We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1. Biblioteca Generation <ol><li> Per costruire librerie di Hsp104 utilizzando soggetto a errori PCR specifico del dominio, prima amplificare il dominio di interesse con un errore DNA polimerasi inclini 20. </li><li> Purificare il prodotto di PCR mediante estrazione gel. </li><li> Eseguire un passo estensione megaprimer utilizzando un protocollo standard di mutagenesi sito-diretta: unire 50 ng modello plasmide, 250 ng megaprimer, 200 mM dNTP, e ad alta fedeltà polimerasi DNA in tampone PCR, e portare a 50 ml volume totale di acqua di grado PCR 20 . Eseguire un programma standard di PCR. NOTA: primer specifici usati varieranno in base alla particolare regione del gene che deve essere amplificato. <ol><li> A seguito di PCR, digerire il DNA dei genitori modello con 1 ml Dpn I restriction enzyme per 2 ore a 37 ° C. </li></ol></li><li> Trasforma la libreria di elettroporazione o altri mezzi a ultracompetent E. coli e purificarlo da miniprep. NOTA: generazione Bibliotecavariabile in base sostanzialmente sugli obiettivi di un dato esperimento. Hsp104 è una grande proteina, quindi non è pratico di randomizzare l&#39;intero gene, che è per questo che utilizziamo errore specifico del dominio incline PCR. Inoltre, la struttura di Hsp104 rimane poco compresa, rendendo la progettazione di librerie diretti impegnativi 21. Le librerie possono essere costruiti utilizzando approcci dirette o casuali per mutagenesi, con l&#39;unica limitazione è che il plasmide ossatura modello deve contenere il gene URA3 per consentire 5-FOA contatore scelta su supporti destrosio. </li></ol> 2. Trasformazione della Biblioteca Hsp104 <ol><li> Integrare il substrato associata a malattia in lievito Hsp104 W303aΔ con un acetato di litio standard di / PEG protocollo di trasformazione 22. Selezionare singole colonie e lo screening per la tossicità per isolare un ceppo con elevata tossicità della malattia substrato 23 associati. NOTA: Utilizzare un ceppo integrato e isolaTé una singola colonia per garantire la parità di espressione in tutte le cellule. Clone substrato malattia in un plasmide permette l&#39;integrazione del gene sotto qualsiasi marcatore diverso uracile (usiamo istidina) e la libreria Hsp104 nel plasmide pAG416GAL (marcatore uracile) 24. Per consentire 5-FOA counterselection, assicurarsi che la libreria è espresso da un plasmide con il marcatore uracile. Altri ceppi di lievito possono anche essere impiegati. Abbiamo notato un simile fenotipo soppressione di tossicità utilizzando W303a e BY4741 ceppi di lievito in entrambi gli sfondi Hsp104 WT e Δ (MEJ e JS, osservazioni non pubblicate). </li><li> Trasforma la libreria Hsp104 in questo ceppo utilizzando lo stesso acetato di litio / PEG protocollo di trasformazione 22. Scale la trasformazione appropriato per mantenere la sequenza spazio della biblioteca e di preservare la dimensione biblioteca predetto. </li><li> Piatto il composto trasformazione su noninducing, piastre selettive (ad esempio, SD-His-Ura) con un numero sufficiente di piastre aassicurare la crescita di un gran numero di colonie. Utilizzare grandi piastre di Petri (150 mm) per ridurre al minimo il numero di piatti necessari. Recupero trasformanti che utilizzano piastre permette l&#39;efficienza di trasformazione da valutare. </li><li> Trasforma Hsp104 WT e controlli negativi vettoriali in parallelo. </li></ol> 3. Screening per la repressione del proteotoxicity <ol><li> Lavare le colonie al largo delle piastre con raffinosio integrato dei media di abbandono (ad esempio SRaff-His-Ura). Utilizzare una pipetta sierologica e applicatori in legno sterili per allentare le colonie dalle piastre. Trasferire i lavaggi liquido in una provetta da 50 ml e agitare accuratamente per separare eventuali grumi di cellule. Diluire a una cultura un po &#39;nuvoloso, OD 600 ~ 0.025. NOTA: Qui serve raffinosio per alleviare le cellule della repressione di glucosio-mediata di induzione, adescamento in tal modo le cellule per l&#39;induzione galattosio-mediata. </li><li> Far crescere la coltura diluita durante la notte nei mezzi di abbandono raffinosio con agitazione a 30 ° C. Far crescere la Hsp104 WT e controlli vettoriali in parallelo. </li><li> La mattina seguente, piatto una serie di concentrazioni su galattosio individuale (ad esempio SGal-His-Ura) piastre (1 ml a 2 ml cultura concentrate in 400 ml di volume totale). Placcatura una vasta gamma di concentrazioni garantirà che un piatto avrà singole colonie. La concentrazione effettiva richiesta dipende dalla tossicità del substrato patologia di interesse. </li><li> Normalizza il vettore e culture Hsp104 WT al diametro esterno 600 della biblioteca e piastra volumi uguali di confrontare la crescita della biblioteca rispetto ai controlli. </li><li> Per valutare selezione rigore, piatto biblioteca in glucosio (repressione) dei media. Incubare le piastre per 2-3 giorni a 30 ° C, fino a quando le colonie appaiono (Figura 2). Valutare le colonie risultanti e confrontare ai controlli. NOTA: Spesso entrambe le colonie grandi e piccole si ottengono, ma nessuna correlazione tra le dimensioni delle colonie e di agireivity si osserva. Schermo questi potenziali colpi utilizzando una tecnica di selezione contatore 5-FOA (Fase 4) per ridurre al minimo i falsi positivi riportati al sequenziamento. </li></ol> 4. 5-FOA Counterselection e Spotting per eliminare i falsi positivi <ol><li> Streak le singole colonie in duplice copia su supporto matrimoniale e letto singolo caduta di tensione (ad esempio, SD-suo-Ura e SD-I suoi piatti) e crescere una notte a 30 ° C. Ripetere con controlli vettoriali e Hsp104 WT. Placcatura su SD-His prima 5-FOA aumenta l&#39;efficienza della fase 5-FOA aumentando la probabilità che le cellule avranno perso il plasmide Hsp104 quando vengono piastrate su 5-FOA. </li><li> Per evitare che le piastre si secchi, avvolgere le piastre in parafilm suoi Ura-SD-e conservare a 4 ° C durante l&#39;esecuzione dello schermo 5-FOA. Queste piastre saranno eventualmente utilizzati per il sequenziamento. </li><li> Streak le colonie dalla SD-I suoi piatti a singole colonie su piastre 5-FOA (YPD supporti + 1 g / L 5-FOA) e incubare per 1-2 giorni a 30 &amp;# 186; C fino singole colonie appaiono. Qui, 5-FOA viene convertito in un prodotto tossico (5-fluorodeossiuridina) in cellule che contengono il gene URA3. Pertanto, eventuali cellule ancora ospitano il plasmide Hsp104 moriranno. </li><li> Streak le singole colonie dalle piastre 5-FOA in duplicato su SD-Ura e SD-I suoi piatti. Test 3 colonie per ogni hit per aumentare la probabilità che almeno una colonia per ogni colpo avrà perso il plasmide Hsp104. Incubare per 1-2 giorni a 30 ° C. Le colonie che hanno perso il plasmide Hsp104 si svilupperà su SD-I suoi piatti, ma non piatti SD-Ura. </li><li> Far crescere le tensioni che hanno perso i plasmidi Hsp104 per individuare saggi. Crescere i ceppi di saturazione in raffinosio abbandono dei media (ad esempio SRaff-His) in 96 Deepwell 2 piastre ml notte a 30 ° C con agitazione. Assicurati di includere i ceppi di controllo 5-FOA trattati. </li><li> Preparare piastre per individuare test. Utilizzando una pipetta multicanale, un&#39;aliquota raffinosio 200 microlitri mezzi di abbandono (ad esempio SRaff-His) perpozzetto di una piastra da 96 pozzetti, riservando colonne 1 e 7 per le culture pulito. Aliquotare 250 ml di ciascuno dei 16 culture saturi nelle colonne 1 e 7 della piastra. </li><li> Serialmente diluire le culture 5 volte in ogni colonna della piastra, mescolando accuratamente con una pipetta multicanale. Rimuovere 50 ml di coltura diluita dalle colonne 6 e 12 per garantire il volume finale di ciascun pozzetto a 200 microlitri. Questo è essenziale per garantire anche spotting. </li><li> Utilizzando uno strumento replicatore 96-bullone (Frogger), individuare le culture in duplicato su SD-Sue e-SGal suoi piatti. Incubare le piastre a 30 ° C per 2-3 giorni. </li><li> Seleziona per sequenziamento ceppi che presentano tossicità sui SGal-sue piastre simile a quella dei controlli. Eliminare positivi come falsi ceppi che non sono tossici come i controlli. 5-FOA è anche spesso tossici propria, così scartare ceppi che presentano un difetto di crescita su SD-sue piastre o che mostrano sostanzialmente maggiore tossicità rispetto al substrato malattie sola ( <strong&gt; Figura 3). </li></ol> 5. Il sequenziamento del Hsp104 Varianti di Colony PCR <ol><li> Raschiare ~ 10 microlitri lievito dalle piastre SD-His-Ura in NaOH 3 ml 20 mM in tubi strip PCR e lisare le cellule di gelo-disgelo. Porre i tubi striscia in un congelatore -80 ° C per 10 min o in azoto liquido, poi incubare a 99 ° C in un termociclatore per 10 min. Diluire i ceppi a 100 ml con acqua di grado PCR. </li><li> Preparare le reazioni PCR: 5 ml modello PCR, 0,5 microlitri 100 mM di ciascun primer, 0,5 ml 10mM dNTP, 0,25 ml polimerasi del DNA in tampone PCR, e portare a 25 ml volume totale di acqua di grado PCR. Primer design per amplificare la regione randomizzato più di circa 100 bp su entrambe le estremità. Minimizzare la dimensione della regione amplificata per aumentare la probabilità di successo di amplificazione. Eseguire un programma standard di PCR. </li><li> Analizzare i campioni mediante elettroforesi su gel di agarosio per confermare l&#39;amplificazione di un prodotto di PCR del siz appropriatae. Ripetere PCR se nessun prodotto è presente. NOTA: il fallimento PCR è in genere a causa di troppo lievito utilizzato nella fase 5.1. </li><li> Preparare i campioni per il sequenziamento del DNA. Rimuovere primer PCR sia mediante PCR di depurazione o uso di un prodotto di PCR pulizia reattivo come esonucleasi I e gamberetti fosfatasi alcalina (ExoSAP-IT) per degradare DNA a singolo filamento. Questo reagente degrada enzimaticamente primer non incorporati e dNTP, consentendo un throughput più elevato. </li><li> Sequenziare i prodotti di PCR. Assicurati di analizzare a fondo cromatogrammi sequenziamento di mutazioni. Le mutazioni possono essere osservati come una miscela di due nucleotidi in un determinato sito (figura 4), ​​come lievito spesso porto più plasmidi. </li></ol>

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The authors have nothing to disclose.

Qui vi presentiamo il nostro approccio per isolare le varianti Hsp104 potenziati che sopprimono la tossicità di substrati associate alla malattia che utilizzano modelli di lievito proteinopathy. Usando questo approccio, grandi librerie di varianti possono essere proiettati in alta velocità, con l&#39;unica limitazione è il numero di varianti che passano schermo secondario 5-FOA. Per eseguire le operazioni descritte in 96 formati bene, abbiamo regolarmente dello schermo fino a 200 colpi alla volta nella fase 5-FOA nel...

Lievito proteinopathy modelli sono stati sviluppati per i disordini di proteine ​​misfolding tra cui la sclerosi laterale amiotrofica (SLA) e la malattia (PD) 1-3 di Parkinson. L&#39;espressione delle proteine ​​TDP-43 e FUS, che misfold nei pazienti con SLA, sono tossici e mislocalize a formare aggregati citoplasmatici in lievito 1,2. Analogamente, l&#39;espressione di α-sinucleina (α-syn), che è implicato nel PD, è tossico e mislocalizes formare aggregati citoplasmatici in lievito 3. Queste caratteristiche ricapitolano fenotipi in pazienti con questi disturbi 4,5. Così, i modelli di lievito forniscono una piattaforma utile per lo screening di proteine ​​o piccole molecole che impediscono o invertire questi fenotipi 2,6-13. Ci interessa lo sviluppo di proteine ​​che sono in grado di invertire aggregazione e tossicità dovuta al TDP-43, FUS, e α-syn. Ci concentriamo su Hsp104, un AAA + proteine ​​da lievito che è l&#39;unica in grado di disaggregare proteine ​​sia fraggregati amorfi OM e amiloide nel lievito, eppure non ha l&#39;omologo umano 14,15. Hsp104 è finemente sintonizzato per disaggregare i prioni di lievito endogene e ha solo limitata capacità di disaggregare substrati implicati nelle malattie neurodegenerative umane, che non ha mai incontra ordinariamente 16,17. Così, ci proponiamo di progettare versioni avanzate di Hsp104 in grado di disaggregare efficacemente questi substrati umani. A tale scopo, costruiamo grandi biblioteche di Hsp104 varianti mediante PCR soggetto a errori; queste librerie possono essere sottoposti a screening utilizzando i modelli di lievito proteinopathy 17. Abbiamo adottato un approccio dominio mirato alla costruzione di screening e biblioteche, come Hsp104 è molto grande 17. Inizialmente concentrati sul dominio centrale (MD) di Hsp104 17, anche se gli approcci simili possono essere impiegati per lo screening altri domini. Questi modelli consentono lo screening per l&#39;attività disaggregase direttamente, al contrario di tecniche alternative come la visualizzazione di superficie, che è riposoricted da utilizzare per il monitoraggio vincolante 18. Il nostro protocollo si basa su due fasi di screening (Figura 1). In primo luogo, vengono selezionati varianti Hsp104 che sopprimono la tossicità del substrato malattia in lievito. Per fare ciò, il Hsp104 varianti e le malattie associate substrato sono cotransformed in lieviti Δ Hsp104. Ci avvaliamo di lievito Hsp104 Δ per esplorare Hsp104 sequenza spazio in assenza di wild-type (WT) Hsp104 17. È importante sottolineare che l&#39;eliminazione di Hsp104 non influisce α-syn, FUS, o TDP-43 della tossicità in lievito, e l&#39;espressione di Hsp104 WT fornisce il minimo soccorso 1,13,17. Il lievito viene poi placcato sulla indurre media per indurre l&#39;espressione di entrambe le proteine. Lievito ospitare varianti Hsp104 che sopprimono la tossicità della crescita substrato conferiscono associata a malattia della colonia. Queste varianti sono state selezionate per ulteriori analisi, mentre le colonie mantenendo le varianti che non sopprimono la tossicità die. Tuttavia,falsi positivi sono un problema sostanziale in questa schermata. Espressione di TDP-43, FUS, e α-syn sono altamente tossici, che crea una forte pressione selettiva per la comparsa di soppressori genetiche spontanee di tossicità correlato alla variante Hsp104 espressi. Così, abbiamo utilizzato uno schermo secondario che è anche relativamente ad alto throughput per eliminare questi soppressori di tossicità non specifici 17. In questa schermata secondaria, lieviti selezionati sono trattati con 5-Fluorootic Acid (5-FOA) per contrastare selezione per il plasmide Hsp104 19. I ceppi sono poi valutati per substrato (TDP-43, FUS o α-syn) tossicità tramite individuazione test per garantire che la tossicità del substrato viene ripristinata dopo la perdita del plasmide Hsp104. Così, lievito in cui tossicità è ripristinato in questa schermata secondaria visualizzata presumibilmente originariamente soppressione tossicità dovuta alla presenza della variante Hsp104. Questi lieviti sono designati come &quot;hit&quot; e il plasmide Hsp104 dovrebbe quindi essererecuperato e sequenziato per identificare le mutazioni nel gene Hsp104 17 (Figura 1). Tutti i colpi devono poi essere riconfermati costruendo la mutazione indipendente utilizzando mutagenesi sito-diretta e poi ripetere il test per la soppressione tossicità. Le potenziali applicazioni di questo protocollo sono ampi. Usando questi metodi, librerie di qualsiasi tipo di proteina potrebbero essere sottoposti a screening per le varianti che sopprimono tossicità di qualsiasi proteina substrato che è tossica in lievito.

<table><tbody><tr><td>GeneMorphII EZClone Domain Mutagenesis Kit</td><td>Agilent</td><td>200552</td><td></td></tr><tr><td>150 mm Petri dishes</td><td>Falcon</td><td>351058</td><td></td></tr><tr><td>5-Fluorootic Acid</td><td>Research Products International</td><td>f10501-5.0</td><td></td></tr><tr><td>96-DeepWell 2 ml Plates</td><td>Eppendorf</td><td>0030 502.302</td><td></td></tr><tr><td>96 bold replicator tool</td><td>V&amp;amp;P Scientific</td><td>vp-404</td><td></td></tr><tr><td>ExoSAP-IT</td><td>Affymetrix</td><td>78200</td><td></td></tr></tbody></table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Abbiamo costruito una libreria di varianti Hsp104 randomizzati nel dominio centrale e proiettato per la soppressione di TDP-43 tossicità. La biblioteca è stata trasformata e placcato su glucosio e galattosio piastre (Figura 2) per valutare la severità dello schermo. Singole colonie sono stati selezionati e ceppi sono stati selezionati contatore utilizzando 5-FOA per eliminare il plasmide Hsp104. Questi ceppi sono stati poi valutati per confermare che la tossicità era dovuta al TDP-43 da solo, senza ...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

L&#39;isolamento potenziato Hsp104 Varianti Utilizzando lievito Proteinopathy modelle

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Research

JoVE Journal

Biology

8.1K Views.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Video: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Sovraespressione di accoppiamento-based Library Screening in lievito

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Ricerca

Genetica

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

Il lievito come telaio per lo sviluppo di saggi funzionali per studiare l'Uomo P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Ricerca sul cancro

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

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

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunologia e infezioni

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purificazione di Hsp104, una proteina Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Biologia

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

I saggi di crescita per valutare la tossicità poliglutamina nel lievito

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Approcci genetici e biochimici per<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; La valutazione di proteine ​​Oligomerizzazione: Il rianodina Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

High throughput Screening per eredità a base di proteine in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

Un metodo per studiare la tossicità e l'aggregazione della α-sinucleina utilizzando un modello di lievito umanizzato

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

L'isolamento potenziato Hsp104 Varianti Utilizzando lievito Proteinopathy modelle

Riepilogo

Esplora Altri Video

Capitoli in questo video

Sovraespressione di accoppiamento-based Library Screening in lievito

Il lievito come telaio per lo sviluppo di saggi funzionali per studiare l'Uomo P53

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

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Purificazione di Hsp104, una proteina Disaggregase

I saggi di crescita per valutare la tossicità poliglutamina nel lievito

Saggi accoppiati per il monitoraggio delle proteine in ripiegamento

Approcci genetici e biochimici per

High throughput Screening per eredità a base di proteine in S. cerevisiae

Un metodo per studiare la tossicità e l'aggregazione della α-sinucleina utilizzando un modello di lievito umanizzato

L'isolamento potenziato Hsp104 Varianti Utilizzando lievito Proteinopathy modelle

Riepilogo

Esplora Altri Video

Capitoli in questo video

Sovraespressione di accoppiamento-based Library Screening in lievito

Il lievito come telaio per lo sviluppo di saggi funzionali per studiare l'Uomo P53

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

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Purificazione di Hsp104, una proteina Disaggregase

I saggi di crescita per valutare la tossicità poliglutamina nel lievito

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento

Approcci genetici e biochimici per

High throughput Screening per eredità a base di proteine in S. cerevisiae

Un metodo per studiare la tossicità e l'aggregazione della α-sinucleina utilizzando un modello di lievito umanizzato

Saggi accoppiati per il monitoraggio delle proteine in ripiegamento