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1.
EMBO J ; 40(10): e104566, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33764556

RESUMO

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context-dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single-strand DNA (ssDNA) transactions, including the ssDNA-binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1-Top3-Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
2.
EMBO Rep ; 22(2): e51121, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33491328

RESUMO

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here, we assemble a dataset from 75 independent phosphoproteomic experiments performed in our laboratory using Saccharomyces cerevisiae. We report 30,902 phosphosites identified from cells cultured in a range of DNA damage conditions and/or arrested in distinct cell cycle stages. To generate a comprehensive resource for the budding yeast community, we aggregate our dataset with the Saccharomyces Genome Database and another recently published study, resulting in over 46,000 budding yeast phosphosites. With the goal of enhancing the identification of functional phosphorylation events, we perform computational positioning of phosphorylation sites on available 3D protein structures and systematically identify events predicted to regulate protein complex architecture. Results reveal hundreds of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic "clashes" predicted to disrupt the interaction. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Fosforilação , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
3.
Mol Cell Proteomics ; 20: 100091, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33971369

RESUMO

Non-T cell activation linker (NTAL) membrane protein depletion from lipid rafts by alkylphospholipids or downregulation by shRNA knockdown decreases cell viability through regulation of the Akt/PI3K pathway in mantle cell lymphoma and acute promyelocytic leukemia cells. Here, we confirmed that the knockdown of NTAL in acute myeloid leukemia (AML) cell lines was associated with decreased cell proliferation and survival. Similarly, a xenograft model using AML cells transduced with NTAL-shRNA and transplanted into immunodeficient mice led to a 1.8-fold decrease in tumor burden. Using immunoprecipitation, LC-MS/MS analysis, and label-free protein quantification, we identified interactors of NTAL in two AML cell lines. By evaluating the gene expression signatures of the NTAL protein interactors using the PREdiction of Clinical Outcomes from Genomic Profiles database, we found that 12 NTAL interactors could predict overall survival in AML, in at least two independent cohorts. In addition, patients with AML exhibiting a high expression of NTAL and its interactors were associated with a leukemic granulocyte-macrophage progenitor-like state. Taken together, our data provide evidence that NTAL and its protein interactors are relevant to AML cell proliferation and survival and represent potential therapeutic targets for granulocyte-macrophage progenitor-like leukemias.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Análise de Sobrevida , Transcriptoma
4.
Sens Actuators B Chem ; 353: 131128, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34866796

RESUMO

The outbreak of the COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome of Coronavirus 2 (SARS-CoV-2), has fueled the search for diagnostic tests aiming at the control and reduction of the viral transmission. The main technique used for diagnosing the Coronavirus disease (COVID-19) is the reverse transcription-polymerase chain reaction (RT-PCR) technique. However, considering the high number of cases and the underlying limitations of the RT-PCR technique, especially with regard to accessibility and cost of the test, one does not need to overemphasize the need to develop new and less expensive testing techniques that can aid the early diagnosis of the disease. With that in mind, we developed an ultrasensitive magneto-assay using magnetic beads and gold nanoparticles conjugated to human angiotensin-converting enzyme 2 (ACE2) peptide (Gln24-Gln42) for the capturing and detection of SARS-CoV-2 Spike protein in human saliva. The technique applied involved the use of a disposable electrochemical device containing eight screen-printed carbon electrodes which allow the simultaneous analysis of eight samples. The magneto-assay exhibited an ultralow limit of detection of 0.35 ag mL-1 for the detection of SARS-CoV-2 Spike protein in saliva. The magneto-assay was tested in saliva samples from healthy and SARS-CoV-2-infected individuals. In terms of efficiency, the proposed technique - which presented a sensitivity of 100.0% and specificity of 93.7% for SARS-CoV-2 Spike protein-exhibited great similarity with the RT-PCR technique. The results obtained point to the application potential of this simple, low-cost magneto-assay for saliva-based point-of-care COVID-19 diagnosis.

5.
Proc Natl Acad Sci U S A ; 116(47): 23518-23526, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31690664

RESUMO

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PR-ubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas Desubiquitinantes/metabolismo , Legionella pneumophila/metabolismo , Diester Fosfórico Hidrolases/metabolismo , ADP-Ribosilação , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ubiquitina , Ubiquitinação , Vacúolos/microbiologia
6.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232817

RESUMO

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial-mesenchymal transition (↑) and PI3K signaling via AKT to mTORC1 (↓ in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (↑ in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis.


Assuntos
Endometriose , Células-Tronco Mesenquimais , Estudos de Casos e Controles , Proliferação de Células , Endometriose/patologia , Feminino , Humanos , Interleucina-6 , Laminina , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Menstruação , Células-Tronco Mesenquimais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteoma , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espectrometria de Massas em Tandem , Transcriptoma , Fator de Crescimento Transformador beta/genética
7.
Mol Cell Proteomics ; 15(3): 906-17, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26764010

RESUMO

Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-ß, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.


Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Histona Desacetilase 1/metabolismo , Proteômica/métodos , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Redes Reguladoras de Genes , Humanos , Células MCF-7 , Invasividade Neoplásica , Proteoma/isolamento & purificação , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Espectrometria de Massas em Tandem
8.
Proteomics ; 17(7)2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28130891

RESUMO

Contrary to what is being said by several colleagues (and even advertised), data-independent analysis/acquisition (DIA) is not a new mass spectrometry acquisition method. Here we draw a timeline of events showing that DIA has been around since the early 2000s.


Assuntos
Peptídeos/análise , Proteômica/métodos , Espectrometria de Massas/métodos , Terminologia como Assunto
9.
Adv Exp Med Biol ; 974: 205-212, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28353237

RESUMO

Patients with psychiatric disorders exhibit dysfunctions in peripheral and central metabolism. This may be a root cause of impaired neuronal function, manifested as changes in mood, behavior, and cognitive capabilities in patients suffering with these conditions. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS)-based targeted proteomic protocol for precise simultaneous quantitation of three glycolytic enzymes in postmortem brain tissue extracts. The SRM-MS approach has several advantages in terms of sensitivity, reproducibility, and reduced sample consumption, compared to traditional MS methods.


Assuntos
Encéfalo/enzimologia , L-Lactato Desidrogenase/análise , Espectrometria de Massas/métodos , Proteínas do Tecido Nervoso/análise , Fosfopiruvato Hidratase/análise , Triose-Fosfato Isomerase/análise , Biomarcadores/análise , Cromatografia de Fase Reversa/métodos , Glicólise , Humanos , Peptídeos/análise , Mudanças Depois da Morte
10.
Nature ; 452(7187): 571-9, 2008 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-18385731

RESUMO

Systematic searches for plasma proteins that are biological indicators, or biomarkers, for cancer are underway. The difficulties caused by the complexity of biological-fluid proteomes and tissue proteomes (which contribute proteins to plasma) and by the extensive heterogeneity among diseases, subjects and levels of sample procurement are gradually being overcome. This is being achieved through rigorous experimental design and in-depth quantitative studies. The expected outcome is the development of panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy. Achieving these objectives requires high-quality specimens with well-matched controls, reagent resources, and an efficient process to confirm discoveries through independent validation studies.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/sangue , Plasma/metabolismo , Proteoma/metabolismo , Proteômica , Biomarcadores Tumorais/análise , Líquidos Corporais/metabolismo , Humanos , Neoplasias/diagnóstico , Neoplasias/patologia
11.
Mol Cell Proteomics ; 11(12): 1898-912, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23001822

RESUMO

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 µm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 µm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five µm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Fosfolipídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Trióxido de Arsênio , Arsenicais/farmacologia , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células , Colesterol/metabolismo , Ativação Enzimática , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Microdomínios da Membrana , Óxidos/farmacologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfolipídeos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Estrutura Terciária de Proteína , Proteoma/análise , Interferência de RNA , RNA Interferente Pequeno
12.
Antiviral Res ; 229: 105968, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-39004311

RESUMO

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Humanos , Animais , SARS-CoV-2/efeitos dos fármacos , COVID-19/virologia , Camundongos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/uso terapêutico , Tratamento Farmacológico da COVID-19 , Antivirais/farmacologia , Antivirais/química , Linhagem Celular , Pneumonia/tratamento farmacológico , Pneumonia/virologia , Pneumonia/prevenção & controle , Pulmão/virologia , Pulmão/patologia , Feminino
13.
Elife ; 122023 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-37523305

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Linfócitos T CD8-Positivos , Linfócitos T Auxiliares-Indutores , Pulmão
14.
Front Immunol ; 13: 948419, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36148232

RESUMO

The autoimmune regulator (AIRE) protein functions as a tetramer, interacting with partner proteins to form the "AIRE complex," which relieves RNA Pol II stalling in the chromatin of medullary thymic epithelial cells (mTECs). AIRE is the primary mTEC transcriptional controller, promoting the expression of a large set of peripheral tissue antigen genes implicated in the negative selection of self-reactive thymocytes. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates K residues of the AIRE SAND domain. Once the AIRE SAND domain is deacetylated, the binding with SIRT1 is undone, allowing the AIRE complex to proceed downstream with the RNA Pol II to the elongation phase of transcription. Considering that the in silico and in vitro binding of the AIRE SAND domain with SIRT1 provides a powerful model system for studying the dominant SAND G228W mutation mechanism, which causes the autoimmune polyglandular syndrome-1, we integrated computational molecular modeling, docking, dynamics between the whole SAND domain with SIRT1, and surface plasmon resonance using a peptide harboring the 211 to 230 residues of the SAND domain, to compare the structure and energetics of binding/release between AIRE G228 (wild-type) and W228 (mutant) SAND domain to SIRT1. We observed that the G228W mutation in the SAND domain negatively influences the AIRE-SIRT1 interaction. The disturbed interaction might cause a disruption in the binding of the AIRE SAND domain with the SIRT1 catalytic site, impairing the AIRE complex to proceed downstream with RNA Pol II.


Assuntos
RNA Polimerase II , Sirtuína 1 , Cromatina , Regulação da Expressão Gênica , Mutação , Peptídeos , Sirtuína 1/genética
15.
Elife ; 112022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35133275

RESUMO

The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteoma , Testículo/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA , Reparo do DNA , Masculino , Meiose , Camundongos Endogâmicos C57BL , Morfolinas/administração & dosagem , Fosforilação , Pirimidinas/administração & dosagem , RNA Mensageiro/metabolismo , Transdução de Sinais , Espermatócitos/metabolismo
16.
Elife ; 112022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35133274

RESUMO

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pareamento Cromossômico , Reparo do DNA , Meiose , Animais , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Testículo/metabolismo
17.
Mol Cell Proteomics ; 8(3): 451-66, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18984577

RESUMO

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.


Assuntos
Encéfalo/metabolismo , Proteínas do Líquido Cefalorraquidiano/metabolismo , Doença de Huntington/líquido cefalorraquidiano , Animais , Proteínas do Líquido Cefalorraquidiano/genética , Perfilação da Expressão Gênica , Humanos , Laboratórios , Camundongos , Especificidade de Órgãos , Proteômica
18.
Biosci Rep ; 41(3)2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33629708

RESUMO

Tau is a microtubule-associated protein (MAP) responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In the present study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either -20 or -80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ∼25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry (MS) analysis in excised bands revealed this ∼25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Criopreservação/métodos , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Humanos , Imuno-Histoquímica/métodos , Proteólise , Ratos , Ratos Wistar , Proteínas tau/toxicidade
19.
Biochim Biophys Acta Proteins Proteom ; 1869(6): 140623, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33607274

RESUMO

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Cistadenocarcinoma Seroso/patologia , Fator de Crescimento Epidérmico/farmacologia , Neoplasias Ovarianas/patologia , Proteômica/métodos , Regulação para Cima , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida , Cistadenocarcinoma Seroso/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Marcação por Isótopo , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Espectrometria de Massas em Tandem
20.
PLoS Med ; 5(6): e123, 2008 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-18547137

RESUMO

BACKGROUND: The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. METHODS AND FINDINGS: Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. CONCLUSIONS: Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Pancreáticas/diagnóstico , Proteoma/metabolismo , Animais , Humanos , Espectrometria de Massas , Camundongos , Neoplasias Pancreáticas/sangue , Proteômica/métodos , RNA Mensageiro/metabolismo
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