Name: Mappatura delle interazioni disfunzionali proteina-proteina nella malattia (Video) | JoVE Journal
Uploaded: 2025-10-24T13:55:00.000+00:00
Duration: 9 min 39 s
Description: Scopri come mappare le interazioni proteina-proteina disfunzionali direttamente da cellule e tessuti nativi utilizzando sonde chimiche e spettrometria di massa. Questo metodo rivela come le malattie riconfigurano le reti cellulari senza ingegneria genetica.

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<table><tbody><tr><td>2-mercaptoetanolo</td><td>Sigma-Aldrich</td><td>M6250</td><td>Tossico, Ardente</td></tr><tr><td>Acetonitrile (ACN) Optima&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>A955-1</td><td>Grado LC/MS (Infiammabile)</td></tr><tr><td>Mezzi di cromatografia ad affinità attivati Affi-Gel 10</td><td>Bio-Rad</td><td>1536099</td><td>Usata per produrre perle in PU e perle di controllo (infiammabili)</td></tr><tr><td>Bicarbonato di ammonio (ABC)</td><td>Sigma-Aldrich</td><td>09830-500G</td><td></td></tr><tr><td>Colonna UHPLC Aurora Ultimate 25x75 C18</td><td>IonOpticks</td><td>AUR3-25075C18-CSI</td><td></td></tr><tr><td>Film autoradiografico</td><td>EWEN-PARKER X-RAY CORP</td><td>EBA45</td><td>Alternativa per il rilevamento del segnale chemiluminescente dopo il Western blot</td></tr><tr><td>Microomogeneizzatore dei tessuti Biomasher</td><td>DWK Kimble Scienze della Vita</td><td>7496250010</td><td>Utilizzato per omogeneizzare tessuto cerebrale congelato per l'estrazione proteica</td></tr><tr><td>Blu di bromofenolo</td><td>Bioreagente Fisher</td><td>BP115-25</td><td></td></tr><tr><td>Centrifuga per tubi da 1,5 mL</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Centrifuga per tubi da 1,5 mL &nbsp;</td><td>Eppendorf</td><td>5417R</td><td>Con controllo della temperatura</td></tr><tr><td>Centrifuga per tubi da 15-50 mL</td><td>Eppendorf</td><td>5810R</td><td>Uso per tubi conici</td></tr><tr><td>Sistema di imaging ChemiDoc MP</td><td>Bio-Rad</td><td>17001402</td><td>Usato per la densitometria gel/blot</td></tr><tr><td>Forno a colonna</td><td>Sonation GmBH, Germania</td><td>ES071/ES072</td><td>Mantiene la colonna nanoLC a 50 &deg; C per una ritenzione costante</td></tr><tr><td>Tubi conici (15 mL)</td><td>Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>Sfere di controllo</td><td>Laboratorio Chiosis</td><td>N/A</td><td>Taldone et al. (PMID:&nbsp; 21459002)</td></tr><tr><td>Colorazione Coomassie G-250 (Bio-sicura)</td><td>Bio-Rad</td><td>1610786</td><td></td></tr><tr><td>Tubi Eppendorf scuri (1,5 mL)</td><td>USA Scientific</td><td>1415-2507</td><td></td></tr><tr><td>Ditiotreitolo (DTT)</td><td>Roche</td><td>10197777001</td><td>Irritante</td></tr><tr><td>Facile nLC 1000</td><td>Thermo Fisher Scientific</td><td>Facile nLC 1000</td><td>Sistema LC utilizzato con Q Exactive HF</td></tr><tr><td>Dischi di estrazione in fase solida Empore</td><td>Compagnia 3M</td><td>2215-C18 (Ottadicelo)</td><td>Utilizzato per preparare gli StageTips per la desalinizzazione dei peptidi; Attivato con metanolo al 100%</td></tr><tr><td>Kit di chemiluminescenza avanzata (ECL)</td><td>Thermo Fisher Scientific</td><td>32209</td><td></td></tr><tr><td>Tubi Eppendorf (1,5 mL)</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Acido Formico (FA) Optima&nbsp;</td><td>Fisher Scientific</td><td>A117-10X1AMP</td><td>Grado LC/MS</td></tr><tr><td>g:Profiler (e111_eg58_p18_f463989d)</td><td>Gruppo BIIT-Tartu & Uuml; likool</td><td>https://biit.cs.ut.ee/gprofiler/gost</td><td>Arricchimento dei percorsi</td></tr><tr><td>Glicerolo</td><td>Bioreagente Fisher</td><td>BP229-1</td><td>Grado in biologia molecolare</td></tr><tr><td>Glicina</td><td>Bioreagente Fisher</td><td>BP381-5</td><td>Polveri cristalline</td></tr><tr><td>Anticorpo secondario coniugato HRP anti-topo (RRID: AB_2619742)</td><td>Sothern Biotech</td><td>1030-05</td><td>Secondario per l'anticorpo H90-10</td></tr><tr><td>Anticorpo secondario anti-coniglio coniugato HRP (RRID: AB_2632593)</td><td>Sothern Biotech</td><td>4010-05</td><td>Secondario per l'HSP90 e alpha; e anticorpi HOP</td></tr><tr><td>Anticorpo secondario HRP coniugato contro il topo (RRID: AB_2716837)</td><td>Sothern Biotech</td><td>3030-05</td><td>Secondario per l'anticorpo HSC70</td></tr><tr><td>Shaker orbitale riscaldato</td><td>Eppendorf</td><td>ThermoMixer C (2231001127)</td><td></td></tr><tr><td>HeLa protein digest standard-Pierce</td><td>Thermo Fisher Scientific</td><td>88329</td><td>Utilizzato per la valutazione del controllo qualità del LC-MS</td></tr><tr><td>Anticorpo HOP (RRID: AB_10828378)</td><td>Segnalazione cellulare</td><td>5670</td><td>Per il controllo qualità delle perle in PU</td></tr><tr><td>Anticorpo HSC70 (RRID: AB_10617277)</td><td>Enzo</td><td>SPA-815</td><td>Per il controllo qualità delle perle in PU</td></tr><tr><td>Anticorpo HSP90 (H90-10) (RRID: AB_854214)</td><td>StressMarq</td><td>SMC-107</td><td>Per il controllo qualità delle perle in PU</td></tr><tr><td>Anticorpo alfa HSP90 (RRID: AB_303423)</td><td>Abcam</td><td>Ab2928</td><td>Per la normalizzazione secondo la Sezione 1.2</td></tr><tr><td>Linea cellulare umana: ASPC1 (RRID:CVCL_0152)</td><td>ATCC</td><td>CRL-1682</td><td>Linea cellulare di controllo, epichaperoma basso/accompagnatore alto</td></tr><tr><td>Linea cellulare umana: MDA-MB-468 (RRID: CVCL_0419)</td><td>ATCC</td><td>HTB-132</td><td>Linea cellulare di controllo, epichaperoma alto/accompagnatore 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>Iodoacetamide (IAA)</td><td>Sigma-Aldrich</td><td>I1149-5G</td><td>Tossico</td></tr><tr><td>Tubi microcentrifughe a basso legame proteico</td><td>Sarstedt</td><td>72.706.600</td><td></td></tr><tr><td>Lys-C proteasi</td><td>Promega&nbsp;</td><td>VA1170</td><td>Grado LC/MS</td></tr><tr><td>Cloruro di magnesio</td><td>Sigma-Aldrich</td><td>63068</td><td>Grado in biologia molecolare</td></tr><tr><td>MaxQuant (v2.1.1.0)</td><td>Istituto Max-Planck di Biochimica</td><td>https://www.maxquant.org/</td><td>Workflow MaxDIA per l'identificazione di peptidi/proteine e la quantificazione basata sull'intensità senza marcatori</td></tr><tr><td>Metanolo</td><td>Fisher Chemical</td><td>A454-4</td><td>Grado HPLC, infiammabile, tossico</td></tr><tr><td>Mini compressa cocktail per inibitori della proteasi (Completa)</td><td>Roche</td><td>4693124001</td><td></td></tr><tr><td>Mini Trans Blot Cellula di Trasferimento Elettroforetica</td><td>Bio-Rad</td><td>1703930</td><td></td></tr><tr><td>Mini-gel per electroforesi & nbsp;</td><td>Thermo Fisher Scientific</td><td>A25977</td><td>Usato per gel prefabbricati Novex</td></tr><tr><td>Cellula di elettroforesi verticale tetra mini-PROTEAN &nbsp;</td><td>Bio-Rad</td><td>1658004</td><td>Utilizzato per gel proteici prefabbricati Mini-PROTEAN o gel colati a mano</td></tr><tr><td>Mini-PROTEAN&nbsp; TGX Precast Protein Gels (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>Utilizzato per stimare la concentrazione peptidica prima dell'iniezione di 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>Utilizzato nel flusso di lavoro diaPASEF</td></tr><tr><td>Sorgente Nanospray Flex</td><td>Thermo Fisher Scientific</td><td>ES071</td><td>Per l'accoppiamento ESI nanospray</td></tr><tr><td>Nuovo obiettivo PicoTip Emmiter/PicoFrit SELF/P</td><td>New Objective Inc.</td><td>PF360-75-10-N-5</td><td>Riempito con una pasta C18 fino a 20 cm di lunghezza</td></tr><tr><td>Membrana di nitrocellulosa</td><td>Cytiva Amersham</td><td>10600006</td><td></td></tr><tr><td>Latte crosio scremato</td><td>Laboratorio Scientifico</td><td>M0841</td><td></td></tr><tr><td>Gel prefabbricati 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>Soluzione salina tamponata con fosfato (PBS)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Compressa cocktail per inibitori della fosfatasi PhosSTOP</td><td>Roche</td><td>4906837001</td><td></td></tr><tr><td>Kit di analisi proteica Pierce BCA</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Contenitori di plastica</td><td>Fisher Scientific</td><td>FB0875711A</td><td></td></tr><tr><td>Oscillatore a piattaforma</td><td>Corning</td><td>6705</td><td></td></tr><tr><td>Cloruro di potassio</td><td>Bioreagente Fisher</td><td>BP366-1</td><td>Cristalli Bianchi</td></tr><tr><td>Alimentatore universale PowerPac</td><td>Bio-Rad</td><td>1645070</td><td></td></tr><tr><td>Standard Proteico Proteico Proteico Proteico Proteico Prestained Precision Plus</td><td>Bio-Rad</td><td>1610375</td><td></td></tr><tr><td>Perle PU-</td><td>Laboratorio Chiosis</td><td>N/A</td><td>Taldone et al. (PMID:&nbsp; 21459002)</td></tr><tr><td>Q Spettrometro di massa HF Orbitrap esattivo</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>Utilizzato per DIA-MS</td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 3 & micro; m</td><td>Dr. Maisch GmbH, Germania</td><td>R13.aq</td><td>substrati silicei per la separazione peptidica; Miscela di polvere/Metanolo (1:3, v/v)</td></tr><tr><td>Unità di rotazione (rotatore end-over-end)</td><td>Barnstead&nbsp; Internazionale</td><td>400110</td><td></td></tr><tr><td>Cartucce Sep-Pak C18 (100 mg)</td><td>Waters Corporation</td><td>WAT023590</td><td></td></tr><tr><td>Dodecile solfato di sodio (SDS)</td><td>Bioreagente Fisher</td><td>BP166-500</td><td>Grado di elettroforesi</td></tr><tr><td>Sonicator</td><td>Branson</td><td>2510</td><td></td></tr><tr><td>Lettore di micropiastre multimodalità SpectraMax Paradigm</td><td>Dispositivi molecolari</td><td>Paradigma SpectraMax</td><td></td></tr><tr><td>Spectronaut</td><td>Biognosys</td><td>https://biognosys.com/software/spectronaut/</td><td>Flusso di lavoro directDIA</td></tr><tr><td>Concentratore SpeedVac</td><td>Thermo Fisher Scientific&nbsp;</td><td>Savant SPD111V</td><td></td></tr><tr><td>Aspirazione a vuoto</td><td>Cardinal Health</td><td>CDL-65651-320</td><td></td></tr><tr><td>Spettrometro di massa TimsTOF HT</td><td>Bruker</td><td>https://www.bruker.com/en/products-and-solutions/mass-spectrometry/timstof/timstof-ht.html</td><td>Utilizzato per l'analisi diaPASEF</td></tr><tr><td>Ottima dell'Acido Trifluoracetico (TFA)</td><td>Sigma-Aldrich</td><td>T6508-10AMP</td><td>Grado LC/MS (Corrosivo, combustibile)</td></tr><tr><td>Tris</td><td>Bioreagente Fisher</td><td>BP152-5</td><td>Polvere cristallina; Grado in biologia molecolare</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>Bioreagente Fisher</td><td>BP1757-500</td><td>Grado in biologia molecolare</td></tr><tr><td>Soluzione salina tris-buffer (20x TBS) tampone</td><td>VWR, Scienze della vita</td><td>97064-338</td><td></td></tr><tr><td>Tris-Glycine-SDS (TGS 10x) in funzione con gel buffer</td><td>Bioreagente Fisher</td><td>BP13414</td><td>Elettroforesi</td></tr><tr><td>Tripsina&nbsp;</td><td>Promega</td><td>V5111</td><td>Grado di sequenziamento</td></tr><tr><td>Tripsina Oro</td><td>Promega</td><td>V5280</td><td></td></tr><tr><td>Preadolescenti 20</td><td>Bioreagente Fisher</td><td>BP337-100</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U0631-500G</td><td></td></tr><tr><td>Vortice</td><td>Fisher Scientific</td><td>02-215-414</td><td></td></tr><tr><td>Acqua</td><td>Fisher Scientific</td><td>W6-1</td><td>Grado LC/MS</td></tr></tbody></table>

mappatura delle interazioni disfunzionali proteina-proteina nella malattia

Mapping Dysfunctional Protein-Protein Interactions in Disease

Mappatura delle interazioni disfunzionali proteina-proteina nella malattia

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

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JoVE Journal

Neuroscience

Neuroscienze

Le sequenze proteiche sintetiche basate su motivi di consenso tipicamente ignorano i residui co-evolutivi, che implicano dipendenze interposizionali (IPD).  Gli IPD possono essere essenziali per l'attività e i progetti che li ignorano possono portare a risultati non ottimali.  Questo protocollo utilizza StickWRLD per identificare gli IPD e aiutare a informare la progettazione razionale delle proteine, ottenendo risultati più efficienti.

Ottimizzazione di proteine ​​sintetiche: identificazione di interposizione Dipendenze Indicazione Strutturalmente e / o residui funzionalmente collegate

Il controllo dell'espressione proteica non è solo essenziale per ogni organismo vivente, ma anche un'importante strategia per studiare le funzioni proteiche nei modelli cellulari. Il protocollo presentato mostra l'applicazione dell'interferenza anticorpale nelle cellule di mammifero, compresi i neuroni primari dell'ippocampo, e dimostra l'uso di ricostruzioni tridimensionali nello studio della funzione delle proteine.

Studiando la funzione delle proteine ​​e il ruolo di Altered espressione della proteina da anticorpo interferenza e ricostruzioni tridimensionali

Questo rapporto descrive i protocolli per misurare i tassi di degradazione delle proteine mal ripiegate mediante western blot o saggi basati sulla fluorescenza. I metodi possono essere applicati all'analisi di altre proteine mal ripiegate e per lo screening ad alto rendimento.

Saggi per la degradazione delle proteine ​​nelle cellule mal ripiegate

Questo protocollo delinea le procedure sperimentali per caratterizzare il genoma cambiamenti nei livelli di modifiche post-traduzionali degli istoni (PTM) che si verificano in relazione la sovraespressione di proteine associate con ALS e malattia del Parkinson in Modelli di Saccharomyces cerevisiae . Dopo la separazione di SDS-PAGE, vengono rilevati livelli PTM istone individuali con gli anticorpi specifici di modifiche tramite Western blotting.

Alterazioni di modificazione post-traduzionale istone caratterizzante nel lievito Neurodegenerative Proteinopathy modelli

L'obiettivo di questo protocollo è quello di delineare la progettazione e le prestazioni degli esperimenti in vivo nella Drosophila melanogaster per valutare le conseguenze funzionali delle varianti genetiche rare associate alle malattie umane.

Studio funzionale in vivo sulle varianti umane rare associate alla malattia utilizzando la Drosophila

Il declino proteostatico è un segno distintivo dell'invecchiamento, facilitando l'insorgenza di malattie neurodegenerative. Delineiamo un protocollo per misurare in modo quantificabile la proteostasi in due diversi tessuti di Caenorhabditis elegans attraverso l'espressione eterologa di ripetizioni di poliglutamina fuse a un reporter fluorescente. Questo modello consente una rapida analisi genetica in vivo della proteostasi.

Quantificazione del declino proteostatico tessuto-specifico nella Caenorhabditis elegans

Per studiare le interazioni chaperone-chaperone e chaperone-substrato, eseguiamo screening di interazione sintetica in Caenorhabditis elegans utilizzando l'interferenza dell'RNA in combinazione con mutazioni lievi o sovraespressione di chaperoni e monitoriamo la disfunzione proteica tessuto-specifica a livello di organismo.

Utilizzo di Caenorhabditis elegans per lo screening delle interazioni chaperone tessuto-specifiche

Vengono presentati un protocollo computazionale, CaseOLAP LIFT, e un caso d'uso per studiare le proteine mitocondriali e le loro associazioni con le malattie cardiovascolari come descritto nei rapporti biomedici. Questo protocollo può essere facilmente adattato per studiare i componenti cellulari e le malattie selezionate dall'utente.

Un approccio basato su Knowledge Graph per chiarire il ruolo delle vie organellari nella malattia attraverso referti biomedici

Noi usiamo magnetoencefalografia (MEG) e elettroencefalografia (EEG) per mappare aree del cervello coinvolte nella elaborazione di semplici stimoli sensoriali.

Mappatura funzionale con EEG e MEG simultaneo

Una delle caratteristiche patologiche di Alzheimer è la formazione di placche di amiloide β neuritiche proteina positivo. In questo protocollo si descrivono due metodi per rilevare placche neuritiche in topi transgenici modello AD: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente thioflavin S.

Rilevazione di placche neuritiche nel modello di malattia di Alzheimer mouse

Scopri come mappare le interazioni proteina-proteina disfunzionali direttamente da cellule e tessuti nativi utilizzando sonde chimiche e spettrometria di massa. Questo metodo rivela come le malattie riconfigurano le reti cellulari senza ingegneria genetica.

Mappatura delle interazioni disfunzionali proteina-proteina nella malattia

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