Chaperones and Proteases of the Plasmodium falciparum Parasite

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Protein homeostasis in the cell is regulated by a wide array of quality control systems consisting of molecular chaperones and proteases. These systems assist newly-translated proteins in folding to their native state and also ensure that misfolded proteins and short-lived regulatory proteins are targeted for degradation. Understanding the cellular roles and mechanisms of function of chaperones and proteases will provide critical insights into the basic physical principles that govern protein folding in the cell. The eukaryotic protozoan parasite Plasmodium falciparum is the causative agent of malaria. It harbors two organelles of prokaryotic origin: the mitochondrion and the apicoplast. The apicoplast is a non-photosynthetic plastid derived from an ancient red algal endosymbiont. Hence, the apicoplast is considered a vestigial plastid and studies on the apicoplast should provide critical insights into the evolution of the plant chloroplast-specific processes. The parasite has a large number of molecular chaperones and proteases. Importantly, several of these chaperones and proteases reside in the apicoplast and are shown to be critical for the development of a functional organelle. To this end, we started a trans-disciplinary research program aimed at biochemically and biophysically characterizing the chaperones and proteases in the P. falciparum apicoplast. Our studies in the current proposed project will specifically concentrate on the Caseinolytic (Clp) chaperone-protease complexes. These complexes are highly conserved across species and perform essential quality control functions by degrading regulatory and misfolded proteins. Initial studies from my group have shown that a PfClpCRP chaperone-protease complex exists in the parasite apicoplast. PfClpC is an ATPase and a member of the AAA+ superfamily, and, hence, contains in its sequence conserved Walker A and B motifs. PfClpC is proposed to form a hexameric complex. PfClpP and PfClpR are paralogs that we propose form a cylindrically-shaped oligomeric protease containing a proteolytic chamber for degradation. While PfClpP has the Ser-His-Asp catalytic triad, these residues are mutated in PfClpR rendering PfClpR the inactive subunit in the PfClpRP oligomer. We propose that the mechanism of function of the PfClpCRP complex involves the binding and unfolding of target proteins by PfClpC ATPase chaperone, which then translocates them into the PfClpRP cylindrical protease for degradation. The proposed project will concentrate on the structural and functional characterization of PfClpCRP. The project has two specific aims: 1. Biochemical and biophysical studies on PfClpCRP. We will initially reconstitute a PfClpRP complex from purified protein components. The oligomeric state, activity, and proteolytic specificity of the complex will be assessed using biophysical approaches. Similarly, PfClpC will be characterized. The ATPase and unfoldase activity of the chaperone will be investigated using model substrates. Subsequently, the mechanism of function of the complete PfClpCRP complex will be investigated. 2. Structural studies on PfClpCRP. Earlier, we had obtained the X-ray crystal structure of PfClpP and PfClpR, and negative stain electron microscopy (EM) 2D images of the separate PfClpP and PfClpR heptamers. We will continue our structural efforts with the long term goal of obtaining a high resolution structure of the PfClpCRP complex by combining X-ray and electron microscopy approaches. Such a comprehensive study of the PfClpCRP system in P. falciparum apicoplast will provide valuable insights into the mechanism of function of this essential chaperone-protease system and the principles that govern protein homeostasis in this vestigial plastid.