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1.
PLoS One ; 15(12): e0243901, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33378376

RESUMO

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2ß) functions as a partial agonist at ErbB4. NRG2ß/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.


Assuntos
Proliferação de Células/genética , Melanoma/genética , Fatores de Crescimento Neural/genética , Receptor ErbB-4/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Mutação com Ganho de Função/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Fosforilação/genética , Receptor ErbB-4/agonistas , Receptor ErbB-4/antagonistas & inibidores , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
2.
PLoS Genet ; 16(8): e1008975, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750056

RESUMO

The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Receptores de Adiponectina/genética , Alelos , Animais , Caenorhabditis elegans/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Mutação com Ganho de Função/genética , Células HEK293 , Humanos , Fluidez de Membrana/genética , Fenótipo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo
3.
Am J Hum Genet ; 107(2): 311-324, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32738225

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.


Assuntos
Aspartato-tRNA Ligase/genética , Mutação com Ganho de Função/genética , Mutação com Perda de Função/genética , Transtornos do Neurodesenvolvimento/genética , Aminoacil-RNA de Transferência/genética , Alelos , Aminoacil-tRNA Sintetases/genética , Linhagem Celular , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Linhagem , RNA de Transferência/genética , Células-Tronco/fisiologia
4.
Bioessays ; 42(10): e2000091, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32786014

RESUMO

Despite claims from prominent scientists that SARS-CoV-2 indubitably emerged naturally, the etiology of this novel coronavirus remains a pressing and open question: Without knowing the true nature of a disease, it is impossible for clinicians to appropriately shape their care, for policy-makers to correctly gauge the nature and extent of the threat, and for the public to appropriately modify their behavior. Unless the intermediate host necessary for completing a natural zoonotic jump is identified, the dual-use gain-of-function research practice of viral serial passage should be considered a viable route by which the novel coronavirus arose. The practice of serial passage mimics a natural zoonotic jump, and offers explanations for SARS-CoV-2's distinctive spike-protein region and its unexpectedly high affinity for angiotensin converting enzyme (ACE2), as well as the notable polybasic furin cleavage site within it. Additional molecular clues raise further questions, all of which warrant full investigation into the novel coronavirus's origins and a re-examination of the risks and rewards of dual-use gain-of-function research.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/etiologia , Infecções por Coronavirus/transmissão , Pneumonia Viral/etiologia , Pneumonia Viral/transmissão , Zoonoses/transmissão , Sequência de Aminoácidos , Animais , Betacoronavirus/crescimento & desenvolvimento , Mutação com Ganho de Função/genética , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Zoonoses/virologia
5.
Proc Natl Acad Sci U S A ; 117(31): 18737-18743, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675245

RESUMO

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral ß-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the ß-barrel assembly machine (BAM), which is composed of one OMP-BamA-and four lipoproteins-BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamA E470K , that bypasses the requirement for BamD. Although bamD::kan bamA E470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.


Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Externa Bacteriana , Proteínas de Escherichia coli , Mutação com Ganho de Função/genética , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
6.
Nature ; 581(7809): 459-464, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461653

RESUMO

Naturally occurring human genetic variants that are predicted to inactivate protein-coding genes provide an in vivo model of human gene inactivation that complements knockout studies in cells and model organisms. Here we report three key findings regarding the assessment of candidate drug targets using human loss-of-function variants. First, even essential genes, in which loss-of-function variants are not tolerated, can be highly successful as targets of inhibitory drugs. Second, in most genes, loss-of-function variants are sufficiently rare that genotype-based ascertainment of homozygous or compound heterozygous 'knockout' humans will await sample sizes that are approximately 1,000 times those presently available, unless recruitment focuses on consanguineous individuals. Third, automated variant annotation and filtering are powerful, but manual curation remains crucial for removing artefacts, and is a prerequisite for recall-by-genotype efforts. Our results provide a roadmap for human knockout studies and should guide the interpretation of loss-of-function variants in drug development.


Assuntos
Genes Essenciais/efeitos dos fármacos , Genes Essenciais/genética , Mutação com Perda de Função/genética , Terapia de Alvo Molecular , Artefatos , Automação , Consanguinidade , Éxons/genética , Mutação com Ganho de Função/genética , Frequência do Gene , Técnicas de Silenciamento de Genes , Heterozigoto , Homozigoto , Humanos , Proteína Huntingtina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doenças Neurodegenerativas/genética , Proteínas Priônicas/genética , Reprodutibilidade dos Testes , Tamanho da Amostra , Proteínas tau/genética
7.
Nat Med ; 26(6): 869-877, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32461697

RESUMO

Human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants) provide natural in vivo models of human gene inactivation and can be valuable indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes1,2. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson's disease3,4, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. While preclinical studies in model organisms have raised some on-target toxicity concerns5-8, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here, we systematically analyze pLoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)9, 49,960 exome-sequenced individuals from the UK Biobank and over 4 million participants in the 23andMe genotyped dataset. After stringent variant curation, we identify 1,455 individuals with high-confidence pLoF variants in LRRK2. Experimental validation of three variants, combined with previous work10, confirmed reduced protein levels in 82.5% of our cohort. We show that heterozygous pLoF variants in LRRK2 reduce LRRK2 protein levels but that these are not strongly associated with any specific phenotype or disease state. Our results demonstrate the value of large-scale genomic databases and phenotyping of human loss-of-function carriers for target validation in drug discovery.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação com Perda de Função/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Bancos de Espécimes Biológicos , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Feminino , Mutação com Ganho de Função/genética , Heterozigoto , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Longevidade/genética , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Fenótipo
8.
Nat Commun ; 11(1): 2086, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350249

RESUMO

Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.


Assuntos
Códon/genética , Mutação/genética , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Epigênese Genética/efeitos dos fármacos , Mutação com Ganho de Função/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos SCID , Modelos Biológicos , Proteínas Mutantes/metabolismo , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Motivos de Nucleotídeos/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/genética , Sulfonamidas/farmacologia , Topotecan/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Int J Hematol ; 112(2): 258-262, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32180118

RESUMO

Signal transducer and activator of transcription 1 gain-of-function (STAT1 GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC). We report the effect of oral ruxolitinib, an inhibitor of Janus kinase (JAK) family tyrosine kinases, on the clinical and immune status of a 3-year-old male with steroid-dependent severe autoimmunity due to a STAT1 GOF T385M mutation. The patient's susceptibility to infection improved with antimicrobial prophylaxis and immunoglobulin replacement therapy, but he continued to exhibit severely disabling symptoms of autoimmunity. More than one-third of patients with STAT1 GOF mutations present with autoimmune manifestations, and this patient's mutation was reported to cause CMC with autoimmunity. We analyzed the interleukin (IL)-17A and IFN-γ levels and immunophenotype by flow cytometry before and during treatment with ruxolitinib. The peripheral IL-17A level did not increase, but the IFN-γ level decreased after 4 months of therapy. The STAT1 phosphorylation level decreased significantly upon stimulation of patient cells with IFN-γ. Clinically, cytomegalovirus reactivation occurred, but was controlled. No other adverse effect was noted. We report the potential of JAK1/2 inhibition with ruxolitinib for both CMC and steroid-dependent autoimmunity. However, long-term administration is necessary, as the effect is not sustained after treatment is discontinued.


Assuntos
Candidíase Mucocutânea Crônica/tratamento farmacológico , Candidíase Mucocutânea Crônica/imunologia , Mutação com Ganho de Função/genética , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Fator de Transcrição STAT1/genética , Autoimunidade , Candidíase Mucocutânea Crônica/genética , Citocinas/metabolismo , Humanos , Lactente , Janus Quinase 1/antagonistas & inibidores , Masculino , Fosforilação , Fator de Transcrição STAT1/metabolismo , Índice de Gravidade de Doença
10.
Biochem Biophys Res Commun ; 523(4): 841-846, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31954514

RESUMO

Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD.


Assuntos
Diferenciação Celular , Polpa Dentária/patologia , Nanismo/genética , Mutação com Ganho de Função/genética , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocondrodisplasias/genética , Células-Tronco/metabolismo , Canais de Cátion TRPV/genética , Adolescente , Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais
11.
Am J Cardiol ; 125(6): 880-886, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31932084

RESUMO

Autosomal dominant hypercholesterolemia results from mutations affecting the low-density lipoprotein receptor pathway, including proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutations (GoFm) and apolipoprotein B (APOB) loss-of-function mutations (LoFm). This study examined the long-term efficacy and safety of alirocumab in patients with PCSK9 GoFm and APOB LoFm who participated in the open-label extension to a Phase 2 double-blind study (NCT01604824). Of the 23 patients who completed the 14-week double-blind period and 8-week follow-up, 21 opted to continue in the open-label extension (PCSK9 GoFm, n = 15; APOB LoFm, n = 6). Patients received alirocumab 150 mg every 2 weeks from week 32 up to 3 years for PCSK9 GoFm and 2 years for APOB LoFm. Mean duration of alirocumab exposure was 129 weeks (median: 144 weeks). After initiation of alirocumab treatment, low-density lipoprotein cholesterol (LDL-C) decreased in both groups. At week 80, mean percent reduction in LDL-C from baseline was 58.0% and 47.1% for PCSK9 GoFm and APOB LoFm groups, respectively. Treatment-emergent adverse events were reported in 19 patients (90.5%); no patients discontinued treatment due to treatment-emergent adverse events. In patients with autosomal dominant hypercholesterolemia and elevated LDL-C levels despite receiving maximally tolerated lipid-lowering therapies, alirocumab 150 mg every 2 weeks resulted in clinically meaningful reductions in LDL-C, sustained through to 3 years and 2 years for patients with PCSK9 GoFm and APOB LoFm, respectively. Alirocumab was generally well tolerated with no unexpected safety concerns.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Apolipoproteína B-100/genética , Mutação com Ganho de Função/genética , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/genética , Mutação com Perda de Função/genética , Pró-Proteína Convertase 9/genética , Adulto , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Aberrações Cromossômicas , Método Duplo-Cego , Feminino , Genes Dominantes , Genótipo , Humanos , Hiperlipoproteinemia Tipo II/sangue , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
12.
Sci Adv ; 6(2): eaax9605, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31934629

RESUMO

Overnutrition results in adiposity and chronic inflammation with expansion of white adipose tissue (WAT). However, genetic factors controlling fat mass and adiposity remain largely undetermined. We applied whole-exome sequencing in young obese subjects and identified rare gain-of-function mutations in CTNNB1/ß-catenin associated with increased obesity risk. Specific ablation of ß-catenin in mature adipocytes attenuated high-fat diet-induced obesity and reduced sWAT mass expansion with less proliferated Pdgfrα+ preadipocytes and less mature adipocytes. Mechanistically, ß-catenin regulated the transcription of serum amyloid A3 (Saa3), an adipocyte-derived chemokine, through ß-catenin-TCF (T-Cell-Specific Transcription Factor) complex in mature adipocytes, and Saa3 activated macrophages to secrete several factors, including Pdgf-aa, which further promoted the proliferation of preadipocytes, suggesting that ß-catenin/Saa3/macrophages may mediate mature adipocyte-preadipocyte cross-talk and fat expansion in sWAT. The identification of ß-catenin as a key regulator in fat expansion and human adiposity provides the basis for developing drugs targeting Wnt/ß-catenin pathway to combat obesity.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Adiposidade , Diferenciação Celular , beta Catenina/genética , Células 3T3-L1 , Tecido Adiposo Branco/metabolismo , Animais , Sítios de Ligação , Proliferação de Células , Dieta Hiperlipídica , Mutação com Ganho de Função/genética , Homeostase , Humanos , Ativação de Macrófagos , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/patologia , Tamanho do Órgão , Células RAW 264.7 , Proteína Amiloide A Sérica/metabolismo , Fatores de Transcrição TCF/metabolismo , Transcrição Genética , Via de Sinalização Wnt
13.
Mol Cells ; 43(1): 34-47, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31896234

RESUMO

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbß, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.


Assuntos
Reabsorção Óssea/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , Animais , Remodelação Óssea , Reabsorção Óssea/genética , Diferenciação Celular , Células Cultivadas , Relógios Circadianos , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Oncogene ; 39(2): 334-355, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31477837

RESUMO

The Hippo pathway has emerged as a key signaling pathway that regulates various biological functions. Dysregulation of the Hippo pathway has been implicated in a broad range of human cancer types. While a number of stimuli affecting the Hippo pathway have been reported, its upstream kinase and extracellular regulators remain largely unknown. Here we performed the first comprehensive gain-of-functional screen for receptor tyrosine kinases (RTKs) regulating the Hippo pathway using an RTK overexpression library and a Hippo signaling activity biosensor. Surprisingly, we found that the majority of RTKs could regulate the Hippo signaling activity. We further characterized several of these novel relationships [TAM family members (TYRO3, AXL, METRK), RET, and FGFR family members (FGFR1 and FGFR2)] and found that the Hippo effectors YAP/TAZ are central mediators of the tumorigenic phenotypes (e.g., increased cell proliferation, transformation, increased cell motility, and angiogenesis) induced by these RTKs and their extracellular ligands (Gas6, GDNF, and FGF) through either PI3K or MAPK signaling pathway. Significantly, we identify FGFR, RET, and MERTK as the first RTKs that can directly interact with and phosphorylate YAP/TAZ at multiple tyrosine residues independent of upstream Hippo signaling, thereby activating their functions in tumorigenesis. In conclusion, we have identified several novel kinases and extracellular stimuli regulating the Hippo pathway. Our findings also highlight the pivotal role of the Hippo pathway in mediating Gas6/GDNF/FGF-TAM/RET/FGFR-MAPK/PI3K signaling during tumorigenesis and provide a compelling rationale for targeting YAP/TAZ in RTK-driven cancers.


Assuntos
Carcinogênese/genética , Mutação com Ganho de Função/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Técnicas Biossensoriais/métodos , Proliferação de Células/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/genética , Fosforilação , Transdução de Sinais/genética , Fatores de Transcrição/genética
15.
Clin Immunol ; 210: 108269, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31683054

RESUMO

Genetic studies have led to identification of an increasing number of monogenic primary immunodeficiency disorders. Monoallelic pathogenic gain-of-function (GOF) variants in NFKBIA, the gene encoding IκBα, result in an immunodeficiency disorder, typically accompanied by anhidrotic ectodermal dysplasia (EDA). So far, 14 patients with immunodeficiency due to NFKBIA GOF mutations have been reported. In this study we report three patients from the same family with immunodeficiency, presenting with recurrent respiratory tract infections, bronchiectasis and viral skin conditions due to a novel pathogenic NFKBIA variant (c.106 T > G, p.Ser36Ala), which results in reduced IκBα degradation. Immunological investigations revealed inadequate antibody responses against vaccine antigens, despite hypergammaglobulinemia. Interestingly, none of the studied patients displayed features of EDA. Therefore, missense NFKBIA variants substituting serine 36 of IκBα, differ from the rest of pathogenic GOF NFKBIA variants in that they cause combined immunodeficiency, even in the absence of EDA.


Assuntos
Mutação com Ganho de Função/genética , Síndromes de Imunodeficiência/diagnóstico , Leucócitos Mononucleares/imunologia , Meningites Bacterianas/diagnóstico , Inibidor de NF-kappaB alfa/genética , Neisseria meningitidis/fisiologia , Papillomaviridae/fisiologia , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/fisiologia , Viroses/imunologia , Adulto , Artrite Juvenil , Azitromicina/uso terapêutico , Bronquiectasia , Proliferação de Células , Células Cultivadas , Criança , Displasia Ectodérmica , Gentamicinas/uso terapêutico , Humanos , Síndromes de Imunodeficiência/genética , Masculino , Meningites Bacterianas/tratamento farmacológico , Linhagem , Infecções por Pseudomonas/tratamento farmacológico , Viroses/diagnóstico , Verrugas , Adulto Jovem
16.
Clin Immunol ; 210: 108316, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31770611

RESUMO

Germline STAT3 gain-of-function (GOF) mutations have been linked to poly-autoimmunity and lymphoproliferation with variable expressivity and incomplete penetrance. Here we studied the impact of 17 different STAT3 GOF mutations on the canonical STAT3 signaling pathway and correlated the molecular results with clinical manifestations. The mutations clustered in three groups. Group 1 mutants showed altered STAT3 phosphorylation kinetics and strong basal transcriptional activity. They were associated with the highest penetrance of lymphoproliferation and autoimmunity. Group 2 mutants showed a strongly inducible transcriptional reporter activity and were clinically less penetrant. Group 3 mutants were mostly located in the DNA binding domain and showed the strongest DNA binding affinity despite a poor transcriptional reporter response. Thus, the GOF effect of STAT3 mutations is determined by a heterogeneous response pattern at the molecular level. The correlation of response pattern and clinical penetrance indicates a significant contribution of mutation-determined effects on disease manifestations.


Assuntos
Mutação com Ganho de Função/genética , Linfócitos/imunologia , Fator de Transcrição STAT3/genética , Autoimunidade/genética , Proliferação de Células , Feminino , Humanos , Masculino , Família Multigênica , Penetrância , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética
17.
Plant Cell Rep ; 39(2): 259-271, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31820142

RESUMO

KEY MESSAGE: Auxin can alter the fertility of bin2-1 plants and depends on the expression of SHY2. Brassinosteroids (BRs) play important roles in plant growth and developmental processes. By systematically evaluating the phenotypes of BR biosynthesis and BR signaling mutants, researchers have reported that BRs positively regulate floral development. In this study, we found that brassinosteroid-insensitive 2 (bin2-1) and short-hypocotyl 2 (shy2-2) mutants exhibited significantly reduced fertility. These mutants had short inflorescences, decreased floral organ length (short petals, stamens, carpels, and stigmas), and short siliques. Exogenous auxin applications could partially rescue the shortened length of the floral organs and siliques of the bin2-1 mutants. Additional experiments revealed that a lack of SHY2 activity increased the fertility of the bin2-1 mutants. A search for downstream affected genes revealed that auxin influences the expression of ARFs and PINs in the bin2-1 mutants, suggesting that auxin plays a major role in the regulation of bin2-1 plant fertility. Thus, BIN2 plays a role in fertility by affecting auxin levels, mainly by altering the expression of SHY2.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Mutação com Ganho de Função/genética , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Proteínas Nucleares/genética , Fenótipo , Desenvolvimento Vegetal , Proteínas Quinases/genética , Transdução de Sinais , Transcriptoma
18.
J Clin Endocrinol Metab ; 105(3)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31820785

RESUMO

CONTEXT: Autosomal dominant hypocalcemia types 1 and 2 (ADH1 and ADH2) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and its signaling partner, the G-protein subunit α 11 (Gα 11), respectively. More than 70 different gain-of-function CaSR mutations, but only 6 different gain-of-function Gα 11 mutations are reported to date. METHODS: We ascertained 2 additional ADH families and investigated them for CaSR and Gα 11 mutations. The effects of identified variants on CaSR signaling were evaluated by transiently transfecting wild-type (WT) and variant expression constructs into HEK293 cells stably expressing CaSR (HEK-CaSR), and measuring intracellular calcium (Ca2+i) and MAPK responses following stimulation with extracellular calcium (Ca2+e). RESULTS: CaSR variants were not found, but 2 novel heterozygous germline Gα 11 variants, p.Gly66Ser and p.Arg149His, were identified. Homology modeling of these revealed that the Gly66 and Arg149 residues are located at the interface between the Gα 11 helical and GTPase domains, which is involved in guanine nucleotide binding, and this is the site of 3 other reported ADH2 mutations. The Ca2+i and MAPK responses of cells expressing the variant Ser66 or His149 Gα 11 proteins were similar to WT cells at low Ca2+e, but significantly increased in a dose-dependent manner following Ca2+e stimulation, thereby indicating that the p.Gly66Ser and p.Arg149His variants represent pathogenic gain-of-function Gα 11 mutations. Treatment of Ser66- and His149-Gα 11 expressing cells with the CaSR negative allosteric modulator NPS 2143 normalized Ca2+i and MAPK responses. CONCLUSION: Two novel ADH2-causing mutations that highlight the Gα 11 interdomain interface as a hotspot for gain-of-function Gα 11 mutations have been identified.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Mutação com Ganho de Função/genética , Hipercalciúria/genética , Hipocalcemia/genética , Hipoparatireoidismo/congênito , Receptores de Detecção de Cálcio/genética , Adulto , Criança , Feminino , Células HEK293 , Humanos , Hipoparatireoidismo/genética , Masculino , Linhagem
19.
Brain ; 143(2): 441-451, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31855252

RESUMO

Heterozygous mutations in the STXBP1 gene encoding the presynaptic protein MUNC18-1 cause STXBP1 encephalopathy, characterized by developmental delay, intellectual disability and epilepsy. Impaired mutant protein stability leading to reduced synaptic transmission is considered the main underlying pathogenetic mechanism. Here, we report the first two cases carrying a homozygous STXBP1 mutation, where their heterozygous siblings and mother are asymptomatic. Both cases were diagnosed with Lennox-Gastaut syndrome. In Munc18-1 null mouse neurons, protein stability of the disease variant (L446F) is less dramatically affected than previously observed for heterozygous disease mutants. Neurons expressing Munc18L446F showed minor changes in morphology and synapse density. However, patch clamp recordings demonstrated that L446F causes a 2-fold increase in evoked synaptic transmission. Conversely, paired pulse plasticity was reduced and recovery after stimulus trains also. Spontaneous release frequency and amplitude, the readily releasable vesicle pool and the kinetics of short-term plasticity were all normal. Hence, the homozygous L446F mutation causes a gain-of-function phenotype regarding release probability and synaptic transmission while having less impact on protein levels than previously reported (heterozygous) mutations. These data show that STXBP1 mutations produce divergent cellular effects, resulting in different clinical features, while sharing the overarching encephalopathic phenotype (developmental delay, intellectual disability and epilepsy).


Assuntos
Encefalopatias/genética , Mutação com Ganho de Função/genética , Proteínas Munc18/genética , Transmissão Sináptica/genética , Animais , Epilepsia/genética , Epilepsia/fisiopatologia , Deficiência Intelectual/genética , Camundongos Knockout
20.
Clin Immunol ; 210: 108311, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31760094

RESUMO

Activated PI3Kδ syndrome (APDS) Type I results from gain-of-function mutations in PIK3CD, which encodes the p110δ subunit of PI3Kδ. Abnormal actin dynamics have been hypothesized to contribute to the lymphopenia associated with this disease but have not been studied in patients with APDS. We report a patient with APDS who had widespread necrotic skin lesions that were responsive specifically to immunosuppressive therapy. EBV-transformed lymphoblastoid cells (EBV-LCLs) from patients with APDS exhibit increased polymerized actin and increased apoptosis, suggesting a contribution of impaired actin dynamics to this disease.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Pele/patologia , Citoesqueleto de Actina/genética , Apoptose , Células Cultivadas , Pré-Escolar , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Mutação com Ganho de Função/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfopenia , Necrose , Doenças da Imunodeficiência Primária/genética
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