title: Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Plasmodium 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Present address: Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, 28760, , Madrid, Spain. lydia.c.mata@gsk.com.|Centro de Investigación Básica, GlaxoSmithKline, Santiago Grisolía 4, 28760, Tres Cantos, Madrid, Spain. lydia.c.mata@gsk.com.|Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain. lydia.c.mata@gsk.com.
authors.forename: Lydia
authors.initials: L
authors.lastname: Mata-Cantero
authors.affiliation: Centro de Investigación Básica, GlaxoSmithKline, Santiago Grisolía 4, 28760, Tres Cantos, Madrid, Spain. concha.cid@gsk.com.
authors.forename: Concepción
authors.initials: C
authors.lastname: Cid
authors.affiliation: Present address: Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, 28760, , Madrid, Spain. maria.g.gomez@gsk.com.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Gomez-Lorenzo
authors.affiliation: Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain. wendyxolalpa@gmail.com.
authors.forename: Wendy
authors.initials: W
authors.lastname: Xolalpa
authors.affiliation: Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain. ailletfabienne@gmail.com.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Aillet
authors.affiliation: Centro de Investigación Básica, GlaxoSmithKline, Santiago Grisolía 4, 28760, Tres Cantos, Madrid, Spain. Julio.J.Martin@gsk.com.
authors.forename: J Julio
authors.initials: JJ
authors.lastname: Martín
authors.affiliation: Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain. msrodriguez@inbiomed.org.
authors.forename: Manuel S
authors.initials: MS
authors.lastname: Rodriguez
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D016778:Malaria, Falciparum / Q000188:drug therapy; D010963:Plasmodium falciparum / Q000187:drug effects*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D015800:Protozoan Proteins / Q000032:analysis*; D057149:Ubiquitinated Proteins / Q000032:analysis*; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D000962:Antimalarials; D015800:Protozoan Proteins; D057149:Ubiquitinated Proteins; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex