues where it is highly expressed, influencing PACG pathogenesis.


Assuntos
Glaucoma de Ângulo Aberto/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Mutação , Adolescente , Adulto , Idoso , Divisão Celular , Núcleo Celular/metabolismo , Olho/metabolismo , Feminino , Glaucoma de Ângulo Aberto/patologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Cinetocoros/metabolismo , Masculino , Pessoa de Meia-Idade , Linhagem , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico
10.
PLoS One ; 15(3): e0229953, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32168507

RESUMO

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by "in silico" reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.


Assuntos
Biologia Computacional , Epilepsia Reflexa/epidemiologia , Epilepsia/epidemiologia , Convulsões/epidemiologia , Estimulação Acústica , Animais , Cricetinae , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/patologia , Epilepsia Reflexa/tratamento farmacológico , Epilepsia Reflexa/genética , Epilepsia Reflexa/patologia , Feminino , Regulação da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Proteína 3 Homóloga a MutS/genética , Mutação/genética , Convulsões/tratamento farmacológico , Convulsões/genética , Convulsões/patologia , Sequenciamento Completo do Exoma
11.
Yakugaku Zasshi ; 140(3): 391-393, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32115558

RESUMO

Recently, aging is becoming an important social problem in many developed countries including Japan. It is socially and universally important to unveil the impact of aging and extend healthy life expectancy. Here we show our recent finding that dedicator of cytokinesis 11 (DOCK11, also known as Zizimin2) may be involved in immunosenescence of B cells. DOCK11 was identified as a guanine nucleotide exchange factor for a small GTPase called cell division cycle 42. Expression of DOCK11 is restricted to lymphoid tissues, and becomes downregulated with age. Thus we examined the involvement of DOCK11 in immunosenescence of B-1a B cells as an example. B-1a cells are the main source of antibodies at steady state, and function as the first line of defense against infection. Although DOCK11 was expressed by B-1a cells, the expression levels declined with age. Furthermore, production of anti-pneumococcal immunoglobulin M antibodies was suppressed in aged mice, and was recovered by adoptive transfer with B-1a cells in a DOCK11-dependent manner. Thus DOCK11 may be involved in immunosenescence of B-1a cells.


Assuntos
Envelhecimento/imunologia , Imunossenescência , Animais , Linfócitos B/imunologia , Citocinese/imunologia , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imunoglobulina M , Camundongos , Estado Nutricional , Streptococcus pneumoniae/imunologia
12.
Proc Natl Acad Sci U S A ; 117(7): 3627-3636, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32019878

RESUMO

The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.


Assuntos
Neoplasias da Mama/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Prenilação de Proteína , Processamento de RNA
13.
Am J Hum Genet ; 106(3): 338-355, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109419

RESUMO

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Estudos de Coortes , Feminino , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Masculino , Fenótipo , Proteínas Serina-Treonina Quinases/química , Homologia de Sequência de Aminoácidos
14.
Nat Commun ; 11(1): 180, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924779

RESUMO

Macropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pinocitose/fisiologia , Linfócitos T/fisiologia , Aminoácidos , Animais , Linfócitos T CD4-Positivos/fisiologia , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Linfócitos T/citologia
15.
BMC Med Genet ; 21(1): 14, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941453

RESUMO

BACKGROUND: The DENND1A gene is one of the most important sites associated with polycystic ovary syndrome (PCOS). We attempted to analyze the correlation between five single nucleotide polymorphisms (SNPs) in the DENND1A gene and the development of PCOS. METHODS: A total of 346 PCOS patients and 225 normal ovulatory women were involved in the case-control study. Clinical variables and hormones were recorded. According to the Hap Map database, five tagging SNPs (rs2479106, rs2768819, rs2670139, rs2536951 and rs2479102) in the DENND1A gene were identified. The TaqMan probe and the PCR-RFLP (restriction fragment length polymorphism) methods were used for revealing these genotypes. TaqMan Genotype Software was used to analyze the alleles of the five SNPs. RESULTS: Linkage disequilibrium and the gene frequency analysis demonstrated that the CCGGG haplotype might increase the risk of PCOS (P = 0.038, OR = 1.89, 95% CI = 1.027-3.481). Significant differences were found in genotypic and allelic distributions at the rs2536951 and rs2479102 loci between PCOS women and controls (P <  0.001). The LH levels and LH/FSH ratios were higher in PCOS patients than in the control group. A detailed analysis revealed that for the rs2479106 locus, these two values were significantly different in the control subjects who had AA, AG and GG genotypes (P = 0.013 and P = 0.007, respectively), and for the rs2468819 locus, these two values were significantly different among the PCOS patients with AA, AG and GG genotypes (P = 0.013 and 0.002, respectively). CONCLUSIONS: The tagging SNPs rs2479106 and rs2468819 in the DENND1A gene are associated with PCOS in the Chinese population, whereas rs2670139, rs2536951 and rs2479102 are not correlated with PCOS in the same population.


Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina/genética , Síndrome do Ovário Policístico/genética , Adulto , Alelos , China/epidemiologia , Feminino , Genótipo , Haplótipos/genética , Humanos , Síndrome do Ovário Policístico/epidemiologia , Síndrome do Ovário Policístico/patologia , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
16.
Genes (Basel) ; 11(1)2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906484

RESUMO

X-linked intellectual disability (XLID) is known to contribute up to 10% of intellectual disability (ID) in males and could explain the increased ratio of affected males observed in patients with ID. Over the past decade, next-generation sequencing has clearly stimulated the gene discovery process and has become part of the diagnostic procedure. We have performed targeted next-generation sequencing of 82 XLID genes on 61 non-related male patients with suggestive non-syndromic XLID. These patients were initially referred to the molecular genetics laboratory to exclude Fragile X Syndrome. The cohort includes 47 male patients with suggestive X-linked family history of ID meaning that they had half-brothers or maternal cousins or uncles affected; and 14 male patients with ID and affected brothers whose mothers show skewed X-inactivation. Sequencing data analysis identified 17 candidate variants in 16 patients. Seven families could be re-contacted and variant segregation analysis of the respective eight candidate variants was performed: HUWE1, IQSEC2, MAOA, MED12, PHF8, SLC6A8, SLC9A6, and SYN1. Our results show the utility of targeted next-generation sequencing in unravelling the genetic origin of XLID, especially in retrospective cases. Variant segregation and additional studies like RNA sequencing and biochemical assays also helped in re-evaluating and further classifying the genetic variants found.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Retardo Mental Ligado ao Cromossomo X/diagnóstico , Retardo Mental Ligado ao Cromossomo X/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Exoma/genética , Síndrome do Cromossomo X Frágil , Genes Ligados ao Cromossomo X/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Complexo Mediador/genética , Monoaminoxidase/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Linhagem , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Estudos Retrospectivos , Trocadores de Sódio-Hidrogênio/genética , Fatores de Transcrição , Sequenciamento Completo do Exoma/métodos
17.
Nat Med ; 26(1): 98-109, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932796

RESUMO

Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.


Assuntos
Anormalidades Múltiplas/patologia , Bancos de Espécimes Biológicos , Fatores de Troca do Nucleotídeo Guanina/genética , Fenômica , Proteínas de Peixe-Zebra/genética , Animais , Comportamento Animal , Condrócitos/patologia , Condrócitos/ultraestrutura , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Modelos Biológicos , Sistema Musculoesquelético/patologia , Osteogênese , Fenótipo , Pró-Colágeno/metabolismo , Transporte Proteico , Via Secretória , Síndrome , Peixe-Zebra
18.
FASEB J ; 34(2): 3267-3288, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908036

RESUMO

TIAM2S, the short form of human T-cell lymphoma invasion and metastasis 2, can have oncogenic effects when aberrantly expressed in the liver or lungs. However, it is also abundant in healthy, non-neoplastic brain tissue, in which its primary function is still unknown. Here, we examined the neurobiological and behavioral significance of human TIAM2S using the human brain protein panels, a human NT2/D1-derived neuronal cell line model (NT2/N), and transgenic mice that overexpress human TIAM2S (TIAM2S-TG). Our data reveal that TIAM2S exists primarily in neurons of the restricted brain areas around the limbic system and in well-differentiated NT2/N cells. Functional studies revealed that TIAM2S has no guanine nucleotide exchange factor (GEF) activity and is mainly located in the nucleus. Furthermore, whole-transcriptome and enrichment analysis with total RNA sequencing revealed that TIAM2S-knockdown (TIAM2S-KD) was strongly associated with the cellular processes of the brain structural development and differentiation, serotonin-related signaling, and the diseases markers representing neurobehavioral developmental disorders. Moreover, TIAM2S-KD cells display decreased neurite outgrowth and reduced serotonin levels. Moreover, TIAM2S overexpressing TG mice show increased number and length of serotonergic fibers at early postnatal stage, results in higher serotonin levels at both the serum and brain regions, and higher neuroplasticity and hyperlocomotion in latter adulthood. Taken together, our results illustrate the non-oncogenic functions of human TIAM2S and demonstrate that TIAM2S is a novel regulator of serotonin level, brain neuroplasticity, and locomotion behavior.


Assuntos
Encéfalo/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Locomoção , Serotonina/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Crescimento Neuronal , Plasticidade Neuronal
19.
Sci Adv ; 6(1): eaay3566, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911948

RESUMO

In this study, we investigated the roles of Epac1 in pathological angiogenesis and its potential as a novel therapeutic target for the treatment of vasoproliferative diseases. Genetic deletion of Epac1 ameliorated pathological angiogenesis in mouse models of oxygen-induced retinopathy (OIR) and carotid artery ligation. Moreover, genetic deletion or pharmacological inhibition of Epac1 suppressed microvessel sprouting from ex vivo aortic ring explants. Mechanistic studies revealed that Epac1 acted as a previously unidentified inhibitor of the γ-secretase/Notch signaling pathway via interacting with γ-secretase and regulating its intracellular trafficking while enhancing vascular endothelial growth factor signaling to promote pathological angiogenesis. Pharmacological administration of an Epac-specific inhibitor suppressed OIR-induced neovascularization in wild-type mice, recapitulating the phenotype of genetic Epac1 knockout. Our results demonstrate that Epac1 signaling is critical for the progression of pathological angiogenesis but not for physiological angiogenesis and that the newly developed Epac-specific inhibitors are effective in combating proliferative retinopathy.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Neovascularização Patológica/genética , Neovascularização Retiniana/genética , Animais , Movimento Celular/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Neovascularização Patológica/patologia , Receptores Notch/genética , Neovascularização Retiniana/patologia , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genética
20.
Pediatr Blood Cancer ; 67(2): e28078, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31724816

RESUMO

Defects of platelet intracellular signaling can result in severe platelet dysfunction. Several mutations in each of the linked genes FERMT3 and RASGRP2 on chromosome 11 causing a Glanzmann-like bleeding phenotype have been identified so far. We report on novel variants in two unrelated pediatric patients with severe bleeding diathesis-one with leukocyte adhesion deficiency type III due to a homozygous frameshift in FERMT3 and the other with homozygous variants in both, FERMT3 and RASGRP2. We focus on the challenging genetic and functional variant assessment and aim to accentuate the risk of obtaining misleading results due to the phenomenon of genetic linkage.


Assuntos
Transtornos Plaquetários/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Transtornos Hemorrágicos/patologia , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Adolescente , Transtornos Plaquetários/genética , Criança , Feminino , Ligação Genética , Transtornos Hemorrágicos/genética , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo , Prognóstico
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