Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials

Malaria Journal

Methodology

Concepción Cid¹  J Julio Martín¹  Maria G Gomez-Lorenzo²  Lydia Mata-Cantero³  Wendy Xolalpa⁴  Manuel S Rodriguez⁴  Fabienne Aillet⁴ 

[1] Centro de Investigación Básica, GlaxoSmithKline, Santiago Grisolía 4, 28760, Tres Cantos, Madrid, Spain;Present address: Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760,, Tres Cantos, Madrid, Spain;Present address: Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760,, Tres Cantos, Madrid, Spain;Centro de Investigación Básica, GlaxoSmithKline, Santiago Grisolía 4, 28760, Tres Cantos, Madrid, Spain;Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain;Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain;

关键词: Plasmodium falciparum;    Malaria;    Ubiquitin proteasome system;    High-throughput screening;    Tandem ubiquitin binding entities;    Cellular assay;    Drug discovery;   

DOI  :  10.1186/s12936-015-0708-1

 received in 2015-01-29, accepted in 2015-04-20,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundThe ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery.MethodsPlasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors.ResultsIn both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition.ConclusionsThe two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

【 授权许可】

Unknown   
© Mata-Cantero et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.