Name: growth assays to assess polyglutamine toxicity in yeast
Uploaded: 2012-03-05T12:00:00
Description: Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Arbejdet i Duennwald laboratoriet er støttet af tilskud fra American Federation for Aldringsforskning (Afar), den arvelige sygdom Foundation (HDF) og William Wood Foundation.

1. Angivelse af Giftige PolyQ-ekspansion proteiner i gær En systematisk analyse har etableret den præcise aminosyre-sekvensen af en polyQ udvidelseskoefficient protein, der kræves for at frembringe toksicitet i gær 7. Dette giftige polyQ udvidelseskoefficient protein indeholder en amino-terminal FLAG-tag, efterfulgt af 17 aminosyrer fra den oprindelige sekvens huntingtin protein, et polyQ region og en carboxy-terminal fusion til et fluorescerende protein (enten GFP eller CFP, se...

<ol>
	<li>Soto, C., Estrada, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+and+neurodegeneration.">Protein misfolding and neurodegeneration.</a> Arch. Neurol. 65, 184-189 (2008).</li><li>Ross, C. A., Tabrizi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+from+molecular+pathogenesis+to+clinical+treatment.">Huntington's disease: from molecular pathogenesis to clinical treatment.</a> Lancet Neurol. 10, 83-98 (2011).</li><li>Zoghbi, H. Y., Orr, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+repeats+and+neurodegeneration.">Glutamine repeats and neurodegeneration.</a> Annu. Rev. Neurosci. 23, 217-247 (2000).</li><li>Meriin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+machinery+is+involved+in+aggregation+of+proteins+with+expanded+polyglutamine+domains.">Endocytosis machinery is involved in aggregation of proteins with expanded polyglutamine domains.</a> FASEB J. 21, 1915-1925 (2007).</li><li>Giorgini, F., Guidetti, P., Nguyen, Q., Bennett, C. S., Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+screen+in+yeast+implicates+kynurenine+3-monooxygenase+as+a+therapeutic+target+for+Huntington+disease.">A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease.</a> Nat. Genet. 37, 526-5231 (2005).</li><li>Duennwald, M. L., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+ERAD+and+ER+stress+are+early+and+specific+events+in+polyglutamine+toxicity.">Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity.</a> Genes Dev. 22, 3308-3319 (2008).</li><li>Duennwald, M. L., Jagadish, S., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flanking+sequences+profoundly+alter+polyglutamine+toxicity+in+yeast.">Flanking sequences profoundly alter polyglutamine toxicity in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11045-1150 (2006).</li><li>Duennwald, M. L., Jagadish, S., Giorgini, F., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+network+of+protein+interactions+determines+polyglutamine+toxicity.">A network of protein interactions determines polyglutamine toxicity.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11051-116 (2006).</li><li>Meriin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington+toxicity+in+yeast+model+depends+on+polyglutamine+aggregation+mediated+by+a+prion-like+protein+Rnq1.">Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1.</a> J. Cell. Biol. 157, 997-1004 (2002).</li><li>Murakami, C., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Yeast+Chronological+Life+Span+by+Outgrowth+of+Aged+Cells.">Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells.</a> J. Vis. Exp. (27), e1156-e1156 (2009).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beer+and+bread+to+brains+and+beyond:+can+yeast+cells+teach+us+about+neurodegenerative+disease.">Beer and bread to brains and beyond: can yeast cells teach us about neurodegenerative disease.</a> Neurosignals. 16, 52-62 (2008).</li><li>Giorgini, F., Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+genetic+modifiers+of+amyloid+toxicity+in+yeast.">Screening for genetic modifiers of amyloid toxicity in yeast.</a> Methods Enzymol. 412, 201-222 (2006).</li><li>Ehrnhoefer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+tea+(-)-epigallocatechin-gallate+modulates+early+events+in+huntingtin+misfolding+and+reduces+toxicity+in+Huntington's+disease+models.">Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models.</a> Hum. Mol. Genet. 15, 2743-2751 (2006).</li><li>Cashikar, A. G., Duennwald, M., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chaperone+pathway+in+protein+disaggregation.+Hsp26+alters+the+nature+of+protein+aggregates+to+facilitate+reactivation+by+Hsp104.">A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104.</a> J. Biol. Chem. 280, 23869-2375 (2005).</li></ol>

Dette håndskrift skitserer tre komplementære eksperimentelle metoder til at måle polyQ-toksicitet i model organisme gær baseret på reduceret vækst af gærceller, der udtrykker giftige polyQ-ekspansion proteiner. Arbejde i gær har tilbudt dybtgående indsigt i de grundlæggende cellulære og molekylære mekanismer af protein misfoldning og dens efterfølgende toksicitet, herunder misfoldning og toksicitet af polyQ-ekspansion proteiner 9,11,12. Eksperimenter baseret på de protokoller, der præsenteres h...

<table border="1"><tbody><tr><td> Navn på instrument </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Frogger (6x8 pins) </td><td> V &amp; P Videnskabelige, San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves USA </td><td> 5101370 </td></tr><tr><td> 100-såvel honeycomb plader </td><td> Growthcurves USA </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Protein fejlfoldning er forbundet med mange humane sygdomme, især neurodegenerative sygdomme, såsom Alzheimers sygdom, Parkinsons sygdom og Huntingtons sygdom 1. Huntingtons sygdom (HD) er forårsaget af unormal vækst af en polyglutamine (polyQ) region i proteinet huntingtin. Den polyQ-ekspanderet huntingtin protein opnår en afvigende kropsbygning (dvs. den misfolds) og forårsager cellulær toksicitet 2. Mindst otte yderligere neurodegenerative sygdomme skyldes polyQ-udvidelser, herunder spinocerebellar ataksi og Kennedys sygdom 3. Modellen organisme gær har lettet betydeligt indsigt i cellulære og molekylære grundlag for polyQ-toksicitet, inklusive virkningen af ​​intra-og inter-molekylære faktorer polyQ-toksicitet, og identifikation af cellulære veje, der er svækkede i celler, som udtrykker polyQ-udvidelse proteiner 3-8. Vigtigtly blev mange aspekter af polyQ-toksicitet, der blev fundet i gær er gengivet i andre eksperimentelle systemer og i nogen udstrækning i prøver fra HD-patienter, hvilket viser betydningen af ​​gærmodel for opdagelsen af ​​grundlæggende mekanismer underbygger polyQ-toksicitet. En direkte og relativt enkel måde at bestemme polyQ-toksicitet i gær er at måle vækstdefekter af gærceller, der udtrykker polyQ-ekspansion proteiner. Dette skrift beskriver tre komplementære eksperimentelle fremgangsmåder til at bestemme polyQ-toksicitet i gær ved at måle væksten af ​​gærceller, der udtrykker polyQ-ekspansion proteiner. De første to eksperimentelle tilgange overvåge gær vækst på plader, den tredje tilgang overvåger væksten af ​​flydende gær kulturer ved hjælp af BioscreenC instrument. Desuden er denne manuskript beskriver eksperimentelle vanskeligheder, der kan opstå ved håndtering af gær polyQ modeller og skitserer strategier, der vil bidrage til at undgå ellerminimere disse problemer. Protokollerne der er beskrevet her, kan anvendes til at identificere og karakterisere genetiske veje og små molekyler, der modulerer polyQ-toksicitet. Desuden kan de beskrevne assays tjener som templates for nøjagtige analyser af toksicitet forårsaget af andre sygdom-associerede fejlfoldede proteiner i gær modeller.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Dette håndskrift beskriver tre komplementære protokoller for at vurdere toksiciteten af ​​polyglutamine (polyQ)-ekspansion proteiner i gær<em&gt; Saccharomyces cerevisiae</em&gt;. Disse protokoller kan let modificeres til at overvåge toksicitet andre fejlfoldede proteiner i gær.

Vækstassays til Vurdere polyglutamine Toksicitet i gær

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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