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

Mata-Cantero L, Cid C, Gomez-Lorenzo MG, Xolalpa W, Aillet F, Martín JJ, Rodriguez MS - Malar. J. (2015)

Bottom Line: 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.A panel of well-known proteasome inhibitors has been used to validate both technologies.This methodology is also applicable to other cellular contexts or pathologies.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Analysis of ubiquitylated proteins along Plasmodium falciparum intra-erythrocytic cycle. A. Schematic representation of the parasite cycle in blood stages. It starts when merozoites invade uRBC resulting in the first phase called ring stage. After 24 hrs, the parasite enters in a phase with high metabolic activity, the trophozoite. A few hours later, at the schizont stage, the parasite produces merozoites that will rupture the RBC membrane, looking for new uRBCs to invade and start a new cycle. B. Western blot analysis with P4D1 antibody shows basal levels of ubiquitylated proteins in rings, trophozoites and schizonts. C. Effect of proteasome inhibitors MG132 (1.15 μM) and epoxomicin (0.26 μM), on the levels of ubiquitylated proteins from rings, trophozoites and schizonts. Atovaquone at 0.02 μM was used as negative control.
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Fig1: Analysis of ubiquitylated proteins along Plasmodium falciparum intra-erythrocytic cycle. A. Schematic representation of the parasite cycle in blood stages. It starts when merozoites invade uRBC resulting in the first phase called ring stage. After 24 hrs, the parasite enters in a phase with high metabolic activity, the trophozoite. A few hours later, at the schizont stage, the parasite produces merozoites that will rupture the RBC membrane, looking for new uRBCs to invade and start a new cycle. B. Western blot analysis with P4D1 antibody shows basal levels of ubiquitylated proteins in rings, trophozoites and schizonts. C. Effect of proteasome inhibitors MG132 (1.15 μM) and epoxomicin (0.26 μM), on the levels of ubiquitylated proteins from rings, trophozoites and schizonts. Atovaquone at 0.02 μM was used as negative control.

Mentions: Plasmodium falciparum divides rapidly during its intra-erythrocytic cycle (see Figure 1A), fulfilling the criteria to be targeted by a UPS inhibitor. Moreover, multiple evidence indicates that the UPS is involved in the parasite cell cycle progression and protein quality control [8,9]. Even though there is a conserved sequence homology between the parasite and human proteasome proteins, there is space for selectivity not only in the proteasome but also at specific components of the UPS such as E3 ligases and DUBs [10-12]. Targeting P. falciparum UPS can provide novel modes of action to overcome the emerging resistance to current treatments, as already demonstrated in vitro [13]. Indeed, proteasome inhibitors can efficiently inhibit P. falciparum-resistant strains at various stages of the cycle in the nanomolar range and with limited toxicity to humans [13-15]. New inhibitors targeting specifically the parasite UPS are desirable [16] to reduce possible side effects.


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

Mata-Cantero L, Cid C, Gomez-Lorenzo MG, Xolalpa W, Aillet F, Martín JJ, Rodriguez MS - Malar. J. (2015)

Analysis of ubiquitylated proteins along Plasmodium falciparum intra-erythrocytic cycle. A. Schematic representation of the parasite cycle in blood stages. It starts when merozoites invade uRBC resulting in the first phase called ring stage. After 24 hrs, the parasite enters in a phase with high metabolic activity, the trophozoite. A few hours later, at the schizont stage, the parasite produces merozoites that will rupture the RBC membrane, looking for new uRBCs to invade and start a new cycle. B. Western blot analysis with P4D1 antibody shows basal levels of ubiquitylated proteins in rings, trophozoites and schizonts. C. Effect of proteasome inhibitors MG132 (1.15 μM) and epoxomicin (0.26 μM), on the levels of ubiquitylated proteins from rings, trophozoites and schizonts. Atovaquone at 0.02 μM was used as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4440562&req=5

Fig1: Analysis of ubiquitylated proteins along Plasmodium falciparum intra-erythrocytic cycle. A. Schematic representation of the parasite cycle in blood stages. It starts when merozoites invade uRBC resulting in the first phase called ring stage. After 24 hrs, the parasite enters in a phase with high metabolic activity, the trophozoite. A few hours later, at the schizont stage, the parasite produces merozoites that will rupture the RBC membrane, looking for new uRBCs to invade and start a new cycle. B. Western blot analysis with P4D1 antibody shows basal levels of ubiquitylated proteins in rings, trophozoites and schizonts. C. Effect of proteasome inhibitors MG132 (1.15 μM) and epoxomicin (0.26 μM), on the levels of ubiquitylated proteins from rings, trophozoites and schizonts. Atovaquone at 0.02 μM was used as negative control.
Mentions: Plasmodium falciparum divides rapidly during its intra-erythrocytic cycle (see Figure 1A), fulfilling the criteria to be targeted by a UPS inhibitor. Moreover, multiple evidence indicates that the UPS is involved in the parasite cell cycle progression and protein quality control [8,9]. Even though there is a conserved sequence homology between the parasite and human proteasome proteins, there is space for selectivity not only in the proteasome but also at specific components of the UPS such as E3 ligases and DUBs [10-12]. Targeting P. falciparum UPS can provide novel modes of action to overcome the emerging resistance to current treatments, as already demonstrated in vitro [13]. Indeed, proteasome inhibitors can efficiently inhibit P. falciparum-resistant strains at various stages of the cycle in the nanomolar range and with limited toxicity to humans [13-15]. New inhibitors targeting specifically the parasite UPS are desirable [16] to reduce possible side effects.

Bottom Line: 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.A panel of well-known proteasome inhibitors has been used to validate both technologies.This methodology is also applicable to other cellular contexts or pathologies.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus