Vogliamo riconoscere il dottor Scott Henderson per un aiuto con microscopia confocale. Questa ricerca è stata sostenuta dalle concessioni dal National Institutes of Health 1RO1CA127501 a WAB, nonché al progetto di finanziamento pilota della Massey Cancer Center e la Scuola di Medicina (VCU) a Microscopia WAB è stato eseguito presso il VCU-Dipartimento. di Neurobiologia e Microscopia Fondo Anatomia, sostenuta, in parte, con il finanziamento NIHNINDS Centro nucleo concedere 5P30NS047463.

Il protocollo si compone di tre fasi principali. Il primo, la clonazione fase del gene di interesse in un vettore di espressione mammifero amino-terminale per le versioni monomerico della PCP e / o YFP sarà solo discusso brevemente. I passi secondo e terzo; trasfezione di DNA vettore in EA.hy926 cellule endoteliali, e di imaging confocale con FRET, sono descritti più approfonditamente di seguito. 1. Costruzione di recettori PCP e YFP Chimerico Recettori di interesse dovrebbero essere clonato amino-terminale di monomerico versioni avanzate dei CFP e YFP. Determinazione del FRET è empirica e critica è dipende dalla lunghezza del linker tra i transmembrana spanning regione e inizio del fluoroforo 1. Pertanto diverse lunghezze differenti linker deve essere esplorata per ogni coppia recettore nuovo sotto inchiesta. <ol><li> Nel hygro pcDNA3.1 R o neo vettore R contenente monomerica C o YFP (questo vettore può essere ottenuto dal nostro laboratorio), il tuo clone del recettore di interesse nel NheI / EcoRI siti. * Nota: se necessario, NheI è compatibile enzimi diversi altri tra cui SpeI e XbaI. </li><li> Trasforma la tua reazione legatura in una appropriata linea cellulare endA1 recA competenti (si usano abitualmente Mach1). Colonie schermo per la presenza di inserire seguita da sequenziamento del DNA per verificare l&#39;assenza di mutazioni. </li><li> All&#39;ottenimento conformazione di successo della clonazione molecolare, preparare trasfezione-grade vettore di DNA in acqua sterilizzata o TE utilizzando Qiagen, o simili, kit del DNA. Valutare la concentrazione e la purezza utilizzando la spettroscopia UV. Le rese del DNA dovrebbe essere nel range di 100-200 mg, che viene normalmente diluito a uno stock di lavoro di 1 mg / mL. </li></ol> 2. Trasfezione delle cellule EA.hy 926 <ol><li> Split EA.hy 926 celle 4, coltivate in DMEM completo-(DMEM +10% siero fetale bovino + Pen. / Strep.) In MatTek coprioggetto (n. 0) 35 piatti mm con 2 ml di mezzo. Si può ottenere conservativo ~ 15 piatti (~ 90% confluenti del giorno successivo) da un piatto di 15 centimetri confluenti. Preparare un piatto di valutare l&#39;espressione del recettore di ogni individuo, così come un piatto per ogni coppia recettore. </li><li> Permettono alle cellule di incubare una notte in 5% CO 2 nell&#39;atmosfera a 37 ° C. Il giorno seguente, le colture dovrebbero essere confluenti ~ 70-90%. </li><li> Il giorno della transfezione, preparare i complessi nucleolipid mescolando 2 mg di DNA chimerico vettore recettore (2 mL) con 8 ml di Fugene6 in 200 ml di pre-riscaldato OptiMEM. Per le coppie di recettori, trasfezione con 1 mg di ogni recettore, per un totale di 2 mg (Vedi anche la discussione sotto). </li><li> Mescolare delicatamente per inversione. </li><li> Continuare a incubare a temperatura ambiente per 30 minuti. </li><li> Durante il periodo di incubazione, preparare le cellule per la trasfezione. Aspirare off mezzi freschi e lavare ogni piastra delicatamente e brevemente con PBS caldo prima l&#39;aggiunta di 2 ml preriscaldata completa-DMEM senza antibiotici (DMEM + FBS). E &#39;importante evitare la presenza di penicillina e streptomicina, in quanto sono molto più tossici per le cellule durante il trattamento con Fugene6. </li><li> Aggiungere la goccia complesso saggio cellule e incubare per ulteriori 20-24 ore a 37 ° C in un 5% di CO 2 nell&#39;atmosfera. </li><li> Dopo 24 ore, con attenzione ai media di aspirare le cellule e pipetta fresca completa-DMEM con antibiotici. A seguito di trasfezione, la vitalità cellulare dovrebbe rimanere relativamente alti, cioè poco o nessun cellule galleggianti devono essere osservati. </li><li> L&#39;espressione genica è tipicamente massima dopo un ulteriore 48-60 ore dopo la trasfezione. Tuttavia, l&#39;imaging è solitamente possibile nel più breve di 24 ore. Esperimenti di durata del corso per l&#39;espressione genica ottimale dovrebbe essere eseguita. </li></ol> 3. Imaging confocale e analisi FRET Vivere l&#39;imaging cellulare è eseguita su un TCS-SP2 laser Leica AOBS microscopio confocale a scansione dotato di diodo blu (405 nm), argon (458, 476, 488, 514 nm). HeNe verde (543 nm), arancio HeNe (594 nm), e rosso HeNe (633 nm), un HCX PI Apo 63x/1.3 na glicerina-immersion lente dell&#39;obiettivo, un motorizzato XY (Märzhäuser), e un ambiente controllato ( temperatura, umidità e CO 2) fase di incubatore (Pecon). Controlli sperimentali sono fondamentali per eliminare fluoroforo cross-talk tra i canali di emissione, nonché per valutare i problemi di espressione e non specifici multimerization fluoroforo. Come tale, trasfezioni fluoroforo singole sono utilizzate per l&#39;impostazione di ogni sessione di immagine. Negativo FRET controlli (con nota recettori non interagenti) dovranno essere eseguite, al fine di determinare lo sfondo FRET che si verifica a causa di espressione oltre. <ol><li> Per eliminare le interferenze tra i canali di emissione per PCP e YFP, posizionare il controllo della PCP espressione nell&#39;incubatore palco. Utilizzando luce bianca, regolare la Z-piastra di concentrarsi sullaalle cellule. </li><li> Impostare la finestra delle impostazioni SP per il rilevamento delle emissioni di PCP a 465-505 nm e YFP di 525-600 nm. </li><li> Tenendo sotto controllo i due canali di emissione, eccitare la PCP a 458 nm. Utilizzando il AOTF, impostare la potenza del laser per eliminare sanguinare apprezzabile rispetto della politica comune della pesca nel canale di emissione YFP. Salva questa impostazione. </li><li> Eseguire lo stesso controllo per YFP crosstalk (utilizzando il controllo YFP espressione) nel canale PCP regolando la potenza di eccitazione a 514 nm per le cellule che esprimono YFP solo. Salva questa impostazione. </li><li> Cellule prossimo luogo co-esprimono PCP e YFP nell&#39;incubatore palco. Utilizzando l&#39;impostazione sequenza per il software Leica confocale, individuare cellule che esprimono livelli simili di PCP e YFP con la localizzazione di membrana adeguato. </li><li> Utilizzando il software Leica accettore fotometabolismo programma, configurare le impostazioni di donatore e accettore al CFP salvate e le impostazioni YFP, rispettabile. </li><li> Zoom sul cellulare, evidenzia un ROI (regione di interesse) in cui la foto-distruzione di YFP deve avvenire sulla membrana cellulare e avviare il programma. </li><li> Per foto-distruzione del accettore, regolare il AOTF al 100% a 514 nm e consentono il laser sbiancante per continuare fino al 70% delle emissioni di YFP è stato impoverito. Per accelerare il candeggio, ROI sono scelti tipicamente nell&#39;asse raster del laser (asse X). </li><li> Durante fotometabolismo, monitorare il movimento cellulare e rifiutare misurazioni ottenute con il movimento apprezzabile nel piano XY, in quanto tali misure possono rapporto falso FRET efficienza. </li><li> Immagini pre-e post-candeggina sono registrati sia per i donatori (PCP) e accettore (YFP) e FRET efficienza viene calcolata come: FRET Eff = (D post-D pre) / D post per tutti i messaggi D&gt; D dove D pre pre e D post è l&#39;intensità donatore prima e dopo photobleaching rispettivamente. </li><li> Software JMP (SAS) è usato per determinare il grado di variabilità sperimentale. </li></ol> 4. Rappresentante Risultati Efficienze di trasfezione sono di solito tra il 30 e il 40%. Nonostante i risultati precedenti da altri, non abbiamo visto che la trasfezione ed espressione di un vettore favorisce quella degli altri. Anzi, spesso ci osservano espressione esclusiva di una fluoroforo chimera o l&#39;altro. Tipico FRET efficienze variano tra sistemi recettoriali. Per il sistema recettore Tie, valori tipici sono 20-28% per le cellule epiteliali e 19-23% nelle cellule endoteliali. Per i controlli negativi, l&#39;efficienza tipica sono al di sotto del 2-3%. Il grado di variabilità si riduce notevolmente con l&#39;esperienza. <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="Figura 1" /> Figura 1. Immagini rappresentative del transfettate EA.hy 926 cellule. A) immagine DIC di EA.hy 926 monostrato cellulare. B) l&#39;immagine di fluorescenza di cellule visualizzati in (A). C) Sovrapposizione di (A) e (B) che dimostrano l&#39;efficienza di trasfezione ~ 20-30%. <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="Figura 2" /> Figura 2. Accettore rappresentante fotometabolismo analisi di FRET si verificano tra CFP e YFP in una cella EA.hy926. La PCP emissione canale (pannelli in alto) e YFP emissione di canale (in basso due pannelli) sono stati monitorati separatamente, prima e dopo photobleaching accettore. Fotodecolorazione esperimenti sono stati limitati a, e FRET valori calcolati da, la regione all&#39;interno della scatola verde. FRET efficienza viene visualizzato come un campo da assoluto alto (rosso-1.0) a bassa (viola-0.0) su una sovrapposizione ingrandita di una PCP / YFP fusa immagine a scopo orientativo solo.

<ol>
	<li>Kim, M., Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+transmembrane+signaling+by+cytoplasmic+domain+separation+in+integrins.">Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.</a> Science. 301, 1720-1725 (2003).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipidmodified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Seegar, T. C. M., Eller, B., Tzvetkova-Robev, D., Kolev, M., Henderson, S. C., Nikolov, D. B., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1-Tie2+interactions+mediate+functional+differences+between+angiopoietin+ligands.">Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.</a> Molecular Cell. 37 (5), 643-655 (2010).</li><li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc Natl Acad Sci U S A. 80 (12), 3734-3737 (1983).</li></ol>

Nessun conflitto di interessi dichiarati.

Ci sono diversi passaggi che sono cruciali per il successo. Il più importante tra questi è il livello relativo di espressione tra i due recettori chimerici. Per aggirare questo problema, si può fare linee cellulari stabili che esprimono entrambe le proteine ​​di interesse, o identificare i rapporti ottimali di DNA vettore per consentire l&#39;espressione equivalente. Allo stesso modo, a causa della non uniforme transfezione in un piatto, i livelli di proteina sarà raramente equivalente tra le cellule. Pertanto, si deve fare attenzione a distinguere &#39;alta&#39; expressors dal &#39;basso&#39;. Quelli che i livelli di &#39;medio&#39; di visualizzazione di proteine ​​tipicamente resa affidabile e riproducibile FRET efficienza. Un metodo nostro laboratorio ha perseguito per alleviare questo problema è l&#39;uso di adeno-virus e Lenti a &#39;anche&#39; l&#39;espressione genica. Inoltre, un metodo alternativo per accettore fotometabolismo per determinare l&#39;efficienza FRET è emissione sensibilizzata. Anche se, nonostante il potenziale di emissione sensibilizzati a controllare singole cellule in tempo reale, abbiamo trovato accettore fotometabolismo ad essere più sensibili e più affidabile. Infine, utilizzando FRET di monitorare le interazioni proteina può essere impegnativo, e richiede una attenta selezione delle lunghezze linker per i recettori di membrana legato. Inoltre, l&#39;aggiunta di C / YFP spesso influenza notevolmente i livelli di espressione della proteina e si traduce in aggregazione nel reticolo endoplasmatico e apparato di Golgi. Tuttavia, per le interazioni transitori o instabile,, FRET è la metodologia ideale da utilizzare.

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<td>DMEM</td>
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<td>11960-069</td>
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<td>Penicillin- Streptomycin</td>
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<td>Invitrogen</td>
<td>15070-063</td>
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<td>N/A</td>
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<td>&nbsp;</td>
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<td>11058-021</td>
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<td>FUGENE 6</td>
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<td>Coverslip Dishes</td>
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imaging protein-protein interactions in vivo

Interazioni proteina-proteina sono una caratteristica di tutti i processi cellulari fondamentali. Tuttavia, molte di queste interazioni sono transitori, o energeticamente deboli, impedendo la loro identificazione e analisi attraverso i tradizionali metodi biochimici come la co-immunoprecipitazione. A questo proposito, le proteine ​​geneticamente codificabili fluorescente (GFP, RFP, ecc) e le loro associate sovrapposizione spettro di fluorescenza hanno rivoluzionato la nostra capacità di monitorare le interazioni deboli in vivo, utilizzando Förster trasferimento di energia di risonanza (FRET) 1-3. Qui, noi dettaglio il nostro uso di un saggio basato su FRET di prossimità per il monitoraggio recettore-recettore sulla superficie delle cellule endoteliali.

Watch this Scientific Journal Video about Imaging Protein-protein Interactions in vivo at JoVE.com

Imaging Protein-protein Interactions in vivo

Questo protocollo descrive come immagine interazioni proteina-proteina mediante un saggio basato su FRET prossimità.

Imaging interazioni proteina-proteina In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.2K Views.  Virginia Commonwealth University. Questo protocollo descrive come immagine interazioni proteina-proteina mediante un saggio basato su FRET prossimità.

Video: Imaging Protein-protein Interactions in vivo

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Ricerca

Biologia

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In vivo di rilevamento di interazioni proteina-proteina su disegno micro, Superfici

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interazioni proteina-proteina visualizzati tramite bimolecular complementazione fluorescenza in protoplasti di tabacco e foglie

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Sviluppo di inibitori di interazioni proteina-proteina attraverso REPLACE: Applicazione alla progettazione e allo sviluppo non ATP inibitori CDK competitivo

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Studio delle interazioni proteina-proteina nella ricerca di autofagia

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Avanzate tecniche di microscopia confocale per studiare le interazioni proteina-proteina e cinetica alle lesioni del DNA

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Analisi di Imaging di complementazione di luciferase in foglie di Nicotiana benthamiana per transitoriamente determinare dinamiche di interazione proteina-proteina

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...