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 Generation <ol><li> Para construir bibliotecas de Hsp104 usando PCR propensa a erros de domínio específico, primeiro amplificar o domínio de interesse com um erro DNA polimerase propensas 20. </li><li> Purifica-se o produto de PCR por extracção de gel. </li><li> Realizar uma etapa de extensão megainiciador utilizando um protocolo padrão de mutagénese dirigida ao local: combinar 50 ng de plasmídeo, modelo, 250 megainiciador ng, 200 uM de dNTPs, e de alta fidelidade da polimerase de ADN em tampão de PCR, e diluir até 50 mL de volume total, com água de grau de PCR 20 . Execute um programa de PCR padrão. NOTA: Os primers específicos utilizados irá variar com base na região particular do gene que está a ser amplificado. <ol><li> Após a PCR, digerir ADN molde parental com 1 ul da enzima de restrição Dpnl durante 2 horas a 37 ° C. </li></ol></li><li> Transformar a biblioteca por eletroporação ou outros meios em ultracompetent E. coli e purificá-lo por miniprep. NOTA: geração Bibliotecavaria substancialmente com base nos objetivos de um determinado experimento. Hsp104 é uma proteína muito grande, por isso é impraticável para embaralhar todo o gene, que é por isso que nós utilizamos erro específico do domínio propenso PCR. Além disso, a estrutura de Hsp104 permanece pouco compreendido, tornando o desenho de bibliotecas dirigidas 21 desafiantes. As bibliotecas podem ser construídas utilizando abordagens dirigidas ou aleatórias para mutagénese, com a única restrição que o plasmídeo de esqueleto modelo deve conter o gene URA3 para permitir 5-FOA contra-selecção em meios de dextrose. </li></ol> 2. Transformação da Biblioteca Hsp104 <ol><li> Integrar o substrato associado à doença em W303aΔ Hsp104 levedura usando um acetato de lítio padrão / PEG protocolo de transformação 22. Selecione colónias individuais e analisá-los em termos de toxicidade para isolar uma cepa com alta toxicidade da doença associada substrato 23. NOTA: Use uma tensão integrado e isolate uma única colônia de garantir a igualdade de expressão em todas as células. Clonar o substrato de doença num plasmídeo que permite a integração do gene em qualquer outro marcador de uracilo (usamos histidina) e a biblioteca de Hsp104 no plasmídeo pAG416GAL (marcador uracilo) 24. Para permitir o 5-FOA counterselection, assegurar que a biblioteca é expressa a partir de um plasmídeo com a marca de uracilo. Outras estirpes de levedura também podem ser empregues. Temos notado um fenótipo supressão toxicidade semelhante usando W303a e BY4741 cepas de leveduras em ambos WT e Δ Hsp104 fundos (MEJ e JS, observações não publicadas). </li><li> Transformar a biblioteca em Hsp104 esta estirpe usando o mesmo acetato de lítio / PEG protocolo de transformação 22. Dimensionar-se a transformação de forma adequada para manter o espaço de sequência da biblioteca e para preservar o tamanho da biblioteca previsto. </li><li> Placa da mistura de transformação para noninducing, placas seletivas (por exemplo SD-His-Ura) usando placas suficientes paraassegurar o crescimento de um grande número de colónias. Use grandes placas de Petri (150 mm) para minimizar o número de placas necessárias. Recuperando transformantes utilizando placas permite eficiência de transformação para ser avaliado. </li><li> Transforme Hsp104 WT e controles negativos vetor em paralelo. </li></ol> 3. Triagem para Supressão de Proteotoxicity <ol><li> Lave as colônias fora as placas usando rafinose complementada media de abandono (por exemplo SRaff-His-URA). Usar uma pipeta serológica estéril e aplicadores de madeira para soltar as colónias das placas. Transferir as lavagens líquidos para um tubo cónico de 50 ml e cuidadosamente vórtice para separar quaisquer aglomerações de células. Diluir a uma cultura um pouco nublado, OD 600 ~ 0,025. NOTA: Aqui rafinose serve para aliviar as células de repressão mediada por glicose de indução, priming assim que as células de indução mediada por galactose. </li><li> Incubar a cultura diluído durante a noite em meio de abandono rafinose com agitação a 30 ºC. Crescer a Hsp104 WT e controle de vetores em paralelo. </li><li> Na manhã seguinte, chapear uma gama de concentrações de galactose Onto individual (por exemplo SGAL-His-Ura) placas (1 ul a 2 ml da cultura concentradas em 400 ul de volume total). Chapeamento de uma ampla gama de concentrações vai garantir que uma placa vai ter colónias isoladas. A concentração real necessário depende da toxicidade do substrato doença de interesse. </li><li> Normalizar o vector e culturas Hsp104 WT para o OD 600 da biblioteca e placa volumes iguais para comparar o crescimento da biblioteca em relação aos controlos. </li><li> Para avaliar a seleção rigor, placa da biblioteca em glicose (reprimir) mídia. Incubar as placas durante 2-3 dias a 30 ° C, até que colónias aparecem (Figura 2). Avaliar as colônias resultantes e comparar com os controles. NOTA: Muitas vezes pequenos e grandes colônias são obtidos, mas nenhuma correlação entre o tamanho da colônia e agirivity é observado. Tela estes potenciais sucessos usando uma técnica de contra-selecção 5-FOA (Passo 4) para minimizar os falsos positivos transitados para sequenciação. </li></ol> 4. 5-FOA Counterselection e Spotting para eliminar falsos positivos <ol><li> Streak fora colónias isoladas em duplicata em suporte de casal e solteiro abandono (por exemplo SD-His-Ura e-Seus SD placas) e crescer durante a noite a 30 ºC. Repita a operação com controle de vetores e Hsp104 WT. Plaqueamento em SD-His antes da 5-FOA aumenta a eficiência do passo 5-FOA, aumentando a probabilidade de que as células terão perdido o plasmídeo Hsp104 quando são semeadas em 5-FOA. </li><li> Para evitar que as placas de secar, enrole a placas em parafilm Seus-SD-Ura e armazenar a 4 ºC durante a realização da tela de 5 FOA. Estas placas venham a ser utilizados para a sequenciação. </li><li> Streak as colônias do SD-Suas placas de colónias isoladas em placas 5-FOA (YPD media + 1g / L 5-FOA) e incubar durante 1-2 dias a 30 &amp;# 186; C até as colónias individuais aparecem. Aqui, 5-FOA é convertido num produto tóxico (5-fluorodesoxiuridina) em células que contêm o gene URA3. Assim, destacando as células portadoras do plasmídeo Hsp104 morrerá. </li><li> Streak as colónias individuais das placas 5-FOA em duplicar em SD-Ura e-Seus SD placas. Teste 3 colónias para cada batida para aumentar a probabilidade de que pelo menos uma colónia de cada um batido terá perdido o plasmídeo de Hsp104. Incubar durante 1-2 dias a 30 ° C. As colónias que perderam o plasmídeo Hsp104 vai crescer em SD-His placas mas não placas de SD-Ura. </li><li> Crescer as tensões que perderam os plasmídeos Hsp104 para detectar ensaios. Crescer as tensões à saturação em rafinose mídia abandono (por exemplo SRaff-His) em 96 Deepwell 2 placas ml durante a noite a 30 ºC com agitação. Certifique-se de incluir as estirpes de controlo 5-FOA tratados. </li><li> Prepare placas para detectar ensaio. Usando uma pipeta de canais múltiplos, alíquota de 200 ul rafinose meios de abandono (por exemplo SRaff-His) porpoço de uma placa de 96 poços, reservando colunas 1 e 7 para as culturas puras. Alíquota de 250 mL de cada uma das 16 culturas saturadas em colunas 1 e 7 da placa. </li><li> Serialmente dilua as culturas de 5 vezes em cada coluna da placa, misturar cuidadosamente com uma pipeta de canais múltiplos. Retirar 50 mL da cultura diluída a partir de colunas 6 e 12 para garantir o volume final de cada poço é 200 ul. Isto é essencial para garantir uma identificação. </li><li> Usando uma ferramenta replicador de 96 parafuso (Frogger), identificar as culturas em duplicado para-Seus SD e SGAL-Seus pratos. Incubar as placas a 30 ºC durante 2-3 dias. </li><li> Selecione para sequenciar cepas que apresentam toxicidade nas SGAL-Suas placas semelhantes aos dos controles. Descarte cepas como falsos positivos que não são tão tóxico como os controles. 5-FOA é também muitas vezes tóxico por si próprio, então descartar cepas que apresentam um defeito de crescimento em-seu SD ou placas que exibem substancialmente maior toxicidade do que o substrato doença sozinho ( <strong&gt; A Figura 3). </li></ol> 5. O seqüenciamento do Hsp104 Variantes por Colony PCR <ol><li> Raspe ~ 10 ul de levedura a partir das placas de SD-His-Ura em NaOH 3 ul 20 mM em tubos de PCR de tira e lisar as células por congelação-descongelação. Colocar os tubos em um congelador tira -80 ºC durante 10 min ou em azoto líquido, depois incubar a 99 ° C num termociclador durante 10 min. Dilui-se as estirpes para 100 ul com água de grau de PCR. </li><li> Prepare reações de PCR: 5 jul modelo PCR, 0,5 ul 100? M de cada iniciador, 0,5 mL de 10 mM dNTPs, 0,25 mL DNA polimerase em tampão PCR, e fazer até 25 mL volume total com PCR água grau. Design Os iniciadores para amplificar a região mais ao acaso, aproximadamente 100 pb em cada extremidade. Minimizar o tamanho da região amplificada para aumentar a probabilidade de uma amplificação bem sucedida. Execute um programa de PCR padrão. </li><li> Analisar amostras por electroforese em gel de agarose para confirmar a amplificação de um produto de PCR do siz adequadoe. Repita PCR se nenhum produto está presente. NOTA: A falha PCR é geralmente devido ao excesso de levedura a ser utilizado na etapa 5.1. </li><li> Preparar amostras para seqüenciamento de DNA. Remover os iniciadores de PCR quer através de purificação de PCR ou utilizando um reagente de limpeza do produto de PCR, tais como a Exonuclease I e fosfatase alcalina de camarão enzima (ExoSAP-TI) para degradar o ADN de cadeia simples. Este reagente enzimático degrada primers não incorporados e dNTPs, permitindo maior rendimento. </li><li> Sequenciar os produtos de PCR. Certifique-se de analisar cuidadosamente cromatogramas de sequenciamento de mutações. As mutações podem ser observadas como uma mistura de dois nucleótidos em um dado local (Figura 4), ​​como de levedura muitas vezes abrigam os plasmídeos múltiplos. </li></ol>

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The authors have nothing to disclose.

Aqui apresentamos nossa abordagem para isolar variantes Hsp104 potencializadas que suprimem a toxicidade de substratos associados à doença, utilizando modelos de levedura proteinopathy. Utilizando esta abordagem, bibliotecas grandes de variantes pode ser rastreada em elevado rendimento, com a única limitação é o número de variantes que passam o ecrã secundário 5-FOA. Ao realizar estes passos em formato de 96 poços, que rotineiramente tela até 200 batidas de cada vez no passo 5-FOA ao longo de 1-2 semanas. O n...

Levedura proteinopathy modelos têm sido desenvolvidos para distúrbios-misfolding proteína incluindo a esclerose lateral amiotrófica (ELA) e a doença de Parkinson (DP) 1-3. Expressão das proteínas TDP-43 e FUS, que misfold em pacientes de ELA, são tóxicos e mislocalize para formar agregados citoplasmáticos em levedura 1,2. Do mesmo modo, a expressão de α-sinucleína (α-syn), que está implicado na doença de Parkinson, é tóxico e mislocalizes para formar agregados citoplasmáticos em 3 de levedura. Esses recursos recapitular fenótipos em pacientes com esses transtornos 4,5. Assim, os modelos de levedura fornecer uma plataforma útil para o rastreio de proteínas ou moléculas pequenas que impedem ou invertem estes fenótipos 2,6-13. Estamos interessados ​​no desenvolvimento de proteínas que são capazes de inverter a agregação e toxicidade devido a TDP-43, FUS, e α-syn. Nós nos concentramos em Hsp104, um AAA + proteína de levedura que é o único capaz de desagregar proteínas tanto fragregados amorfos om e amilóide em leveduras, ainda não tem homólogo humano 14,15. Hsp104 é afinado para desagregar prions levedura endógenos e tem apenas capacidade limitada para desagregar substratos implicados em doenças neurodegenerativas humanas, que nunca encontra ordinariamente 16,17. Assim, nosso objetivo é projetar versões aprimoradas de Hsp104 que são capazes de desagregar eficazmente estes substratos humanos. Para isso, construímos grandes bibliotecas de Hsp104 variantes utilizando PCR propenso a erros; essas bibliotecas podem ser rastreados usando os modelos de levedura proteinopathy 17. Adotamos uma abordagem orientada-domínio para construção e pesquisa de bibliotecas, como Hsp104 é muito grande 17. Nós centrou-se inicialmente no domínio médio (DM) de Hsp104 17, embora as abordagens semelhantes podem ser empregues para o rastreio de outros domínios. Esses modelos permitem o rastreio de actividade disaggregase directamente, ao contrário de técnicas alternativas, tais como exposição de superfície, que é restoricted usar para monitorar a ligação 18. O protocolo baseia-se em dois passos de triagem (Figura 1). Em primeiro lugar, as variantes de Hsp104 que suprimem a toxicidade do substrato doença em levedura são seleccionados. Para fazê-lo, a Hsp104 variantes e associado à doença substrato são co-transformados em levedura Δ Hsp104. Nós empregamos Δ Hsp104 levedura para explorar Hsp104 seqüência espaço na ausência de tipo selvagem (WT) Hsp104 17. Importante, supressão de Hsp104 não afecta α-syn, FUS, ou TDP-43 toxicidade em levedura, e expressão de Hsp104 WT fornece salvamento mínima 1,13,17. A levedura é então plaqueadas em meios de indução para induzir a expressão de ambas as proteínas. Levedura abrigar variantes Hsp104 que suprimem a toxicidade do associado à doença de crescimento substrato conferem da colônia. Estas variantes são seleccionados para posterior análise, mantendo ao mesmo tempo as colónias variantes que não suprimem a toxicidade da matriz. No entanto,falsos positivos são um problema substancial na tela. Expressão de TDP-43, FUS, e α-syn são altamente tóxicos, o que cria uma forte pressão selectiva para o aparecimento de supressores genéticas espontâneas de toxicidade relacionada com a variante Hsp104 ser expresso. Assim, temos utilizado uma tela secundária que também é relativamente alto rendimento para eliminar esses supressores de toxicidade inespecíficos 17. Nesta tela secundária, leveduras selecionadas são tratados com 5-Fluorootic ácido (5-FOA) para combater a seleção para o plasmídeo Hsp104 19. As estirpes são então avaliadas para substrato (TDP-43, FUS, ou α-sin) toxicidade via ensaio de mancha para assegurar que a toxicidade do substrato é restaurada após a perda do plasmídeo Hsp104. Assim, de levedura, em que a toxicidade é restaurada neste ecrã secundário originalmente apresentado presumivelmente supressão de toxicidade devido à presença da variante da Hsp104. Estas leveduras são designados como &#39;hits&#39; e do plasmídeo Hsp104 deve, então, serrecuperados e sequenciados para identificar as mutações no gene de Hsp104 17 (Figura 1). Qualquer resultado deve ser reconfirmada em seguida, através da construção da mutação, independentemente, utilizando mutagénese dirigida ao local e, em seguida, para a supressão de novo teste de toxicidade. As potenciais aplicações para este protocolo são amplas. Usando estes métodos, bibliotecas de qualquer tipo de proteína pode ser rastreada para as variantes que suprimem a toxicidade da proteína de qualquer substrato que é tóxico em levedura.

<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.

Construímos uma biblioteca de variantes de Hsp104 randomizados no domínio médio e rastreados-lo para a supressão de TDP-43 toxicidade. A biblioteca foi transformada e plaqueada em placas de glicose e galactose (Figura 2) para avaliar a severidade da tela. Colónias individuais foram seleccionadas e as estirpes foram contra-seleccionadas utilizando 5-FOA para eliminar o plasmídeo Hsp104. Estas estirpes foram então avaliadas para confirmar que a toxicidade foi devido a TDP-43 sozinho, sem as variant...

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.

Isolando potencializado Hsp104 variantes usando levedura 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 ...

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

Research

JoVE Journal

Biology

8.1K visualizações.  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.

Vídeo: 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.

Fermento como um chassi para desenvolver ensaios funcionais para estudar p53 humano

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 ...

Pesquisa do câncer

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.

Caracterizando a histona modificação pós-traducional alterações nos modelos de Neurodegenerative Proteinopathy de levedura

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.

Uma abordagem comparativa para caracterizar a paisagem de Patógeno-Hospedeiro interações proteína-proteína

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

Imunologia e Infecção

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.

Estudos genéticos de proteínas de reparo do DNA Humano Utilizando Levedura como um sistema modelo

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

Biologia

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.

Purificação da Hsp104, um Disaggregase Protein

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.

Ensaios de crescimento para avaliar a toxicidade poliglutamina em Levedura

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.

Ensaios para Monitoramento juntamente redobramento de proteínas em<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.

Abordagens genéticas e bioquímicas para<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; Avaliação de oligomerização da proteína: A Case Study rianodina Receptor

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.

Elevado-throughput Screening para herança à base de proteínas no 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.

Um método para estudar a toxicidade e agregação da α-sinucleína usando um modelo de levedura humanizado

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

Isolando potencializado Hsp104 variantes usando levedura Proteinopathy Models

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