Name: isolating potentiated hsp104 variants using yeast proteinopathy models
Uploaded: 2014-11-11T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

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 Generación <ol><li> Para construir bibliotecas de Hsp104 utilizando PCR propensa a error de dominio específico, primero amplificar el dominio de interés con un error de ADN polimerasa propensa 20. </li><li> Se purificó el producto de PCR mediante extracción en gel. </li><li> Realizar un paso de extensión megacebador utilizando un protocolo estándar de mutagénesis dirigida al sitio: combinar plásmido molde 50 ng, 250 ng megacebador, 200 mM dNTPs, y de alta fidelidad de ADN polimerasa en un tampón de PCR, y diluir a 50 l volumen total con agua de grado PCR 20 . Ejecutar un programa de PCR estándar. NOTA: cebadores específicos usados ​​variará dependiendo de la región particular del gen que se va a amplificar. <ol><li> Tras la PCR, digerir ADN molde parental con 1 l de enzima de restricción Dpn I durante 2 horas a 37 ° C. </li></ol></li><li> Transformar la biblioteca por electroporación u otros medios en Ultracompetent E. coli y purificarla por miniprep. NOTA: generación Bibliotecavaría dependiendo sustancialmente a los objetivos de un determinado experimento. Hsp104 es una proteína muy grande, por lo que es poco práctico para aleatorizar todo el gen, por lo que utilizamos error de dominio específico PCR propensa. Además, la estructura de Hsp104 sigue siendo poco conocida, por lo que el diseño de bibliotecas dirigidas desafiantes 21. Las bibliotecas pueden ser construidos usando enfoques dirigidos o aleatorios a mutagénesis, con la única restricción es que el esqueleto plasmídico plantilla debe contener el gen URA3 para permitir la 5-FOA selección del contador en medios de dextrosa. </li></ol> 2. Transformación de la Biblioteca Hsp104 <ol><li> Integrar el sustrato asociado a la enfermedad en W303aΔ Hsp104 levadura utilizando un / protocolo de transformación PEG acetato de litio estándar 22. Seleccione colonias individuales y detectar ellos para la toxicidad para aislar una cepa con alta toxicidad de la enfermedad sustrato 23 asociados. NOTA: Utilice una cepa integrado y isolaTé a sola colonia para garantizar la igualdad de expresión en todas las células. Clonar el sustrato de la enfermedad en un plásmido que permite la integración del gen bajo cualquier marcador que no sea uracilo (usamos histidina) y la biblioteca Hsp104 en el plásmido pAG416GAL (marcador de uracilo) 24. Para permitir la 5-FOA contraselección, asegúrese de que la biblioteca se expresa a partir de un plásmido con el marcador de uracilo. Otras cepas de levadura también pueden ser empleados. Hemos tomado nota de un fenotipo de supresión de toxicidad similar usando W303a y BY4741 cepas de levadura en ambos WT y Δ Hsp104 fondos (MEJ y JS, observaciones no publicadas). </li><li> Transformar la biblioteca Hsp104 en esta cepa usando la misma acetato de litio / protocolo de transformación PEG 22. Aumentar la escala de la transformación apropiada para mantener el espacio de secuencia de la biblioteca y para preservar el tamaño de la biblioteca predicho. </li><li> Placa de la mezcla de transformación en noninducing, placas selectivas (por ejemplo, SD-Su-Ura) con suficientes platos aasegurar el crecimiento de un gran número de colonias. El uso de grandes placas de Petri (150 mm) para minimizar el número de placas necesarias. La recuperación de los transformantes utilizando placas permite la eficiencia de transformación que deben evaluarse. </li><li> Transformar Hsp104 WT y controles negativos vector en paralelo. </li></ol> 3. La detección de represión de Proteotoxicity <ol><li> Lávese las colonias de las placas utilizando rafinosa complementado los medios de comunicación de abandono (por ejemplo Sraff-Su-Ura). Utilice una pipeta serológica y aplicadores de madera estériles para aflojar las colonias de las placas. Transferir los lavados líquidos a un tubo cónico de 50 ml y agitar a fondo para separar los grumos de células. Diluir a una cultura ligeramente nublado, OD 600 ~ 0,025. NOTA: Aquí rafinosa sirve para aliviar las células de la represión mediada por la glucosa de la inducción, las células de cebado por lo tanto para la inducción mediada por galactosa. </li><li> Mantener el cultivo diluido durante la noche en los medios de comunicación de abandono rafinosa con agitación a 30 ºC. Crecer el Hsp104 WT y controles vectoriales en paralelo. </li><li> A la mañana siguiente, la placa de un rango de concentraciones Onto galactosa individual (por ejemplo SGal-Su-Ura) placas (1 mu l a 2 ml concentrado cultura en 400 l de volumen total). Plating una amplia gama de concentraciones se asegurará de que una placa tendrá colonias individuales. La concentración real requerida depende de la toxicidad del sustrato enfermedad de interés. </li><li> Normalizar el vector y las culturas Hsp104 WT a la OD 600 de la biblioteca y la placa volúmenes iguales al comparar el crecimiento de la biblioteca con respecto a los controles. </li><li> Para evaluar la selección rigor, la placa de la biblioteca en la glucosa (represión) los medios de comunicación. Incubar las placas durante 2-3 días a 30 ° C, hasta que las colonias aparecen (Figura 2). Evaluar las colonias resultantes y comparar con los controles. NOTA: A menudo se obtienen ambas colonias grandes y pequeñas, pero no hay correlación entre el tamaño de la colonia y actuarse observa ividad. Pantalla estos posibles éxitos utilizando una técnica de selección de contador 5-FOA (Paso 4) para minimizar los falsos positivos prorrogados para secuenciación. </li></ol> 4. 5-FOA contraselección y Spotting para eliminar falsos positivos <ol><li> Streak cabo colonias individuales en duplicado sobre doble y una individual (por ejemplo, los medios de comunicación de abandono SD-Su-Ura y SD-Sus placas) y crecer durante la noche a 30 ºC. Repita el procedimiento con los controles vectoriales y Hsp104 WT. Plating en SD-His antes de la 5-FOA aumenta la eficacia de la etapa 5-FOA mediante el aumento de la probabilidad de que las células se han perdido el plásmido Hsp104 cuando se sembraron en 5-FOA. </li><li> Para evitar que las placas se seque, envuelva la placas en parafina Sus-Ura-SD y almacenar a 4 ° C en el desempeño de la pantalla de 5-FOA. Estas placas eventualmente ser utilizados para la secuenciación. </li><li> Racha de las colonias de la SD-Sus placas a colonias individuales en placas 5-FOA (YPD medios + 1g / L 5-FOA) y se incuba durante 1-2 días a 30 y# 186; C hasta que aparezca colonias individuales. Aquí, 5-FOA se convierte en un producto tóxico (5-fluorodesoxiuridina) en las células que contienen el gen URA3. Por lo tanto, las células que albergan el plásmido todavía Hsp104 morirán. </li><li> Racha de las colonias aisladas de las placas 5-FOA en duplicado sobre SD-Ura y SD-Sus placas. Prueba 3 colonias para cada golpe para aumentar la probabilidad de que al menos una colonia de cada golpe se habrá perdido el plásmido Hsp104. Incubar durante 1-2 días a 30 ° C. Las colonias que han perdido el plásmido Hsp104 crecerá en SD-Sus placas pero no las placas SD-ura. </li><li> Crecer las cepas que han perdido los plásmidos Hsp104 para observar los ensayos. Crecer las cepas a la saturación en rafinosa medios de deserción escolar (por ejemplo Sraff-Su) en 96 pozos profundos 2 placas ml durante la noche a 30 ° C con agitación. Asegúrese de incluir las cepas de control de 5-FOA tratados. </li><li> Preparar placas para la detección de ensayo. Con una pipeta multicanal, alícuota de 200 l rafinosa medios de deserción escolar (por ejemplo Sraff-His) porpocillo de una placa de 96 pocillos, reservando las columnas 1 y 7 para las culturas aseados. Alícuota de 250 l de cada uno de los 16 cultivos saturados en las columnas 1 y 7 de la placa. </li><li> En serie diluir las culturas 5 veces en cada columna de la placa, mezclando completamente con una pipeta multicanal. Retire 50 l de cultivo diluido de las columnas 6 y 12 para garantizar el volumen final de cada pocillo es de 200 l. Esto es esencial para asegurar una formación de manchas. </li><li> Con una herramienta de replicador de 96 perno (Frogger), manchar las culturas por duplicado en SD-His y SGal-Sus placas. Incubar las placas a 30 ° C durante 2-3 días. </li><li> Seleccionar para secuenciar cepas que exhiben toxicidad en los SGal-His placas similares a la de los controles. Deseche cepas como falsos positivos que no son tan tóxicos como los controles. 5-FOA es también a menudo tóxicos por sí mismo, por lo deseche cepas que exhiben un defecto de crecimiento en SD-Sus placas o que presentan sustancialmente mayor toxicidad que el sustrato (enfermedad por sí sola <strong&gt; Figura 3). </li></ol> 5. La secuenciación del Hsp104 variantes mediante PCR de colonias <ol><li> Raspe ~ 10 l de levadura de las placas SD-His-ura en NaOH 3 l 20 mM en tubos de PCR de tira y lisar las células por congelación-descongelación. Colocar los tubos de la tira en un congelador -80 ºC durante 10 min o en nitrógeno líquido, a continuación, se incuba a 99 ºC en un termociclador durante 10 min. Diluir las cepas de 100 l con agua de calidad PCR. </li><li> Prepare las reacciones de PCR: 5 l plantilla de PCR, 0,5 mu l de 100 mM de cada cebador, 0,5 l de 10 mM dNTPs, DNA polimerasa 0,25 l en tampón de PCR, y completar hasta 25 l volumen total con agua grado PCR. Diseño cebadores para amplificar la región aleatorizada más aproximadamente 100 pb en cada extremo. Minimizar el tamaño de la región amplificada para aumentar la probabilidad de éxito de la amplificación. Ejecutar un programa de PCR estándar. </li><li> Analizar las muestras mediante electroforesis en gel de agarosa para confirmar la amplificación de un producto de PCR de la siz apropiadoe. Repita la PCR si hay producto está presente. NOTA: el fracaso de PCR suele ser debido a un exceso de levadura que se utiliza en el paso 5.1. </li><li> Preparar las muestras para la secuenciación de ADN. Retire cebadores de PCR, ya sea por purificación de PCR o usando un reactivo de limpieza producto de PCR tales como exonucleasa I y fosfatasa alcalina de camarón enzima (ExoSAP-IT) para degradar el ADN de cadena sencilla. Este reactivo degrada enzimáticamente los cebadores no incorporados y dNTPs, lo que permite un mayor rendimiento. </li><li> Secuenciar los productos de PCR. Asegúrese de analizar a fondo cromatogramas de secuenciación para detectar mutaciones. Las mutaciones pueden ser observadas como una mezcla de dos nucleótidos en un sitio dado (Figura 4), ​​como la levadura a menudo albergar múltiples plásmidos. </li></ol>

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The authors have nothing to disclose.

Aquí presentamos nuestro enfoque para aislar variantes Hsp104 potenciadas que suprimen la toxicidad de los sustratos de las enfermedades asociadas que utilizan modelos proteinopatía levadura. Usando este enfoque, las grandes bibliotecas de variantes se pueden cribar en alto rendimiento, con la única limitación es el número de variantes que pasan la pantalla secundaria 5-FOA. Mediante la realización de estos pasos en formato de 96 pocillos, se examinan habitualmente hasta 200 hits en un momento en el paso 5-FOA en ...

Modelos proteinopatía de levadura se han desarrollado para los trastornos de proteínas misfolding incluyendo esclerosis lateral amiotrófica (ALS) y la enfermedad de Parkinson (PD) 1-3. Expresión de las proteínas TDP-43 y FUS, que se pliegan inadecuadamente en pacientes con ELA, son tóxicos y mislocalize para formar agregados citoplasmáticos en la levadura 1,2. Del mismo modo, la expresión de α-sinucleína (α-syn), que está implicado en la EP, es tóxico y mislocalizes para formar agregados citoplasmáticos en la levadura 3. Estas características recapitulan fenotipos en los pacientes con estos trastornos 4,5. Por lo tanto, los modelos de levadura proporcionan una plataforma útil para la detección de proteínas o moléculas pequeñas que prevenir o revertir estos fenotipos 2,6-13. Estamos interesados ​​en el desarrollo de las proteínas que son capaces de revertir la agregación y la toxicidad debido a la TDP-43, FUS, y α-syn. Nos centramos en Hsp104, un AAA + proteína de la levadura que es el único capaz de desagregar las proteínas tanto fragregados amorfos om y amiloide en la levadura, sin embargo, no tiene homólogo humano 14,15. Hsp104 está bien afinado para desagregar los priones de levadura endógenos y sólo tiene una capacidad limitada para desagregar sustratos implicados en enfermedades neurodegenerativas humanas, que nunca encuentra con ordinariamente 16,17. Por lo tanto, nuestro objetivo es diseñar versiones mejoradas de Hsp104 que son capaces de desagregar eficazmente estos sustratos humanos. Para ello, construimos grandes bibliotecas de variantes Hsp104 usando PCR propensa a errores; estas bibliotecas se pueden cribar utilizando los modelos proteinopatía levadura 17. Hemos adoptado un enfoque de dominio orientados a la construcción y selección de bibliotecas, como Hsp104 es muy grande 17. Al principio nos centramos en el dominio del medio (MD) de Hsp104 17, aunque los enfoques similares pueden ser empleados para detectar otros dominios. Estos modelos permiten la detección de la actividad disaggregase directamente, en contraposición a las técnicas alternativas tales como la presentación en superficie, que es el descansoricted utilizar para el seguimiento de la unión 18. Nuestro protocolo se basa en dos pasos de detección (Figura 1). En primer lugar, se seleccionan variantes Hsp104 que suprimen la toxicidad del sustrato enfermedad en la levadura. Para ello, la Hsp104 variantes y la enfermedad asociada sustrato se cotransformación de la levadura Hsp104 Δ. Empleamos Δ Hsp104 levadura para explorar Hsp104 secuencia espacio en ausencia de tipo salvaje (WT) Hsp104 17. Es importante destacar que, la supresión de Hsp104 no afecta α-syn, FUS, o TDP-43 toxicidad en la levadura, y la expresión de Hsp104 WT proporciona mínima de rescate 1,13,17. La levadura está chapada a continuación, en la inducción de los medios de comunicación para inducir la expresión de ambas proteínas. La levadura que alberga variantes Hsp104 que suprimen la toxicidad del crecimiento confieren sustrato asociado a la enfermedad de la colonia. Estas variantes se seleccionan para su posterior análisis, mientras que el mantenimiento de colonias variantes que no suprimen troquel toxicidad. Sin embargo,falsos positivos son un problema importante en esta pantalla. Expresión de TDP-43, FUS, y α-syn son altamente tóxicos, que crea una fuerte presión selectiva para la aparición de supresores genéticos espontáneos de la toxicidad no relacionada con la variante de Hsp104 ser expresado. Por lo tanto, hemos utilizado una pantalla secundaria que es también relativamente de alto rendimiento para eliminar estos supresores de toxicidad no específica 17. En esta pantalla secundaria, levadura seleccionada se tratan con Fluorootic 5-ácido (5-FOA) para contrarrestar seleccione para el plásmido Hsp104 19. Las cepas se evaluaron entonces por el sustrato (TDP-43, FUS, o α-syn) toxicidad a través de la detección de ensayo para asegurarse de que la toxicidad del sustrato se restaura después de la pérdida del plásmido Hsp104. Por lo tanto, la levadura en la que la toxicidad se restaura en esta pantalla secundaria presumiblemente originalmente muestra la supresión de toxicidad debido a la presencia de la variante de Hsp104. Estas levaduras se designan como &quot;hits&quot; y el plásmido Hsp104 debe entonces serrecuperado y secuenciado para identificar las mutaciones en el gen Hsp104 17 (Figura 1). Cualquier resultado debe entonces ser reconfirmados mediante la construcción de la mutación de forma independiente mediante mutagénesis dirigida al sitio y luego volver a probar para la supresión de la toxicidad. Las aplicaciones potenciales de este protocolo son amplias. Usando estos métodos, las bibliotecas de cualquier tipo de proteína podrían ser examinados para variantes que suprimen la toxicidad de cualquier proteína sustrato que es tóxico en la levadura.

<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.

Hemos construido una biblioteca de variantes Hsp104 aleatorios en el dominio medio y se tamiza para la supresión de la TDP-43 toxicidad. La biblioteca se transformó y colocó en placas sobre placas de glucosa y galactosa (Figura 2) para evaluar la severidad de la pantalla. Se seleccionaron colonias individuales y las cepas se seleccionaron usando contador de 5-FOA para eliminar el plásmido Hsp104. Estas cepas se evaluaron entonces para confirmar que la toxicidad era debido a la TDP-43 solo, sin las v...

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.

Aislar Potenciada Hsp104 Variantes Usando levadura Modelos proteinopatía

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 vistas.  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

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.

Levadura como chasis para el desarrollo de ensayos funcionales para estudiar P53 humanos

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 ...

Investigación

Investigación sobre el cáncer

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.

Caracterización de la histona modificación post-translacional alteraciones en modelos neurodegenerativos Proteinopathy de levadura

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

Genética

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 enfoque comparativo para caracterizar el paisaje del Huésped-Patógeno interacciones proteína-proteína

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

Inmunología e Infección

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biología

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.

La purificación de Hsp104, una proteína Disaggregase

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

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.

Los ensayos de crecimiento para evaluar la toxicidad de poliglutaminas en la levadura

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.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<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.

Los enfoques genéticos y bioquímicos para<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; Evaluación de la oligomerización de proteínas el receptor de rianodina Estudio de caso

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.

Proyección de alto rendimiento para la herencia basados en proteína en 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 método para estudiar la toxicidad y agregación de α-sinucleína utilizando un modelo de levadura humanizado

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

Aislar Potenciada Hsp104 Variantes Usando levadura Modelos proteinopatía

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