Chapter title |
Proteostasis
|
---|---|
Chapter number | 29 |
Book title |
Proteostasis
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3756-1_29 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3754-7, 978-1-4939-3756-1
|
Authors |
Garcia, G, Santos, C Nunes do, Menezes, R, G. Garcia, C. Nunes do Santos, R. Menezes |
Editors |
Rune Matthiesen |
Abstract |
The association between altered proteostasis and inflammatory responses has been increasingly recognized, therefore the identification and characterization of novel compounds with anti-inflammatory potential will certainly have a great impact in the therapeutics of protein-misfolding diseases such as degenerative disorders. Although cell-based screens are powerful approaches to identify potential therapeutic compounds, establishing robust inflammation models amenable to high-throughput screening remains a challenge. To bridge this gap, we have exploited the use of yeasts as a platform to identify lead compounds with anti-inflammatory properties. The yeast cell model described here relies on the high-degree homology between mammalian and yeast Ca(2+)/calcineurin pathways converging into the activation of NFAT and Crz1 orthologous proteins, respectively. It consists of a recombinant yeast strain encoding the lacZ gene under the control of Crz1-recongition elements to facilitate the identification of compounds interfering with Crz1 activation through the easy monitoring of β-galactosidase activity. Here, we describe in detail a protocol optimized for high-throughput screening of compounds with potential anti-inflammatory activity as well as a protocol to validate the positive hits using an alternative β-galactosidase substrate. |
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