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1.
Am J Respir Cell Mol Biol ; 58(6): 706-716, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29232161

RESUMO

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.


Assuntos
Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Adulto , Idoso , Feminino , Frequência do Gene , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Estabilidade Proteica , Proteólise , alfa 1-Antitripsina/química
2.
EMBO Mol Med ; 14(3): e14552, 2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35174975

RESUMO

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.


Assuntos
Antineoplásicos , Neoplasias Encefálicas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Barreira Hematoencefálica , Neoplasias Encefálicas/tratamento farmacológico , Camundongos , Recidiva Local de Neoplasia , Proteômica
3.
Nat Med ; 28(4): 752-765, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35411077

RESUMO

Whole-brain radiotherapy (WBRT) is the treatment backbone for many patients with brain metastasis; however, its efficacy in preventing disease progression and the associated toxicity have questioned the clinical impact of this approach and emphasized the need for alternative treatments. Given the limited therapeutic options available for these patients and the poor understanding of the molecular mechanisms underlying the resistance of metastatic lesions to WBRT, we sought to uncover actionable targets and biomarkers that could help to refine patient selection. Through an unbiased analysis of experimental in vivo models of brain metastasis resistant to WBRT, we identified activation of the S100A9-RAGE-NF-κB-JunB pathway in brain metastases as a potential mediator of resistance in this organ. Targeting this pathway genetically or pharmacologically was sufficient to revert the WBRT resistance and increase therapeutic benefits in vivo at lower doses of radiation. In patients with primary melanoma, lung or breast adenocarcinoma developing brain metastasis, endogenous S100A9 levels in brain lesions correlated with clinical response to WBRT and underscored the potential of S100A9 levels in the blood as a noninvasive biomarker. Collectively, we provide a molecular framework to personalize WBRT and improve its efficacy through combination with a radiosensitizer that balances therapeutic benefit and toxicity.


Assuntos
Neoplasias Encefálicas , Melanoma , Neoplasias Encefálicas/secundário , Irradiação Craniana , Humanos , Melanoma/radioterapia
4.
Mol Neurobiol ; 58(12): 6153-6169, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34458961

RESUMO

The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and ßIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.


Assuntos
Segmento Inicial do Axônio/metabolismo , Citoesqueleto/metabolismo , Forminas/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Camundongos , Microtúbulos/metabolismo
5.
J Clin Virol ; 65: 62-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25766991

RESUMO

BACKGROUND: Toll-like receptor-3 (TLR3) is a cellular receptor that may recognize double-stranded RNA (dsRNA) from viruses, resulting in production of proinflammatory cytokines and interferons, which are important for the adaptive immune response. OBJECTIVES: To analyze the association between Toll-like receptor-3 (TLR3) polymorphisms (rs3775291 and rs13126816) and virologic response to pegylated interferon-alpha plus ribavirin (pegIFNα/RBV) therapy in HIV/HCV coinfected patients. STUDY DESIGN: We performed a retrospective study in 321 naïve patients treated with pegIFNα/RBV. Genotyping was performed by using the GoldenGate(®) assay with VeraCode(®). The outcome variables were early virologic response (EVR) and sustained virologic response (SVR). RESULTS: In a multivariate analysis, rs3775291 A allele decreased the likelihood of achieving EVR (aOR = 0.20; p = 0.018) and SVR (aOR = 0.38; p = 0.024). Regarding rs13126816, the percentage of EVR decreased with each minor A allele (p = 0.034) in HCV-GT2/3 patients, although no significant association was obtained in the multivariate analysis (p = 0.076). Regarding TLR3 haplotypes (comprised of rs3775291 and rs13126816), GT2/3 patients with AA haplotype had decreased odds of achieving EVR (p = 0.030), whereas GG haplotype increased the likelihood (p = 0.018). Regarding SVR, GG haplotype carriers had increased odds of achieving SVR (p = 0.019, p = 0.043 and p = 0.070 for all, GT2/3 and GT1/4 patients, respectively). Besides, GT1/4 patients with GA haplotype had lower odds of achieving SVR (p = 0.039). CONCLUSIONS: Our study shows the first evidence that two TLR3 polymorphisms (rs3775291 and rs13126816) seem to be related to the HCV therapy response in HCV/HIV coinfected patients.


Assuntos
Antivirais/uso terapêutico , Coinfecção/virologia , Infecções por HIV/complicações , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Receptor 3 Toll-Like/genética , Adulto , Alelos , Coinfecção/tratamento farmacológico , Feminino , Genótipo , Infecções por HIV/virologia , Haplótipos , Humanos , Interferon-alfa/uso terapêutico , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Ribavirina/uso terapêutico
6.
PLoS One ; 6(2): e16787, 2011 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-21364986

RESUMO

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.


Assuntos
Ciclopentanos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Prostaglandinas/farmacologia , Proteínas Repressoras/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção
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