我々は、共焦点顕微鏡との助けのために博士スコットヘンダーソンの認知を行う予定。この研究は、医学のマジーがんセンターと学校（VCU）からWABの顕微鏡へのWABへの保健1RO1CA127501の国立研究所からの補助金だけでなく、パイロットプロジェクトの資金調達によってサポートされていましたVCU - DEPTで行われました。 NIHNINDSセンターコアグラント5P30NS047463からの資金で、一部では、サポートされている神経生物学と解剖顕微鏡施設、の。

プロトコルは、次の3つの主要手順で構成されています。 CFP及び/またはYFPの単量体のバージョンへのアミノ末端哺乳類発現ベクターに目的遺伝子の最初の、ステップクローニングにのみ簡単に説明します。 2番目と3番目の手順は、FRETを持つEA.hy926内皮細胞へのベクターのDNAのトランスフェクション、および共焦点イメージング、以下でもっと詳しく説明されています。 1。 CFPとYFPキメラ受容体の構築興味の受容体は、CFPとYFPの単量体の強化バージョンにアミノ末端クローニングすることがあります。 FRETの決定は経験的であり、蛍光体1の膜貫通領域と開始の間のリンカーの長さに大きく依存です。したがって、リンカのいくつかの異なる長さを調査中のそれぞれの新しい受容体のペアのために検討する必要があります。 <ol><li>単量体のC -またはYFPを（このベクトルは私たちの研究室から入手可能）を含むであるpcDNA3.1 hygro RまたはネオRベクターに 、NheI部位/ EcoRI部位に興味のある受容体のクローンを作成する。 *注：必要に応じて、NheI部位をSpeIとXbaIでを含む互換性のあるいくつかの他の酵素である。 </li><li>適切なendA1 recAのコンピテントセルの行（我々が日常的にMACH1を使用する）にあなたのライゲーション反応を変換する。変異の有無を確認するためにDNA配列決定に続いて、挿入の有無を画面のコロニー。 </li><li>成功した分子クローニングのコンフォメーションを取得後、キアゲン社、または類似の、DNAのキットを使用して滅菌水やTEでトランスフェクショングレードのベクターDNAを準備する。 UV分光法を用いて濃縮し、純度を評価する。 DNAの典型的な収量は、日常の1μg/μLの作業用ストックに希釈して100から200μgの範囲内でなければなりません。 </li></ol> 2。 EA.hy 926細胞のトランスフェクション<ol><li>マテックカバースリップ（№0）を2mLの培地で35 mmディッシュに完全な- DMEM（DMEM +10％ウシ胎児血清+ Pen. /連鎖球菌。）で成長させたEA.hy 926セル4を 、分割。あなたは控えめに15センチメートルコンフルエント皿から〜15皿（〜90％コンフルエントに翌日）を取得することができます。個々の受容体の発現だけでなく、各受容体のペアのための一皿を評価するための一皿を準備します。 </li><li>細胞は37℃、5％CO 2雰囲気下で一晩インキュベートすることができます℃に翌日、培養物は、〜70〜90％コンフルエントにする必要があります。 </li><li>トランスフェクション当日に、予め温めておいたOPTIMEM 200μLのFugene6 8μLでキメラ受容体のベクターDNA（2μL）2μgを混合することによりnucleolipid複合体を準備する。受容体のペアの場合は、2μgの（また、以下の説明を参照）の合計については、各受容体の1μgのフェ。 </li><li>静かに転倒混和します。 </li><li> 30分間室温でインキュベートし続ける。 </li><li>潜伏期間中に、トランスフェクションのためにセルを準備します。新鮮な培地をオフに吸引し、抗生物質を含まない2mLの予め温めておいた完全な- DMEM（DMEM + FBS）を添加する前に温かいPBSで穏やかにし、簡単に各プレートを洗浄する。彼らはFugene6による治療中にかなり多くの有毒な細胞になるとして、ペニシリンとストレプトマイシンの存在を避けることが重要です。 </li><li>細胞への賢明な複雑なドロップを追加し、37℃で、追加の20〜24時間インキュベート℃、5％CO 2雰囲気でCを。 </li><li> 24時間後、慎重に抗生物質を持つ細胞とピペット新鮮な完全な- DMEMから培地を吸引除去する。トランスフェクション後、細胞生存率が比較的高いままにする、無浮遊細胞にはほとんどが観察されるべきである、すなわち。 </li><li>遺伝子発現は、追加の48から60時間後にトランスフェクション後、通常は最大です。しかし、画像は24時間可能な限り短く、一般的に実現可能です。最適な遺伝子発現のタイムコース実験を実行する必要があります。 </li></ol> 3。共焦点イメージングとFRETの解析ライブセルイメージングは​​青色ダイオード（波長405nm）、アルゴン（458、476、488、514 nm）を装備したライカTCS - SP2 AOBS共焦点レーザー走査型顕微鏡で実行されます。グリーンHeNeレーザ（543 nm）を、オレンジ色のヘリウムネオン（594 nm）、および赤色ヘリウムネオン（633 nm）のレーザー、HCX PIアポ63x/1.3 NAグリセリン浸対物レンズ、電動XYステージ（Märzhäuser）、及び環境制御（温度、湿度、およびCO 2）ステージインキュベーター（PeCon）。実験的なコントロールには、蛍光体の発光チャネルと同様に評価する式の問題や非特異的蛍光体の多量体間のクロストークを除去するために重要です。このように、単一の蛍光体のトランスフェクションは、すべての画像のセッションをセットアップするために利用されています。負の（既知のnoninteracting受容体を使用して）コントロールをFRETは、過剰発現が原因で発生するそのFRETのバックグラウンドを決定するために、実行する必要があります。 <ol><li> CFPとYFPの排出チャンネル間のクロストークを排除するために、ステージインキュベーターにCFP発現のコントロールを配置します。白色光を使用して、に焦点を当てるZ -プレートを調整細胞へ。 </li><li> 465から505 nmおよび525から600 nmのYFPへのCFPの発光検出のためにSPのウィンドウの設定を行います。 </li><li>両方の放出チャネルを監視しながら、458 nmのCFPを励起する。 AOTFを使用し、YFPの放出チャネルにCFPの上のかなりの出血を除去するためにレーザパワーを設定します。この設定を保存します。 </li><li>のみYFPを発現する細胞は514 nmで励起パワーを調整することによって、CFPのチャネルでYFPクロストーク（YFPの発現制御を使用して）に対して同じ制御を行います。この設定を保存します。 </li><li>次の場所細胞ステージインキュベーターにCFPとYFPを共発現。ライカ共焦点のソフトウェアのための順序設定を使用して、適切な膜局在化とCFPとYFPの同様なレベルを発現する細胞を見つけます。 </li><li>ライカのソフトウェアのアクセプターフォトブリーチングプログラムを使用して、立派に、保存したCFPとYFPの設定にドナーとアクセプターの設定を設定します。 </li><li>セルにドラッグズーム、YFPの写真破壊が細胞膜で発生し、プログラムを開始することになっているROI（関心領域）を強調表示します。 </li><li>アクセプターの光破壊の場合は、514 nmで100％にAOTFを調整し、YFPの排出量の70％が枯渇されるまで継続する漂白レーザーを許可する。漂白加速させるために、ROIは通常、レーザーのラスターの軸（X軸）に選ばれている。 </li><li>これらの測定値が偽のFRETの効率を報告できるように光退色時には、細胞の動きを監視し、XY平面でかなりの運動で得られた測定値を拒否する。 </li><li>事前事後の漂白剤のイメージはドナー（CFP）とアクセプター（YFP）の両方に記録し、効率は次のように計算されますFRETがされています：すべてのD のポスト &gt; D プリ D より前のためEffが =（D 後 D プリ ）/ D 後のFRETとD のポストは前に、それぞれ光退色後のドナーの強度です。 </li><li> JMPソフトウェア（SAS）は、実験的なばらつきの程度を決定するために使用されます。 </li></ol> 4。代表的な結果トランスフェクション効率は30〜40％の間、通常です。他のユーザーがこれまでの知見にもかかわらず、我々は一つのベクターのトランスフェクションと発現が、他のことを支持すること見ていない。実際、我々は頻繁にキメラまたはもう一つ蛍光団の排他的な発現を観察。典型的には効率が受容体のシステム間で異なるFRETの。タイ受容体システムの場合、典型的な値は、上皮細胞のための20から28パーセントと内皮細胞の19から23パーセントです。陰性コントロールの場合、典型的な効率は以下の2から3パーセントです。ばらつきの程度は、経験を大幅に減少します。 <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="図1" /> 図1。トランスフェクトEA.hy 926細胞の代表的な画像。EA.hy 926細胞単層の）DIC画像。 B）に表示されているセル（）の蛍光画像を。のC）オーバーレイ（）と（B）〜20-30％のトランスフェクション効率を実証。 <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="図2" /> 図2。代表的な受容体は、EA.hy926セルにCFPとYFPの間で発生するFRETの解析を光退色。CFP放出チャネル（上部パネル）とYFP放出チャネル（下の2パネル）には、事前に別々に監視し、後のアクセプターが退色した。退色実験に限定され、緑色のボックス内の領域、から計算された値のFRETていた。 FRETの効率は、オリエンテーションの目的のために画像をマージCFP / YFPの拡大オーバーレイの低（紫- 0.0）に高いから絶対的な範囲（赤- 1.0）として表示されます。

<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>

利害の衝突は宣言されません。

成功への重要ないくつかのステップがあります。その中で最も顕著には2つのキメラ受容体間の発現の相対的なレベルです。この問題を回避するためには、関心の両方のタンパク質を発現する安定細胞株を作る、または同等の発現を可能にするベクターDNAの最適な比率を識別することができる。同様に、料理全体で不均一なトランスフェクションのために、タンパク質レベルではほとんどの細胞の間で同等できなくなります。そのため、注目は&quot;低&quot;から&quot;ハイ&quot;expressorsを区別するために支払わなければなりません。タンパク質の表示&quot;平均的&quot;レベルは、通常、信頼性と再現性が得られること、それらは効率のFRET。私たちの研究室ではこの問題を緩和するために追求してきた一つの方法は、&#39;even&#39;は遺伝子発現へのアデノとレンティ-ウイルスの使用です。また、アクセプターフォトブリーチングを決定するための代替方法では効率が感作された排出であるFRETの。されているとはいえ、リアルタイムで単一のセルを監視するために増感発光の可能性にもかかわらず、我々はアクセプターフォトブリーチングより高感度と信頼性が高いことがを発見した。 最後に、タンパク質相互作用を監視するためのFRETの使用は困難な場合、および膜結合型受容体のためのリンカーの長さを慎重に選択する必要がありますすることができます。さらに、C / YFPの加算は、しばしば非常に小胞体とゴルジ体に集約におけるタンパク質の発現レベルとの結果に影響を与えます。しかし、一時的な、または不安定な相互作用のために、FRETは、利用することが理想的な方法論です。

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imaging protein-protein interactions in vivo

タンパク質 - タンパク質相互作用はすべての本質的な細胞プロセスの特徴です。しかし、これらの相互作用の多くはそのような共免疫沈降法などの従来の生化学的手法でその識別と分析を防止する、一過性、または精力的に弱いです。この点では、遺伝的にコード化可能蛍光タンパク質（GFP、RFPなど）とそれに関連付けられた重複する蛍光スペクトルは、フェルスター共鳴エネルギー移動（FRET）1-3を用いて生体内で弱い相互作用を監視するために我々の能力に革命をもたらしています。ここで、我々は詳細内皮細胞表面上の受容体 - 受容体相互作用を監視するためのFRETに基づく近接アッセイの使用量を。

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

Imaging Protein-protein Interactions in vivo

このプロトコルは、FRETに基づく近接アッセイを使用してどのようにイメージタンパク質間相互作用について説明します。

イメージングタンパク質間相互作用 in vivoで</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 ビュー.  Virginia Commonwealth University. このプロトコルは、FRETに基づく近接アッセイを使用してどのようにイメージタンパク質間相互作用について説明します。

ビデオ: 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.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

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

生物学

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.

マイクロパターン化表面でのタンパク質間相互作用の in vivo検出

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.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

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.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

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.

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

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.

タンパク質 - タンパク質相互作用の阻害剤の開発を通じてREPLACE：アプリケーションの設計と開発非ATP競合CDK阻害剤に

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.

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</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.

オートファジー研究でタンパク質間相互作用の研究

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.

タンパク質-タンパク質相互作用や DNA 損傷速度論を研究する共焦点の顕微鏡検査の技術を高度な

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.

ベンサミアーナ タバコ葉蛋白質蛋白質の相互作用ダイナミクスの一過性の決定のための試金補完ルシフェラーゼ イメージング

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