Name: evaluation of the impact of protein aggregation on cellular oxidative stress in yeast
Uploaded: 2018-06-23T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast at JoVE.com

1酵母文化や蛋白発現。
注: a β 亜種展示位置 19 (Phe19) Aβ42 ペプチド (図 1 a) の 1 つ残基変異による異なる相対集計性癖。これらのペプチドの亜種は緑色蛍光タンパク質 (GFP) 集計記者 (図 1)29として機能するとタグ付けされます。
<ol><li>BY4741 親の背景 (マタ彼3Δ1ルー2Δ0に会った15Δ0 <...

<ol>
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病気の広い範囲は、携帯電話預金6,7,8,33への誤って折りたたまれたタンパク質の蓄積にリンクされます。アカウント タンパク質濃度を考慮していない、計算のアプローチを使用してこれらの病気の発症を引き起こす分子メカニズムを解明するため多くの努力がなされたまたは体外に近づく?...

プロテオステイシスはセルのフィットネスと老化のプロセスの基本的な決定要因です。ネットワーク、正しいリフォールディング conformers の誤って折りたたまれたタンパク質のシャペロンおよびその対象となるタンパク質がいくつか保存されているメカニズム1 を確保することを目的と高度な品質管理によって細胞の蛋白質の恒常性を維持します。 ,2,3,4,5。多数の調査では、発症および人間の病気の広い範囲とのプロテオステイシス、タンパクにつながる障害の進行の間のリンクをサポートします。例えば、タンパク質の堆積物の存在の多くの神経変性疾患、アルツハイマー病、パーキンソン病などとハンチントン病6,7、8、病理学的特徴であります。prionogenic 病と非変性アミロイドーシス9。それは、凝集反応の初期のオリゴマーと protofibrillar のアセンブリが混雑した細胞環境の10の他の蛋白質と異常な相互作用を確立する細胞毒性の主な成分エリシターであることが示唆されています。また、その毒性効果11,12を伝播する、細胞間タンパク質介在物 (PI) を送信できます。したがって、それは、PI の形成可能性があります確かに危険な集約された種の存在を処理または主要な副作用のない蓄積があるなどこのセルに特定の場所に制限する解毒機構を構成するかもしれない13,14。
標準的な体外生化学的アプローチは、凝集反応およびそのプロパティ15,16種に重要な洞察を提供しています。しかし、これらの試金で使用される条件は、細胞内で発生する明確に異なると、したがって、その生理学的な妥当性を質問します。タンパク質品質管理、オートファジーは、細胞の酸化還元状態の17,18真核生物19,20,21 の規制など携帯電話経路の注目すべき保全のため ,22,23, 出芽酵母酵母(出芽酵母) 蛋白質の集合の分子決定要因を研究する特権を持つ単純な細胞モデルとして浮上しています。関連する生物学的環境24,25,26で関連付けられた細胞毒性影響。
タンパク質凝集傾向は、本質的に主なシーケンスでエンコード機能です。識別と集計推進の力の評価に基づくアミロイド様構造の形成を予測できるため、ポリペプチド27の地域。しかし、タンパク質の体外集計プロパティを予測するバイオインフォマティクス アルゴリズムの成功にもかかわらずこれらの性癖が生体内で細胞毒性影響に変換する方法を予測には程遠いです。体系的に特定の蛋白質の集合の状態とその関連付けられている細胞の損傷間のリンクに対応する研究はこの計算の制限を回避するために役立つことがあります。この接続は、単一の残渣でのみが異なるが、凝集体内性癖28の連続した範囲を表示するアミロイド β ペプチド Aβ42 の亜種の大規模なセットを利用して、本研究で扱われます。特に、酵母細胞の凝集を起こしやすい蛋白質によって誘発される酸化的損傷の会計構造の種を識別するために FC ベースのアプローチを説明します。方法論は、シンプルさ、高処理能力と正確な定量的測定など多くの利点を提供します。このアプローチでは、PI プレイ酸化ストレスに対する保護の役割を確認可能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast cells BY4741&#160;</td>
			<td>ATCC</td>
			<td>201388</td>
			<td>Genotype: MATa his3&#916;1 leu2&#916;0 met15&#916;0 ura3&#916;0</td>
		</tr>
		<tr>
			<td>pESC(-Ura) plasmid&#160;</td>
			<td>Agilent Genomics</td>
			<td>217454</td>
			<td>Yeast expression plasmid with a Gal promotor. Selectable marker URA3</td>
		</tr>
		<tr>
			<td>Yeast Synthetic Drop-out Medium Supplements</td>
			<td>Sigma</td>
			<td>Y1501</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base Without Amino Acids</td>
			<td>Sigma</td>
			<td>Y0626</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Sigma</td>
			<td>R7630</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma</td>
			<td>G0750</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP3991&#160;</td>
			<td>Solution 10X</td>
		</tr>
		<tr>
			<td>CellROX Deep Red Reagent&#160;</td>
			<td>Life Technologies</td>
			<td>C10422</td>
			<td>Free radical cell-permeant fluorescent sensor, non-fluorescent while in a reduced state, and exhibits bright fluorescence upon oxidation by reactive oxygen species (ROS), with absorption/emission maxima at 644/665 nm.&#160;</td>
		</tr>
		<tr>
			<td>Y-PER protein extraction reagent&#160;</td>
			<td>Thermo Scientific</td>
			<td>78990</td>
			<td>Liquid cell lysis buffer</td>
		</tr>
		<tr>
			<td>Acrylamide/Bis-acrylamide</td>
			<td>Sigma</td>
			<td>A6050</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Bradford dye reagent</td>
			<td>Bio-Rad&#160;</td>
			<td>5000205</td>
			<td>Dye reagent for one-step determination of protein concentration</td>
		</tr>
		<tr>
			<td>&#946;-amyloid antibody 6E10&#160;</td>
			<td>BioLegend</td>
			<td>803001</td>
			<td>Mouse IgG1. The epitope lies within amino acids 3-8 of beta amyloid (EFRHDS).</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP conjugate&#160;</td>
			<td>Bio-Rad</td>
			<td>1721011</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Immobilon-P, PVDF</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminata forte</td>
			<td>Merk</td>
			<td>WBLUF0100</td>
			<td>Premixed, ready to use chemiluminescent HRP detection reagent</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride solution (PMSF)</td>
			<td>Sigma</td>
			<td>93482</td>
			<td>Protease inhibitor. Dissolved at 0.1 M in ethanol</td>
		</tr>
		<tr>
			<td>FACSCanto flow cytometer&#160;</td>
			<td>BD Biosciences</td>
			<td>657338</td>
			<td>Equipped with a 488&#160;nm blue laser for the detection of GFP, and 635&#160;nm red laser / 530/30 nm BP filter and 660/20 BP filter</td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Electrophoresis Transfer cell</td>
			<td>Bio-Rad</td>
			<td>1703930</td>
			<td>Protein transference system</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Handcast Systems</td>
			<td>Bio-Rad</td>
			<td>1658000FC</td>
			<td>Electrophoresis system</td>
		</tr>
	</tbody>
</table>

evaluation of the impact of protein aggregation on cellular oxidative stress in yeast

タンパク質のフォールディングとアミロイドの立体構造に集約は、発症といくつかの神経変性疾患の進行に関連しています。しかし、少し情報はまだどのように不溶性タンパク質について集計はその毒性で生体を発揮します。細菌や酵母などの単純な原核生物と真核生物のモデル生物は、細胞内アミロイド線維形成、集計の伝達、および毒性の背後にあるメカニズムの私達の現在の理解に大きく貢献しています。このプロトコルでは、酵母の使用は、タンパク質凝集体の形成と細胞の酸化ストレスに与える影響との関係を分析するモデルとして記述されます。メソッドは、フローサイトメトリー (FC) を使用して、式から生じる細胞の酸化損傷の定量化とアミロイド蛋白質の細胞内の可溶性/集計状態の検出を兼ね備えています。この方法は、簡単、迅速、かつ定量的です。研究は、彼らのそれぞれの組み込み集計性癖とアミロイド β ペプチド亜種の大規模なセットによって引き起こされる細胞の酸化ストレスを関連付けることによってテクニックを示しています。

このプロトコルでは、Phe19 がすべて天然蛋白質構成アミノ酸28変異されてどこ Aβ42 ペプチドの 20 個の商品のコレクションを使用する方法について説明します。これらの蛋白質の集計の理論的な性癖は、2 つの異なるバイオ情報アルゴリズム (AGGRESCAN とタンゴ31,32) を使用して分析できます。両方のケースで?...

Watch this Scientific Journal Video about Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast at JoVE.com

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

酵母細胞の酸化ストレスに対するタンパク質凝集の影響の評価

蛋白質の集合は、細胞の酸化ストレスを引き出します。このプロトコルでは、アミロイド蛋白質の細胞内の状態およびフローサイトメトリーを使用して、それらに関連付けられている酸化ストレスを監視する方法について説明します。アミロイド β ペプチドの水溶性で集計しやすい亜種の挙動を調べるためのアプローチが使用されます。

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Cryomilled哺乳動物細胞からのタンパク質複合体の親和性のキャプチャ

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

カエノラブディティス ・ エレガンスにおけるハイスループット化合物スクリーニングのための簡単な方法

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

蛋白質の相互作用を比較するバッチの酵母 2 ハイブリッド スクリーン

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

体外自己組織化人工脂質膜上のタンパク質パターンの再構成

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

力の単一分子分光法のための蛋白質の共有結合固定

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

タンパク質の折り畳み、トランスポート、および放射性パルス追跡による生体細胞の劣化の解析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

膜上消化技術を用いてのタンパク質・タンパク質相互作用の評価

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

酸化ストレス中のフィブロネクチン上皮細胞の細胞接着と広がりのダイナミクスを調べる

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

出芽酵母サッカロミセスセレビシエにおけるサブソキアドタンパク質の局在化の評価

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...