Abstract
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.
Highlights
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
Assessment of the levels of ubiquitylated proteins at different parasite stages Plasmodium falciparum intra-erythrocytic cycle, which includes rings, trophozoites and schizonts stages (Figure 1A) was used to set up the assays as it is related with its pathogenesis and is the primary target of anti-malarial drug development
To determine the stage containing the largest accumulation of ubiquitylated proteins after treatment with a UPS inhibitor, two different proteasome inhibitors were used: the well-known MG132 and epoxomicin. infected red blood cells (iRBCs) with P. falciparum at different stages were treated for 1 hr with these proteasome inhibitors
Summary
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 protein modification process, known as conjugation, requires three sequential steps that start with the activation of the C-terminal glycine residue of the ubiquitin by an ubiquitin-activating enzyme (E1), which forms a thiolester linkage with ubiquitin. The process can be reverted by the action of deubiquitylating enzymes (DUBs) that are responsible for the dynamic equilibrium of the system
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