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1.
PLoS Biol ; 18(7): e3000755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644996

RESUMO

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Multimerização Proteica , Movimento Celular , Humanos , Integrinas/metabolismo , Células K562 , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
2.
Proc Natl Acad Sci U S A ; 117(31): 18600-18607, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32703806

RESUMO

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.


Assuntos
Proteínas Fúngicas , Proteínas de Neoplasias , Piroptose/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Neurospora crassa/metabolismo
3.
SAR QSAR Environ Res ; 31(6): 439-455, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32539470

RESUMO

The human breast cancer resistance protein (BCRP), one of the members of the large ATP binding cassette (ABC) transporter superfamily, is crucial for resistance against chemotherapeutic agents. Currently, it has been emerged as one of the best biological targets for the designing of small molecule drugs capable of eliminating multidrug resistance in breast cancer. In order to gain insights into the relationship between the molecular structure of compounds and the ABCG2 inhibition, a multi-QSAR approach using different methods was performed on a dataset of 294 ABCG2 inhibitors with diverse scaffolds. The best models obtained by different chemometric methods have the following statistical characteristics: Monte Carlo Optimization-based QSAR (sensitivity = 0.905, specificity = 0.6255, accuracy = 0.756, and MCC = 0.545), Bayesian classification model (sensitivity = 0.735, specificity = 0.775, and concordance = 0.757); structural and physicochemical interpretation analysis-random forest method (balance accuracy = 0.750, sensitivity = 0.810, and specificity = 0.700). Additionally, structural fingerprints modulating the ABCG2 inhibitory properties were identified from the best models of each method and also validated with each other. The current modelling study is an attempt to get a deep insight into the different important structural fingerprints modulating ABCG2 inhibition.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Mapeamento de Nucleotídeos , Relação Quantitativa Estrutura-Atividade , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Teorema de Bayes , Estrutura Molecular , Método de Monte Carlo , Proteínas de Neoplasias/química
4.
Science ; 368(6494)2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32299851

RESUMO

Cytotoxic lymphocyte-mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)-positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon-γ (IFN-γ) up-regulates GSDMB expression and promotes pyroptosis. GSDMB is highly expressed in certain tissues, particularly digestive tract epithelia, including derived tumors. Introducing GZMA-cleavable GSDMB into mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte-killing mechanism, which may enhance antitumor immunity.


Assuntos
Granzimas/metabolismo , Células Matadoras Naturais/imunologia , Proteínas de Neoplasias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/imunologia , Linfócitos T Citotóxicos/enzimologia , Animais , Granzimas/química , Células HEK293 , Humanos , Interferon gama , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Domínios Proteicos , Proteólise
5.
Nat Commun ; 11(1): 1039, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098964

RESUMO

Genetically encoded photoswitches have enabled spatial and temporal control of cellular events to achieve tailored functions in living cells, but their applications to probe the structure-function relations of signaling proteins are still underexplored. We illustrate herein the incorporation of various blue light-responsive photoreceptors into modular domains of the stromal interaction molecule 1 (STIM1) to manipulate protein activity and faithfully recapitulate STIM1-mediated signaling events. Capitalizing on these optogenetic tools, we identify the molecular determinants required to mediate protein oligomerization, intramolecular conformational switch, and protein-target interactions. In parallel, we have applied these synthetic devices to enable light-inducible gating of calcium channels, conformational switch, dynamic protein-microtubule interactions and assembly of membrane contact sites in a reversible manner. Our optogenetic engineering approach can be broadly applied to aid the mechanistic dissection of cell signaling, as well as non-invasive interrogation of physiological processes with high precision.


Assuntos
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Optogenética/métodos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Cálcio/metabolismo , Criptocromos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ativação do Canal Iônico , Proteínas Luminescentes/genética , Mutação , Proteínas de Neoplasias/química , Neoplasias/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Molécula 1 de Interação Estromal/química , Relação Estrutura-Atividade
6.
Sci Rep ; 10(1): 1290, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992775

RESUMO

Cancer stem cells (CSCs) have the ability to self-renew and induce drug resistance and recurrence in colorectal cancer (CRC). As current chemotherapy doesn't eliminate CSCs completely, there is a need to identify novel agents to target them. We investigated the effects of cucurbitacin B (C-B) or I (C-I), a natural compound that exists in edible plants (bitter melons, cucumbers, pumpkins and zucchini), against CRC. C-B or C-I inhibited proliferation, clonogenicity, induced G2/M cell-cycle arrest and caspase-mediated-apoptosis of CRC cells. C-B or C-I suppressed colonosphere formation and inhibited expression of CD44, DCLK1 and LGR5. These compounds inhibited notch signaling by reducing the expression of Notch 1-4 receptors, their ligands (Jagged 1-2, DLL1,3,4), γ-secretase complex proteins (Presenilin 1, Nicastrin), and downstream target Hes-1. Molecular docking showed that C-B or C-I binds to the ankyrin domain of Notch receptor, which was confirmed using the cellular thermal shift assay. Finally, C-B or C-I inhibited tumor xenograft growth in nude mice and decreased the expression of CSC-markers and notch signaling proteins in tumor tissues. Together, our study suggests that C-B and C-I inhibit colon cancer growth by inhibiting Notch signaling pathway.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Simulação de Acoplamento Molecular , Receptores Notch , Transdução de Sinais/efeitos dos fármacos , Triterpenos , Animais , Neoplasias do Colo/química , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Domínios Proteicos , Receptores Notch/química , Receptores Notch/metabolismo , Triterpenos/química , Triterpenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
FASEB J ; 34(2): 2227-2237, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31916632

RESUMO

Cyanidin-3-glucoside (C3G) is a natural pigment, found in many colorful fruits and vegetables. It has many health benefits, including anti-inflammation, cancer prevention, and anti-diabetes. Although C3G is assumed to be an antioxidant, it has been reported to affect cell-matrix adhesions. However, the underlying molecular mechanism is unknown. Here, we show that the expression of talin1, a key regulator of integrins and cell adhesions, negatively correlated with the survival rate of colon cancer patients and that depletion of talin1 inhibited 3D spheroid growth in colon cancer cells. Interestingly, C3G bound to talin and promoted the interaction of talin with ß1A-integrin. Molecular docking analysis shows that C3G binds to the interface of the talin-ß-integrin complex, acting as an allosteric regulator and altering the interaction between talin and integrin. Moreover, C3G promoted colon cancer cell attachment to fibronectin. While C3G had no significant effect on colon cancer cell proliferation, it significantly inhibited 3D spheroid growth in fibrin gel assays. Since C3G has no or very low toxicity, it could be potentially used for colon cancer prevention or therapy.


Assuntos
Antocianinas/farmacocinética , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo , Glucosídeos/farmacocinética , Proteínas de Neoplasias , Talina , Animais , Células CHO , Técnicas de Cultura de Células , Neoplasias do Colo/química , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cricetinae , Cricetulus , Células HCT116 , Humanos , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Talina/química , Talina/metabolismo
8.
Molecules ; 24(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842510

RESUMO

Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.


Assuntos
Antineoplásicos/química , Galectina 3/química , Ouro/química , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/química , Porfirinas/química , Galectina 3/metabolismo , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo
9.
Soft Matter ; 15(48): 9829-9839, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31728468

RESUMO

Membrane curvature has recently been recognized as an active regulator of cellular function, with several protein families identified as sensors and generators of membrane curvature. Amongst them, the inverse Bin/Amphiphysin/Rvs (I-BAR) domain family has been implicated in the sensing and generation of membrane structures with negative membrane curvature e.g. filopodia or dendritic spines. However, to date, quantitative biophysical investigations of I-BAR domains have mostly taken place in reconstitution. Here, we use fluorescence microscopy to quantitatively investigate membrane curvature sensing and generation by I-BARs in filopodia of living cells. As a model system, we selected two prototypic members of the I-BAR family, the insulin receptor substrate p53 and missing-in-metastasis. Our data demonstrated how I-BARs sense negative membrane curvature in the complex environment of live cells by revealing a dependence on membrane curvature for both their binding affinity to membranes and their saturation density. The non-monotonic dependence of protein sorting with negative membrane curvature allowed us to apply previously developed thermodynamic models to provide estimates of the effective intrinsic curvature and bending rigidity of the two I-BARs bound at the plasma membrane. Our results agree with studies performed on the insulin receptor substrate p53 in reconstitution. To quantitate membrane curvature generation by I-BARs we measured how their overexpression reduces the peak and the width of the size distribution of filopodia, resulting in filopodia populations with smaller and more uniform diameters. Our findings provide a quantitative biophysical insight in the ability of I-BARs to sense and generate negative membrane curvature in the crowded environment of living cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Membrana Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Modelos Biológicos , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Pseudópodes/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Células PC12 , Domínios Proteicos , Ratos
10.
Molecules ; 24(22)2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703384

RESUMO

Circular RNAs (circRNAs) are extensively expressed in cells and tissues, and play crucial roles in human diseases and biological processes. Recent studies have reported that circRNAs could function as RNA binding protein (RBP) sponges, meanwhile RBPs can also be involved in back-splicing. The interaction with RBPs is also considered an important factor for investigating the function of circRNAs. Hence, it is necessary to understand the interaction mechanisms of circRNAs and RBPs, especially in human cancers. Here, we present a novel method based on deep learning to identify cancer-specific circRNA-RBP binding sites (CSCRSites), only using the nucleotide sequences as the input. In CSCRSites, an architecture with multiple convolution layers is utilized to detect the features of the raw circRNA sequence fragments, and further identify the binding sites through a fully connected layer with the softmax output. The experimental results show that CSCRSites outperform the conventional machine learning classifiers and some representative deep learning methods on the benchmark data. In addition, the features learnt by CSCRSites are converted to sequence motifs, some of which can match to human known RNA motifs involved in human diseases, especially cancer. Therefore, as a deep learning-based tool, CSCRSites could significantly contribute to the function analysis of cancer-associated circRNAs.


Assuntos
Bases de Dados de Proteínas , Aprendizado Profundo , Proteínas de Neoplasias , Neoplasias , RNA Circular , RNA Neoplásico , Proteínas de Ligação a RNA , Análise de Sequência de Proteína , Sítios de Ligação , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , RNA Circular/química , RNA Circular/genética , RNA Circular/metabolismo , RNA Neoplásico/química , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
11.
Klin Onkol ; 32(Supplementum 3): 56-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627707

RESUMO

BACKGROUND: Ubiquitination is a vital posttranslational protein modification involved in the regulation of many eukaryotic signalling pathways. Aberrant ubiquitin signalling is known to be a molecular causality of certain cancer, neurodegenerative, immune system or cardiovascular diseases. The recent development of mass spectrometry methods enables qualitative and quantitative ubiquitination analysis in biological material from cancer patients. Research of ubiquitination may clarify the molecular cause of aberrant changes in the protein level of tumour suppressors or oncogenes. PURPOSE: We aim to explain the meaning and importance of ubiquitination in certain molecular processes taking place in the human body. We hereby emphasise the connection between ubiquitination and malignant processes. A literature search is followed by introducing our mass spectrometry platform intended for ubiquitin identification via diglycyl remnants in the CHIP protein sequence. The aim is to introduce tandem mass spectrometry identification of ubiquitin modification, ubiquitination tandem mass spectra validation and the time-dependent manner of CHIP ubiquitination to the reader. CONCLUSION: A literature search familiarises the reader with known mechanisms of aberrant ubiquitination in malignant diseases. A successfully optimised mass spectrometry platform could serve as a potent tool for determining ubiquitin position in proteins that are a part of real tumour samples.


Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Transdução de Sinais , Ubiquitinação
12.
Adv Exp Med Biol ; 1172: 189-205, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628657

RESUMO

Gasdermin is a recently identified family of pore-forming proteins consisting of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC), Gasdermin D (GSDMD), Gasdermin E (GSDME), and DFNB59. Gasdermin D (GSDMD) is a downstream effector of inflammasomes, which are supramolecular complexes that activate inflammatory caspases (-1, -4, and -5 in human and -1 and -11 in mouse). GSDMD contains a functionally important N-terminal domain (GSDMD-N), a C-terminal domain, and a linker in between that is recognized and cleaved by the activated inflammatory caspases. Upon cleavage, the GSDMD-N fragments translocate on the membrane and oligomerize to form membrane-embedded pores after specifically binding to acidic lipids such as phosphatidylinositol phosphates (PIPs), phosphatidic acid (PA), phosphatidylserine (PS), and cardiolipin. The pore exhibits strong membrane-disrupting cytotoxicity in mammalian cells by disrupting the osmotic potential and also serves as a gate for extracellular release of mature IL-1ß and IL-18 during pyroptosis. In this chapter, we review our current understanding of GSDM proteins in physiological and pathological cell death, with more focused discussions on its structural basis for GSDM activation and pore formation.


Assuntos
Proteínas de Neoplasias , Piroptose , Animais , Caspases/metabolismo , Ativação Enzimática , Humanos , Inflamassomos , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Relação Estrutura-Atividade
13.
Nanoscale ; 11(41): 19520-19528, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31573595

RESUMO

The therapeutic efficacy of anti-cancer nanomedicines is generally constrained due to limited accumulation in the solid tumors. In this study, we developed a biomimetic nano-carrier to enhance the chemo-therapeutic efficacy of doxorubicin and icotinib in a chemo-resistant non-small cell lung cancer (NSCLC) cell line harboring a mutation in the epidermal growth factor receptor (EGFR). The unique nanomedicine was prepared by coating with targeting cancer cell membrane proteins as highly specific ligands. The resulting biomimetic nanoparticles were highly stable and exhibited superior homologous targeting ability in vitro compared with control groups. In a mouse EGFR-mutated NSCLC xenograft model, intravenous injection of the biomimetic nanomedicine led to a high tumour inhibition rate (87.56%). Histopathological analysis demonstrated that the biomimetic nanomedicine had minimal side effects. Taken together, a cancer cell membrane-based biomimetic drug carrier can significantly enhance drug accumulation and improve therapeutic efficacy in cancers.


Assuntos
Materiais Biomiméticos/química , Resistencia a Medicamentos Antineoplásicos , Nanocompostos/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/uso terapêutico , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Mutação , Nanocompostos/toxicidade , Nanomedicina , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Transplante Heterólogo
14.
Proc Natl Acad Sci U S A ; 116(47): 23534-23541, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31591207

RESUMO

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.


Assuntos
Neoplasias da Mama/patologia , Autorrenovação Celular/fisiologia , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/fisiologia , Superóxido Dismutase/fisiologia , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Neoplasias da Mama/metabolismo , Reprogramação Celular , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Peróxido de Hidrogênio/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/enzimologia , Invasividade Neoplásica , Proteínas de Neoplasias/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/química
15.
PLoS Comput Biol ; 15(9): e1006789, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527881

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor. Molecular heterogeneity is a hallmark of GBM tumors that is a barrier in developing treatment strategies. In this study, we used the nonsynonymous mutations of GBM tumors deposited in The Cancer Genome Atlas (TCGA) and applied a systems level approach based on biophysical characteristics of mutations and their organization in patient-specific subnetworks to reduce inter-patient heterogeneity and to gain potential clinically relevant insights. Approximately 10% of the mutations are located in "patches" which are defined as the set of residues spatially in close proximity that are mutated across multiple patients. Grouping mutations as 3D patches reduces the heterogeneity across patients. There are multiple patches that are relatively small in oncogenes, whereas there are a small number of very large patches in tumor suppressors. Additionally, different patches in the same protein are often located at different domains that can mediate different functions. We stratified the patients into five groups based on their potentially affected pathways that are revealed from the patient-specific subnetworks. These subnetworks were constructed by integrating mutation profiles of the patients with the interactome data. Network-guided clustering showed significant association between the groups and patient survival (P-value = 0.0408). Also, each group carries a set of signature 3D mutation patches that affect predominant pathways. We integrated drug sensitivity data of GBM cell lines with the mutation patches and the patient groups to analyze the possible therapeutic outcome of these patches. We found that Pazopanib might be effective in Group 3 by targeting CSF1R. Additionally, inhibiting ATM that is a mediator of PTEN phosphorylation may be ineffective in Group 2. We believe that from mutations to networks and eventually to clinical and therapeutic data, this study provides a novel perspective in the network-guided precision medicine.


Assuntos
Neoplasias Encefálicas/genética , Análise por Conglomerados , Biologia Computacional/métodos , Glioblastoma/genética , Mutação/genética , Neoplasias Encefálicas/epidemiologia , Mapeamento Cromossômico , Glioblastoma/epidemiologia , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Medicina de Precisão/métodos
16.
Proc Natl Acad Sci U S A ; 116(40): 19952-19962, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31515451

RESUMO

ANO1 (TMEM16A) is a Ca2+-activated Cl- channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here, we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2 Three of these sites captured 85% of all ANO1-PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found 3 regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same 3 sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid side chains and phosphate/hydroxyl groups on PI(4,5)P2 Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl- ions. We propose a model consisting of a network of 3 PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.


Assuntos
Anoctamina-1/química , Cálcio/química , Proteínas de Neoplasias/química , Fosfatidilinositol 4,5-Difosfato/química , Sítios de Ligação , Cátions , Citosol/metabolismo , Eletrofisiologia , Células HEK293 , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Técnicas de Patch-Clamp , Fosfolipídeos/química , Relação Estrutura-Atividade
17.
Proc Natl Acad Sci U S A ; 116(41): 20404-20410, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548401

RESUMO

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.


Assuntos
Neoplasias Colorretais/metabolismo , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas de Neoplasias/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Conformação Proteica
18.
Nucleic Acids Res ; 47(19): 10086-10103, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31529049

RESUMO

The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.


Assuntos
Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/química , Células HEK293 , Código das Histonas/genética , Histonas/química , Humanos , Metiltransferases/genética , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas de Neoplasias/química , Fosforilação/genética , Protamina Quinase/química , Protamina Quinase/genética , Proteínas Serina-Treonina Quinases/química , Células Satélites de Músculo Esquelético/metabolismo , Análise de Sequência de RNA
19.
J Recept Signal Transduct Res ; 39(3): 253-263, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31517548

RESUMO

Type 2 diabetes mellitus is a metabolic disorder that requires potent therapeutic approaches. The FAM3B is a cytokine-like protein also referred to as PANcreatic-DERrived factor (PANDER) which mainly exists in pancreatic islets. In the process of identifying potential inhibitors with the aid of structure-based method PANDER protein is identified as a novel therapeutic target against type 2 diabetes mellitus as it involved in the development of type 2 diabetes by negatively regulating the pancreatic ß-cell function and insulin sensitivity in the liver. In the present study, the 3d model of target protein FAM3B was generated by homology modeling technique using the MODELLER9.9 program. The assessment of the structural stability of the 3d model was established by energy minimization technique. The structural quality was evaluated with standard validating protocols. Binding regions of the target protein has been determined by literature and SiteMap tool. In the current study of research, the FAM3B model was subjected to molecular screening with the Asinex-elite database of 14849 output molecules using the Glide virtual screening module in the Schrodinger suite. The final XP descriptor output of 14 molecules was analyzed and prioritized based on molecular interactions at the FAM3B active site. The docking score, binding free energies (Prime MM/GBSA) and bioavailability were undertaken into the consideration to identify lead inhibitors. The identified lead compounds were checked for ADME properties all falling within the permeable ranges. The analysis of results gave the insight to develop the novel therapeutic strategies against type 2 diabetes mellitus.


Assuntos
Citocinas/antagonistas & inibidores , Citocinas/química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Domínios Proteicos , Estrutura Secundária de Proteína , Software , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Termodinâmica
20.
Proc Natl Acad Sci U S A ; 116(38): 18962-18970, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31462496

RESUMO

Large-scale exome sequencing of tumors has enabled the identification of cancer drivers using recurrence-based approaches. Some of these methods also employ 3D protein structures to identify mutational hotspots in cancer-associated genes. In determining such mutational clusters in structures, existing approaches overlook protein dynamics, despite its essential role in protein function. We present a framework to identify cancer driver genes using a dynamics-based search of mutational hotspot communities. Mutations are mapped to protein structures, which are partitioned into distinct residue communities. These communities are identified in a framework where residue-residue contact edges are weighted by correlated motions (as inferred by dynamics-based models). We then search for signals of positive selection among these residue communities to identify putative driver genes, while applying our method to the TCGA (The Cancer Genome Atlas) PanCancer Atlas missense mutation catalog. Overall, we predict 1 or more mutational hotspots within the resolved structures of proteins encoded by 434 genes. These genes were enriched among biological processes associated with tumor progression. Additionally, a comparison between our approach and existing cancer hotspot detection methods using structural data suggests that including protein dynamics significantly increases the sensitivity of driver detection.


Assuntos
Biologia Computacional/métodos , Genômica/métodos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/genética , Bases de Dados Genéticas , Exoma/genética , Humanos , Mutação , Conformação Proteica , Reprodutibilidade dos Testes , Fluxo de Trabalho
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