Name: Mapeando interações proteína-proteína disfuncionais na doença (Vídeo) | JoVE Journal
Uploaded: 2025-10-24T13:55:00.000+00:00
Duration: 9 min 39 s
Description: Aprenda a mapear interações proteína-proteína disfuncionais em tecidos nativos usando sondas químicas e espectrometria de massas. Este método permite analisar redes celulares alteradas por doenças sem engenharia genética.

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<li>Sharma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+the+mechanism+of+epichaperome+modulation+by+zelavespib%3A+Biochemical+insights+on+target+occupancy+and+extended+residence+time+at+the+site+of+action%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Unraveling the mechanism of epichaperome modulation by zelavespib: Biochemical insights on target occupancy and extended residence time at the site of action.</a> Biomedicines. 11 (10), 2599(2023).</li>
<li>Guzman, U. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultra-fast+label-free+quantification+and+comprehensive+proteome+coverage+with+narrow-window+data-independent+acquisition%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.</a> Nat Biotechnol. 42 (12), 1855-1866 (2024).</li>
</ol>

<table><tbody><tr><td>2-mercaptoetanol</td><td>Sigma-Aldrich</td><td>M6250</td><td>Tóxico, Combustível</td></tr><tr><td>Acetonitrilo (ACN) Optima&nbsp;</td><td>Fisher Scientific & nbsp;</td><td>A955-1</td><td>Grau LC/MS (Inflamável)</td></tr><tr><td>Mídia de cromatografia de afinidade ativada Affi-Gel 10</td><td>Bio-Rad</td><td>1536099</td><td>Usado para fabricar contas de PU e contas de controle (Inflamável)</td></tr><tr><td>Bicarbonato de Amônio (ABC)</td><td>Sigma-Aldrich</td><td>09830-500G</td><td></td></tr><tr><td>Coluna UHPLC Aurora Ultimate 25x75 C18</td><td>IonOpticks</td><td>AUR3-25075C18-CSI</td><td></td></tr><tr><td>Filme autoradiográfico</td><td>EWEN-PARKER X-RAY CORP</td><td>EBA45</td><td>Alternativa para a detecção de sinais quimioluminescentes após Western blot</td></tr><tr><td>Biomasher micro homogeneizador de tecidos</td><td>DWK Ciências da Vida Kimble</td><td>7496250010</td><td>Usado para homogeneizar tecido cerebral congelado para extração de proteínas</td></tr><tr><td>Azul de bromofenol</td><td>Biorreagente Fisher</td><td>BP115-25</td><td></td></tr><tr><td>Centrífuga para tubos de 1,5 mL</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Centrífuga para tubos de 1,5 mL &nbsp;</td><td>Eppendorf</td><td>5417R</td><td>Com controle de temperatura</td></tr><tr><td>Centrífuga para tubos de 15-50 mL</td><td>Eppendorf</td><td>5810R</td><td>Uso para tubos cônicos</td></tr><tr><td>Sistema de imagem ChemiDoc MP</td><td>Bio-Rad</td><td>17001402</td><td>Usado para densitometria de gel/blot</td></tr><tr><td>Forno de coluna</td><td>Sonation GmBH, Alemanha</td><td>ES071/ES072</td><td>Mantém a coluna nanoLC em 50 e graus; C de retenção consistente</td></tr><tr><td>Tubos cônicos (15 mL)</td><td>Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>Esferas de controle</td><td>Laboratório de Quiose</td><td>N/A</td><td>Taldone et al. (PMID:&nbsp; 21459002)</td></tr><tr><td>Coloração Coomassie G-250 (Bio-segura)</td><td>Bio-Rad</td><td>1610786</td><td></td></tr><tr><td>Tubos Eppendorf escuros (1,5 mL)</td><td>USA Scientific</td><td>1415-2507</td><td></td></tr><tr><td>Ditiotreitol (DTT)</td><td>Roche</td><td>10197777001</td><td>Irritante</td></tr><tr><td>NLC 1000 fácil</td><td>Thermo Fisher Scientific</td><td>NLC 1000 fácil</td><td>Sistema LC usado com Q Exactive HF</td></tr><tr><td>Discos de extração em fase sólida Empore</td><td>Companhia 3M</td><td>2215-C18 (Octadecílo)</td><td>Usado para preparar StageTips para dessalinização de peptídeos; Ativado com 100% de metanol</td></tr><tr><td>Kit de quimioluminescência aprimorada (ECL)</td><td>Thermo Fisher Scientific</td><td>32209</td><td></td></tr><tr><td>Tubos Eppendorf (1,5 mL)</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Ácido Fórmico (FA) Optima&nbsp;</td><td>Fisher Scientific</td><td>A117-10X1AMP</td><td>Grau LC/MS</td></tr><tr><td>g:Profiler (e111_eg58_p18_f463989d)</td><td>Grupo BIIT-Tartu & Uuml; likool</td><td>https://biit.cs.ut.ee/gprofiler/gost</td><td>Enriquecimento de vias</td></tr><tr><td>Glicerol</td><td>Biorreagente Fisher</td><td>BP229-1</td><td>Grau em biologia molecular</td></tr><tr><td>Glicina</td><td>Biorreagente Fisher</td><td>BP381-5</td><td>Pó cristalino</td></tr><tr><td>Anticorpo secundário conjugado com HRP anti-camundongo (RRID: AB_2619742)</td><td>Sothern Biotech</td><td>1030-05</td><td>Secundário para o anticorpo H90-10</td></tr><tr><td>Anticorpo secundário conjugado com HRP anti-coelho (RRID: AB_2632593)</td><td>Sothern Biotech</td><td>4010-05</td><td>Secundário para o HSP90 e alfa; e anticorpos HOP</td></tr><tr><td>Anticorpo secundário conjugado com HRP anti-rato de cabra (RRID: AB_2716837)</td><td>Sothern Biotech</td><td>3030-05</td><td>Secundário para o anticorpo HSC70</td></tr><tr><td>Agitador orbital aquecido</td><td>Eppendorf</td><td>TermoMisturador C (2231001127)</td><td></td></tr><tr><td>HeLa proteína digerir standard-Pierce</td><td>Thermo Fisher Scientific</td><td>88329</td><td>Utilizado para avaliação de QC LC-MS</td></tr><tr><td>Anticorpo HOP (RRID: AB_10828378)</td><td>Sinalização de Celular</td><td>5670</td><td>Para controle de qualidade de contas de PU</td></tr><tr><td>Anticorpo HSC70 (RRID: AB_10617277)</td><td>Enzo</td><td>SPA-815</td><td>Para controle de qualidade de contas de PU</td></tr><tr><td>Anticorpo HSP90 (H90-10) (RRID: AB_854214)</td><td>StressMarq</td><td>SMC-107</td><td>Para controle de qualidade de contas de PU</td></tr><tr><td>Anticorpo alfa HSP90 (RRID: AB_303423)</td><td>Abcam</td><td>Ab2928</td><td>Para normalização conforme a Seção 1.2</td></tr><tr><td>Linha celular humana: ASPC1 (RRID:CVCL_0152)</td><td>ATCC</td><td>CRL-1682</td><td>Linha celular de controle, epichaperoma baixo/acompanhante alto</td></tr><tr><td>Linha celular humana: MDA-MB-468 (RRID: CVCL_0419)</td><td>ATCC</td><td>HTB-132</td><td>Linha celular de controle, epichaperoma alto/acompanhante alto</td></tr><tr><td>ImageJ (v1.54p)</td><td>Wayne Rasband</td><td>https://imagej.net/ij/</td><td>Densitometria</td></tr><tr><td>Iodoacetamida (IAA)</td><td>Sigma-Aldrich</td><td>I1149-5G</td><td>Tóxico</td></tr><tr><td>Tubos microcentrífugas de baixa ligação a proteínas</td><td>Sarstedt</td><td>72.706.600</td><td></td></tr><tr><td>Protease Lys-C</td><td>Promega&nbsp;</td><td>VA1170</td><td>Grau LC/MS</td></tr><tr><td>Cloreto de Magnésio</td><td>Sigma-Aldrich</td><td>63068</td><td>Grau em biologia molecular</td></tr><tr><td>MaxQuant (v2.1.1.0)</td><td>Instituto Max-Planck de Bioquímica</td><td>https://www.maxquant.org/</td><td>Fluxo de trabalho MaxDIA para identificação de peptídeos/proteínas e quantificação baseada em intensidade sem marcação</td></tr><tr><td>Metanol</td><td>Fisher Chemical</td><td>A454-4</td><td>Grau HPLC, inflamável, tóxico</td></tr><tr><td>Mini comprimido de coquetel inibidor de protease (Completo)</td><td>Roche</td><td>4693124001</td><td></td></tr><tr><td>Célula de Transferência Eletroforética Mini Trans Blot</td><td>Bio-Rad</td><td>1703930</td><td></td></tr><tr><td>Mini-gel de eletroforese & nbsp;</td><td>Thermo Fisher Scientific</td><td>A25977</td><td>Usado para géis pré-moldados Novex</td></tr><tr><td>Célula de Eletroforese Tetra Vertical Mini-PROTEAN &nbsp;</td><td>Bio-Rad</td><td>1658004</td><td>Usado para géis proteicos pré-moldados Mini-PROTEAN ou géis fundidos à mão</td></tr><tr><td>Mini-PROTEAN&nbsp; Gels de proteína pré-fabricados TGX (4&ndash; 20%)</td><td>Bio-Rad</td><td>4561094</td><td></td></tr><tr><td>NanoDrop</td><td>Agilent</td><td>BioTek Sinergia H1</td><td>Usado para estimar a concentração de peptídeos antes da injeção de LC-MS</td></tr><tr><td>NanoElute 2</td><td>Bruker</td><td>https://flextra.hu/images/keszulekek/daltonics/nanoelute-brosura_eng.pdf</td><td>Usado no fluxo de trabalho diaPASEF</td></tr><tr><td>Fonte Nanospray Flex</td><td>Thermo Fisher Scientific</td><td>ES071</td><td>Para acoplamento ESI de nanospray</td></tr><tr><td>Novo Objetivo PicoTip Emmiter/PicoFrit SELF/P</td><td>New Objective Inc.</td><td>PF360-75-10-N-5</td><td>Compactado com pasta C18 até 20 cm de comprimento</td></tr><tr><td>Membrana de nitrocelulose</td><td>Cytiva Amersham</td><td>10600006</td><td></td></tr><tr><td>Leite desnatado</td><td>Laboratório Científico</td><td>M0841</td><td></td></tr><tr><td>Géis pré-fabricados Novex 8% Tris-Glycine</td><td>Thermo Fisher Scientific</td><td>XP00080BOX</td><td></td></tr><tr><td>NP-40</td><td>Roche</td><td>11754599001</td><td></td></tr><tr><td>Soro salino tamponado com fosfato (PBS)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Comprimido de coquetel inibidor de fosfatase PhosSTOP</td><td>Roche</td><td>4906837001</td><td></td></tr><tr><td>Kit de ensaio de proteína Pierce BCA</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Recipientes plásticos</td><td>Fisher Scientific</td><td>FB0875711A</td><td></td></tr><tr><td>Balanço plataforma</td><td>Corning</td><td>6705</td><td></td></tr><tr><td>Cloreto de potássio</td><td>Biorreagente Fisher</td><td>BP366-1</td><td>Cristais Brancos</td></tr><tr><td>Fonte de Alimentação Universal PowerPac</td><td>Bio-Rad</td><td>1645070</td><td></td></tr><tr><td>Proteína Proteica Caleidoscópica Precision Plus Proteína Padrão Prestained</td><td>Bio-Rad</td><td>1610375</td><td></td></tr><tr><td>Contas de PU-</td><td>Laboratório de Quiose</td><td>N/A</td><td>Taldone et al. (PMID:&nbsp; 21459002)</td></tr><tr><td>Q Espectrômetro de massa HF Exativo Orbitrap</td><td>Thermo Fisher Scientific</td><td>https://assets.thermofisher.com/TFS-Assets/CMD/brochures/BR-64052-LC-MS-Q-Exactive-HF-Orbitrap-BR64052-EN.pdf</td><td>Usado para DIA-MS</td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 3 & micro; m</td><td>Dr. Maisch GmbH, Alemanha</td><td>R13.aq</td><td>Mídia de sílica para separação peptídica; Pasta de pó/Metanol (1:3, v/v)</td></tr><tr><td>Unidade de rotação (rotador de ponta a ponta)</td><td>Barnstead&nbsp; Internacional</td><td>400110</td><td></td></tr><tr><td>Cartuchos Sep-Pak C18 (100 mg)</td><td>Waters Corporation</td><td>WAT023590</td><td></td></tr><tr><td>Dodecilsulfato de sódio (SDS)</td><td>Biorreagente Fisher</td><td>BP166-500</td><td>Grau de eletroforese</td></tr><tr><td>Sonicator</td><td>Branson</td><td>2510</td><td></td></tr><tr><td>Leitor de Microplacas Multi-Modo SpectraMax Paradigm</td><td>Dispositivos Moleculares</td><td>Paradigma SpectraMax</td><td></td></tr><tr><td>Spectronaut</td><td>Biognosys</td><td>https://biognosys.com/software/spectronaut/</td><td>Fluxo de trabalho directDIA</td></tr><tr><td>Concentrador SpeedVac</td><td>Thermo Fisher Scientific&nbsp;</td><td>Savant SPD111V</td><td></td></tr><tr><td>Vácuo de sucção</td><td>Saúde Cardinal</td><td>CDL-65651-320</td><td></td></tr><tr><td>Espectrômetro de massa TimsTOF HT</td><td>Bruker</td><td>https://www.bruker.com/en/products-and-solutions/mass-spectrometry/timstof/timstof-ht.html</td><td>Usado para análise diaPASEF</td></tr><tr><td>Óptimos do Ácido Trifluoracético (TFA)</td><td>Sigma-Aldrich</td><td>T6508-10AMP</td><td>Grau LC/MS (Corrosivo, combustível)</td></tr><tr><td>Tris</td><td>Biorreagente Fisher</td><td>BP152-5</td><td>Pó cristalino; Grau em biologia molecular</td></tr><tr><td>Tris, pH 6,8 (1 M)</td><td>Teknova</td><td>T1068</td><td></td></tr><tr><td>Tris, pH 7,5 (1 M)</td><td>Biorreagente Fisher</td><td>BP1757-500</td><td>Grau em biologia molecular</td></tr><tr><td>Buffer de soro fisiológico trís-tamponado (20x colheres de sopa)</td><td>VWR, Ciências da vida</td><td>97064-338</td><td></td></tr><tr><td>Tris-Glicina-SDS (TGS 10x) funcionando com tampão de gel</td><td>Biorreagente Fisher</td><td>BP13414</td><td>Eletroforese</td></tr><tr><td>Tripsina&nbsp;</td><td>Promega</td><td>V5111</td><td>Classificação de sequenciamento</td></tr><tr><td>Tripsina Ouro</td><td>Promega</td><td>V5280</td><td></td></tr><tr><td>Tween 20</td><td>Biorreagente Fisher</td><td>BP337-100</td><td></td></tr><tr><td>Ureia</td><td>Sigma-Aldrich</td><td>U0631-500G</td><td></td></tr><tr><td>Vórtice</td><td>Fisher Scientific</td><td>02-215-414</td><td></td></tr><tr><td>Água</td><td>Fisher Scientific</td><td>W6-1</td><td>Grau LC/MS</td></tr></tbody></table>

mapeando interações proteína-proteína disfuncionais na doença

Mapping Dysfunctional Protein-Protein Interactions in Disease

Mapeando interações proteína-proteína disfuncionais na doença

Protein-protein interaction (PPI) networks are dynamically remodeled in disease, yet most systems biology approaches focus on changes in protein ...

mapping-dysfunctional-protein-protein-interactions-in-disease

Research

JoVE Journal

Neuroscience

Neurociência

As sequências de proteínas sintéticas baseadas em motivos de consenso normalmente ignoram resíduos co-evolutivos, que implicam dependências interposicionais (IPDs).  Os IPDs podem ser essenciais para a atividade, e projetos que os desconsideram podem resultar em resultados abaixo do ideal.  Este protocolo usa StickWRLD para identificar IPDs e ajudar a informar o design racional de proteínas, resultando em resultados mais eficientes.

Otimização de proteínas sintéticas: Identificação de interposição Dependências Indicating Estruturalmente e / ou resíduos funcionalmente ligadas

Controlar a expressão de proteínas não é apenas essencial para todos os organismos vivos, mas também uma estratégia importante para investigar as funções das proteínas em modelos celulares. O protocolo apresentado mostra a aplicação da interferência de anticorpos em células de mamíferos, incluindo neurônios primários do hipocampo, e demonstra o uso de reconstruções tridimensionais no estudo da função proteica.

Estudar a função da proteína eo papel da expressão proteína alterada pela interferência de anticorpos e reconstruções tridimensionais

Este relatório descreve protocolos para medir as taxas de degradação de proteínas mal dobradas por ensaios baseados em western blot ou fluorescência. Os métodos podem ser aplicados à análise de outras proteínas mal dobradas e para triagem de alto rendimento.

Os ensaios para a degradação de proteínas em células deformadas

Este protocolo descreve procedimentos experimentais para caracterizar todo o genoma de alterações nos níveis de modificações borne-translational do histone (PTM) ocorrendo em relação a superexpressão de proteínas associadas com a ALS e a doença de Parkinson em Modelos de Saccharomyces cerevisiae . Após a separação de SDS-PAGE, níveis PTM histona individuais são detectados anticorpos específicos de modificação através da mancha ocidental.

Caracterizando a histona modificação pós-traducional alterações nos modelos de Neurodegenerative Proteinopathy de levedura

O objetivo deste protocolo é delinear o desenho e o desempenho de experimentos in vivo em Drosophila melanogaster para avaliar as conseqüências funcionais de variantes genéticas raras associadas a doenças humanas.

Estudo funcional in vivo de variantes humanas raras associadas à doença usando Drosophila

O declínio proteostático é uma marca do envelhecimento, facilitando o aparecimento de doenças neurodegenerativas. Delineamos um protocolo para medir quantificadamente a proteostase em dois tecidos elegans caenorhabditis diferentes através da expressão heteróloga de repetições de poliglutamina fundidas a um repórter fluorescente. Este modelo permite uma rápida análise genética in vivo da proteostase.

Quantificando o declínio proteostático específico do tecido em elegans caenorhabditis

Para estudar as interações acompanhante-acompanhante e acompanhante-substrato, realizamos telas de interação sintética em elegans caenorhabditis usando interferência de RNA em combinação com mutações leves ou sobre-expressão de acompanhantes e monitorar disfunção proteica específica do tecido no nível do organismo.

Usando elegans caenorhabditis para tela para interações de acompanhantes específicas de tecido

Um protocolo computacional, CaseOLAP LIFT, e um caso de uso são apresentados para investigar proteínas mitocondriais e suas associações com doenças cardiovasculares, conforme descrito em relatórios biomédicos. Este protocolo pode ser facilmente adaptado para estudar componentes celulares e doenças selecionadas pelo usuário.

Uma Abordagem de Gráfico de Conhecimento para Elucidar o Papel das Vias Organelares na Doença via Relatórios Biomédicos

Usamos magnetoencefalografia (MEG) e eletroencefalografia (EEG) para mapear áreas do cérebro envolvidas no processamento de estímulos sensoriais simples.

Mapeamento funcional com MEG e EEG em simultâneo

Uma das características patológicas da doença de Alzheimer é a formação de amilóide β proteína placas neuríticas positivo. Neste protocolo, descrevemos dois métodos para detectar placas neuríticas em camundongos transgênicos modelo AD: detecção imunohistoquímica utilizando o método ABC e DAB e detecção fluorescente usando o método thioflavin S coloração.

Detecção de placas neuríticas no rato da doença de Alzheimer Modelo

Aprenda a mapear interações proteína-proteína disfuncionais em tecidos nativos usando sondas químicas e espectrometria de massas. Este método permite analisar redes celulares alteradas por doenças sem engenharia genética.

Mapeando interações proteína-proteína disfuncionais na doença

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