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Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum
Published on: December 4, 2015
The multifaceted histone chaperone RbAp46/48 in Plasmodium falciparum: structural insights, production, and
Manjeri Kaushik1, Ashima Nehra1, Surendra Kumar Gakhar1
1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, 124 001, India.
Abstract:
RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7_0110700 and PF3D7_1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 β-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7_0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology.
Insights
Researchers characterized Plasmodium falciparum RbAp46/48 (PfRbAp46/48), a histone chaperone essential for chromatin structure. The study confirmed its nuclear localization and interaction with histone H4, advancing understanding of its role in parasite biology.
Area of Science:
- Molecular Biology
- Parasitology
- Chromatin Biology
Background:
- RbAp46/RBBP7 and RbAp48/RBBP4 are crucial WD40-repeat histone chaperones involved in chromatin structure maintenance.
- RbAp48 is a key subunit of the chromatin assembly factor-1 (CAF-1) complex, also known as CAF-1C.
- In silico analysis revealed conserved features and species-specific differences in RbAp46/48 homologs across various organisms.
Purpose of the Study:
- To clone, express, and characterize the putative RbAp46/48 homolog (PF3D7_0110700) from Plasmodium falciparum, termed PfRbAp46/48.
- To investigate the expression pattern and subcellular localization of PfRbAp46/48 during asexual blood stages.
- To determine the interaction of purified PfRbAp46/48 with histone H4.
Main Methods:
- Cloning of P. falciparum PF3D7_0110700 into a pTEM11 vector with a 6xHistidine tag.
- Over-expression in Escherichia coli B834 cells and purification using Ni-NTA affinity and gel permeation chromatography.
- Analysis of protein expression across asexual blood stages, subcellular localization studies, and in vitro histone interaction assays.
Main Results:
- PfRbAp46/48 was successfully cloned, expressed, and purified as a recombinant protein (rPfRbAp46/48).
- The protein was detected in all three asexual blood stages of the parasite and localized to the nucleus.
- Direct interaction between purified rPfRbAp46/48 and histone H4 was demonstrated.
Conclusions:
- The characterization of PfRbAp46/48 provides insights into the function of RbAp46/48 proteins in Plasmodium falciparum.
- Nuclear localization and histone H4 interaction highlight the potential role of PfRbAp46/48 in chromatin dynamics and regulation within the parasite.
- This study contributes to understanding the fundamental biological processes governed by histone chaperones in malaria parasites.
