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1.
Methods Mol Biol ; 2477: 293-309, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524124

RESUMO

Especially in eukaryotes, the N-terminal acetylation status of a protein reveals translation initiation sites and substrate specificities and activities of N-terminal acetyltransferases (NATs). Here, we discuss a bottom-up proteomics protocol for the enrichment of N-terminal peptides via strong cation exchange chromatography. This protocol is based on depleting internal tryptic peptides from proteome digests through their retention on strong cation exchangers, leaving N-terminally acetylated/blocked peptides enriched among the nonretained peptides. As such, one can identify novel N-terminal proteoforms and quantify the degree of N-terminal protein acetylation.


Assuntos
Proteoma , Proteômica , Acetilação , Cromatografia , Peptídeos/química , Proteômica/métodos
2.
Proc Natl Acad Sci U S A ; 119(11): e2118220119, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35254915

RESUMO

SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.


Assuntos
Brassinosteroides/metabolismo , Ácidos Indolacéticos/metabolismo , Proteoma , Transdução de Sinais , Aminopiridinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteômica/métodos , Succinatos/metabolismo
4.
FEBS J ; 2022 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-35066984

RESUMO

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity.

5.
Cytotherapy ; 24(2): 213-222, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34696961

RESUMO

Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA. The authors have developed MIDRIXNEO, a personalized mRNA-loaded dendritic cell vaccine targeting tumor neoantigens, which is currently being evaluated in a phase 1 clinical study in lung cancer patients. To facilitate this study, the authors set up a Good Manufacturing Practice (GMP)-compliant production process for the manufacture of small batches of personalized neoantigen-encoding mRNA. In this article, the authors describe the complete mRNA production process and the extensive quality assessment to which the mRNA is subjected. Validation runs have shown that the process delivers mRNA of reproducible, high quality. This process is now successfully applied for the production of neoantigen-encoding mRNA for the clinical evaluation of MIDRIXNEO. To the authors' knowledge, this is the first time that a GMP-based production process of patient-tailored neoantigen mRNA has been described.


Assuntos
Vacinas Anticâncer , Neoplasias Pulmonares , Neoplasias , Antígenos de Neoplasias/genética , Humanos , Imunoterapia , Neoplasias/genética , Neoplasias/terapia , Peptídeos , RNA Mensageiro/genética
6.
J Mol Biol ; 434(2): 167397, 2022 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-34896361

RESUMO

Actin is a hallmark protein of the cytoskeleton in eukaryotic cells, affecting a range of cellular functions. Actin dynamics is regulated through a myriad of actin-binding proteins and post-translational modifications. The mammalian actin family consists of six different isoforms, which vary slightly in their N-terminal (Nt) sequences. During and after synthesis, actins undergo an intricate Nt-processing that yields mature actin isoforms. The ubiquitously expressed cytoplasmic ß-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, ß-actin was also reported to be Nt-arginylated by arginyltransferase 1 (ATE1) after further peptidase-mediated processing, yielding RDDI-. To characterize in detail the Nt-processing of actin, we used state-of-the-art proteomics. To estimate the relative cellular levels of Nt-modified proteoforms of actin, we employed NAA80-lacking cells, in which actin was not Nt-acetylated. We found that targeted proteomics is superior to a commercially available antibody previously used to analyze Nt-arginylation of ß-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated ß-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Nt-arginylation of the initially cleaved ß-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified ß-actin (DDI-) as well as cleaved and Nt-acetylated ß-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of ß-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation.


Assuntos
Acetiltransferases/metabolismo , Actinas/química , Actinas/metabolismo , Aminoaciltransferases/metabolismo , Acetilação , Acetiltransferases/genética , Aminoaciltransferases/genética , Animais , Citoplasma/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica
7.
Front Cell Infect Microbiol ; 11: 735416, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34804992

RESUMO

RNF213 is a large, poorly characterized interferon-induced protein. Mutations in RNF213 are associated with predisposition for Moyamoya disease (MMD), a rare cerebrovascular disorder. Recently, RNF213 was found to have broad antimicrobial activity in vitro and in vivo, yet the molecular mechanisms behind this function remain unclear. Using mass spectrometry-based proteomics and validation by real-time PCR we report here that knockdown of RNF213 leads to transcriptional upregulation of MVP and downregulation of CYR61, in line with reported pro- and anti-bacterial activities of these proteins. Knockdown of RNF213 also results in downregulation of DDAH1, which we discover to exert antimicrobial activity against Listeria monocytogenes infection. DDAH1 regulates production of nitric oxide (NO), a molecule with both vascular and antimicrobial effects. We show that NO production is reduced in macrophages from RNF213 KO mice, suggesting that RNF213 controls Listeria infection through regulation of DDAH1 transcription and production of NO. Our findings propose a potential mechanism for the antilisterial activity of RNF213 and highlight NO as a potential link between RNF213-mediated immune responses and the development of MMD.


Assuntos
Doença de Moyamoya , Óxido Nítrico , Adenosina Trifosfatases/genética , Animais , Predisposição Genética para Doença , Camundongos , Proteoma , Ubiquitina-Proteína Ligases/genética
8.
Nat Commun ; 12(1): 5772, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599178

RESUMO

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.


Assuntos
Adenosina Trifosfatases/metabolismo , Anti-Infecciosos/metabolismo , Citocinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Células A549 , Animais , Enterovirus/fisiologia , Células HEK293 , Células HeLa , Herpesvirus Humano 1/fisiologia , Humanos , Interferon Tipo I/metabolismo , Gotículas Lipídicas/metabolismo , Listeria monocytogenes/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Ligação Proteica , Multimerização Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Células THP-1 , Ubiquitina/metabolismo
9.
Nat Immunol ; 22(11): 1416-1427, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34663977

RESUMO

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.


Assuntos
COVID-19/imunologia , Citocinas/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , SARS-CoV-2/fisiologia , Ubiquitinas/metabolismo , Diferenciação Celular , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Citocinas/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Evasão da Resposta Imune , Imunidade Inata , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Células-Tronco Pluripotentes/citologia , Ubiquitinação , Ubiquitinas/genética , Zika virus/fisiologia , Infecção por Zika virus/imunologia
10.
J Extracell Vesicles ; 10(10): e12122, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34429857

RESUMO

Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Fracionamento Químico/métodos , Vesículas Extracelulares/química , Lipoproteínas/análise , Plasma/química , Proteoma , Biomarcadores/análise , Neoplasias da Mama/sangue , Neoplasias da Mama/química , Cromatografia em Gel , Feminino , Infecções por HIV/sangue , Humanos , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/química
11.
Front Immunol ; 12: 720765, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34447387

RESUMO

During infection, pathogen sensing and cytokine signaling by the host induce expression of antimicrobial proteins and specialized post-translational modifications. One such protein is ISG15, a ubiquitin-like protein (UBL) conserved among vertebrates. Similar to ubiquitin, ISG15 covalently conjugates to lysine residues in substrate proteins in a process called ISGylation. Mice deficient for ISGylation or lacking ISG15 are strongly susceptible to many viral pathogens and several intracellular bacterial pathogens. Although ISG15 was the first UBL discovered after ubiquitin, the mechanisms behind its protective activity are poorly understood. Largely, this stems from a lack of knowledge on the ISG15 substrate repertoire. To unravel the antiviral activity of ISG15, early studies used mass spectrometry-based proteomics in combination with ISG15 pulldown. Despite reporting hundreds of ISG15 substrates, these studies were unable to identify the exact sites of modification, impeding a clear understanding of the molecular consequences of protein ISGylation. More recently, a peptide-based enrichment approach revolutionized the study of ubiquitin allowing untargeted discovery of ubiquitin substrates, including knowledge of their exact modification sites. Shared molecular determinants between ISG15 and ubiquitin allowed to take advantage of this technology for proteome-wide mapping of ISG15 substrates and modification sites. In this review, we provide a comprehensive overview of mass spectrometry-based proteomics studies on protein ISGylation. We critically discuss the relevant literature, compare reported substrates and sites and make suggestions for future research.


Assuntos
Imunidade Inata , Processamento de Proteína Pós-Traducional , Proteoma , Proteômica , Ubiquitinas/metabolismo , Citocinas/metabolismo , Ligação Proteica , Proteômica/métodos , Especificidade por Substrato
12.
Cancer Res ; 81(16): 4218-4229, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34215617

RESUMO

Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency-driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. SIGNIFICANCE: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.


Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Meningioma/genética , Mutação , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Biologia Computacional , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteoma , Semaforinas/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Ativação Transcricional , Ubiquitina/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas ras/metabolismo
13.
Function (Oxf) ; 2(4): zqab029, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34179788

RESUMO

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

14.
EMBO Rep ; 22(6): e52626, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34009726

RESUMO

Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.


Assuntos
Proteômica , Espectrometria de Massas
15.
Trends Microbiol ; 29(11): 1034-1045, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34030969

RESUMO

Antimicrobial resistance is an increasing global threat and alternative treatments substituting failing antibiotics are urgently needed. Vaccines are recognized as highly effective tools to mitigate antimicrobial resistance; however, the selection of bacterial antigens as vaccine candidates remains challenging. In recent years, advances in mass spectrometry-based proteomics have led to the development of so-called immunopeptidomics approaches that allow the untargeted discovery of bacterial epitopes that are presented on the surface of infected cells. Especially for intracellular bacterial pathogens, immunopeptidomics holds great promise to uncover antigens that can be encoded in viral vector- or nucleic acid-based vaccines. This review provides an overview of immunopeptidomics studies on intracellular bacterial pathogens and considers future directions and challenges in advancing towards next-generation vaccines.


Assuntos
Vacinas Bacterianas , Antígenos de Bactérias , Espectrometria de Massas , Proteômica
16.
Sci Rep ; 11(1): 3176, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542435

RESUMO

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, lacking effective therapy. Many TNBCs show remarkable response to carboplatin-based chemotherapy, but often develop resistance over time. With increasing use of carboplatin in the clinic, there is a pressing need to identify vulnerabilities of carboplatin-resistant tumors. In this study, we generated carboplatin-resistant TNBC MDA-MB-468 cell line and patient derived TNBC xenograft models. Mass spectrometry-based proteome profiling demonstrated that carboplatin resistance in TNBC is linked to drastic metabolism rewiring and upregulation of anti-oxidative response that supports cell replication by maintaining low levels of DNA damage in the presence of carboplatin. Carboplatin-resistant cells also exhibited dysregulation of the mitotic checkpoint. A kinome shRNA screen revealed that carboplatin-resistant cells are vulnerable to the depletion of the mitotic checkpoint regulators, whereas the checkpoint kinases CHEK1 and WEE1 are indispensable for the survival of carboplatin-resistant cells in the presence of carboplatin. We confirmed that pharmacological inhibition of CHEK1 by prexasertib in the presence of carboplatin is well tolerated by mice and suppresses the growth of carboplatin-resistant TNBC xenografts. Thus, abrogation of the mitotic checkpoint by CHEK1 inhibition re-sensitizes carboplatin-resistant TNBCs to carboplatin and represents a potential strategy for the treatment of carboplatin-resistant TNBCs.


Assuntos
Carboplatina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Pirazinas/farmacologia , Pirazóis/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nature ; 589(7842): 448-455, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33328637

RESUMO

FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.


Assuntos
Caderinas/deficiência , Transição Epitelial-Mesenquimal/genética , Deleção de Genes , Metástase Neoplásica/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Proteômica , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Quinases da Família src/metabolismo
19.
J Biol Chem ; 296: 100182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33310703

RESUMO

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Adenosina Trifosfatases/genética , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Mitoguazona/farmacologia , Mutação , Sequenciamento Completo do Genoma/métodos
20.
Sci Adv ; 6(27): eaaz9072, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32923585

RESUMO

RNA 3' end processing provides a source of transcriptome diversification which affects various (patho)-physiological processes. A prime example is the transcript isoform switch that leads to the read-through expression of the long non-coding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. NEAT1_2 is required for assembly of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that modulate this event, we identified factors involved in the 3' end processing of polyadenylated RNA and components of the Integrator complex. Perturbation experiments established that, by promoting the cleavage of NEAT1_2, Integrator forces NEAT1_2 to NEAT1_1 isoform switching and, thereby, restrains PS assembly. Consistently, low levels of Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study establishes that Integrator regulates PS biogenesis and a link between Integrator, cancer biology, and chemosensitivity, which may be exploited therapeutically.

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