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Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum
Published on: December 4, 2015
Erythrocytic stage-dependent regulation of oligomerization of Plasmodium ribosomal protein P2
Sudipta Das1, Rajagopal Sudarsan, Subramanian Sivakami
1Department of Biological Sciences, Tata Institute of Fundamental Research, 400005 Mumbai, India.
Insights
Plasmodium falciparum protein P2 (PfP2) undergoes development-specific oligomerization, suggesting nonribosomal roles. This process, crucial for export to the infected erythrocyte surface, involves complex interactions beyond simple disulfide bonds.
Area of Science:
- Molecular Biology
- Parasitology
- Biophysics
Background:
- The eukaryotic 60S ribosomal stalk includes P0, P1, and P2 proteins.
- Plasmodium falciparum protein P2 (PfP2) exhibits nonribosomal functions and is exported to the infected erythrocyte surface.
- PfP2 oligomerization is linked to its export and occurs during specific developmental stages.
Purpose of the Study:
- To investigate the biophysical properties of Plasmodium falciparum P2 (PfP2).
- To understand the mechanism and regulation of PfP2 oligomerization.
- To explore the structure-function relationship of PfP2 during its export to the infected erythrocyte surface.
Main Methods:
- Recombinant PfP2 (rPfP2) protein expression and characterization.
- Sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) treatment to assess oligomerization.
- Circular dichroism (CD) analysis to determine protein secondary structure.
- Synchronized Plasmodium falciparum erythrocytic cultures and analysis at different post-merozoite invasion (PMI) time points.
- Tetramethylrhodamine (TMR) labeling for protein tracking.
Main Results:
- Recombinant PfP2 (rPfP2) exhibits SDS-resistant oligomerization, partially reducible by DTT.
- Oligomerization persists even after cysteine mutation or C-terminal deletion, indicating non-disulfide bond involvement.
- PfP2 exists as a ribosomal complex (∼96 kDa) at 18 and 30 h PMI, sensitive to SDS.
- At 30 h PMI, large SDS-sensitive aggregates (>600 kDa) and smaller complexes (60-80 kDa) are observed.
- DTT- and SDS-resistant oligomerization of TMR-labeled rPfP2 occurs only with parasite extracts from 24-36 h PMI, requiring multiple proteins.
Conclusions:
- PfP2 oligomerization is a complex, development-specific process in Plasmodium.
- The oligomerization mechanism involves factors beyond simple disulfide bonds and C-terminal interactions.
- Understanding PfP2 oligomerization regulation is key to elucidating its novel structure-function relationship and export mechanism.
Abstract:
The eukaryotic 60 S-ribosomal stalk consists of P0, P1, and P2 proteins, which associate in a pentameric structure (P1(2)-P0-P2(2)). The Plasmodium falciparum protein P2 (PfP2) appears to play nonribosomal roles. It gets exported to the infected erythrocyte (IE) surface at 30 h post-merozoite invasion (PMI), concomitant with extensive oligomerization. Here we present certain biophysical properties of PfP2. Recombinant P2 (rPfP2) protein showed SDS-resistant oligomerization, which could be significantly abolished under reducing conditions. However, the protein continued to oligomerize even when both cysteine residues were mutated, and with up to 40 amino acids (aa) deleted from the C-terminal end. CD analysis of P2 showed largely α-helical and random coil domains. The SDS- and DTT-resistant oligomerization was studied further as it occurred in a development-specific manner in Plasmodium. In a synchronized erythrocytic culture of P. falciparum, the PfP2 protein was detected as part of the ribosomal complex (∼96 kDa) at 18 and 30 h PMI, and was SDS sensitive. However, at 30 h, large amounts of SDS-sensitive aggregates of >600 kDa were also seen. At 30 h PMI, each of the parasites, IE cytosol and IE ghost contained 60-80-kDa PfP2 complexes, which resolved to a single 65-kDa species on SDS-PAGE. Tetramethylrhodamine-labeled rPfP2 protein exhibited DTT- and SDS-resistant oligomerization when treated with P. falciparum parasite extracts only from 24 to 36 h PMI, and multiple proteins appeared to be required for this oligomerization. Understanding the regulation of oligomerization of PfP2 may help in the elucidation of the novel structure-function relationship in the export of PfP2 to the red cell surface.
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