CHAPTER 3 Bioinformatic studies and antigenic peptide selection for the putative
3.3 Discussion
3.3.1 Identification of a putative Plasmodium spp. copper transport protein and
characterised copper-dependent proteins. This yielded positive results for some, but not all, of
0 10 20 30 40 50 60 70
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
0.8 0.9 1 1.1 1.2
Amino acid number
Hydrophilicity and surface probability Flexibility
0 10 20 30 40 50 60 70
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
0.8 0.9 1 1.1 1.2
Amino acid number
Hydrophilicity and surface probability Flexibility
B.
A.
KTC
NKG
the sequences used (Table 3.1). Perhaps one of the more important proteins identified was a subunit of a cytochrome-c oxidase complex that requires copper for the formation of the active protein (Table 3.1). In the presence of oxygen, energy production in eukaryotic cells is predominantly generated by mitochondrial oxidative phosphorylation (Iwata et al., 1995). This process is driven by an electron-transport chain composed of a number of complex mitochondrial membrane proteins, of which cytochrome-c oxidase is the terminal enzyme (Cobine et al., 2006a). Deficiencies in cytochrome-c oxidase activity have been linked to a number of human diseases including encephalomyopathies, Leigh syndrome, hypertrophic cardiomyopathies and fatal lactic acidosis (Diaz, 2010). This highlights the importance of this enzyme to human cells and suggests the possibility that this enzyme is likely to be important in Plasmodium metabolism. This is supported by the identification of Cox11, Cox17, Cox19 and Sco1 metallochaperone orthologs, which transfer and insert copper into the cytochrome-c oxidase complex. The identification of a copper-requiring S-adenosyl-L-homocysteine hydrolase ortholog was also significant since this protein has been established as being important to parasite survival since enzyme inhibition interferes with parasite proliferation (Bujnicki et al., 2003; Creedon et al., 1994). Taken together, these results suggest a parasite requirement for copper.
A physiological role for copper, in the Plasmodium parasite, has previously been suggested from studies using the intracellular copper chelator neocuproine. Treatment of an in vitro parasite culture with nanomolar concentrations of neocuproine inhibited parasite transition from rings to trophozoites (Rasoloson et al., 2004). However, the mechanism by which the parasite acquires copper has not yet been established. Two possible mechanisms exist. The first is copper acquisition via parasite ingestion and digestion of host erythrocyte Cu/Zn SOD (Rasoloson et al., 2004), whilst the other is via a dedicated copper transport protein, similar to that identified in yeast and mammalian cells (Dancis et al., 1994; Lee et al., 2000; Lee et al., 2002). Following extensive research, characteristic sequence features have been identified for this family of proteins. The most notable features include a methionine-rich amino terminus, three transmembrane domains and essential MX3M and GX3G motifs in the second and third transmembrane domains respectively (Aller et al., 2004; Puig et al., 2002a) (Figure 1.5). A BLASTp search of PlasmoDB identified candidate copper transporter sequences for eight species of the Plasmodium parasite. Alignment of the retrieved sequences with sequences of known copper transporters identified the essential and largely definitive MX3M and GX3G motifs in all eight Plasmodium species (Figure 3.1). Methionine motifs were also identified in the predicted amino terminal domain of these proteins. These motifs are important for protein function (Eisses and Kaplan, 2005; Guo et al., 2004; Puig et al., 2002a) but are not unique to the copper transport proteins.
A topological analysis of each sequence identified the presence of three putative membrane- spanning domains (Figure 3.2). This too is considered a definitive feature of the copper transport proteins (Dancis et al., 1994). It is, however, important to note that three transmembrane domains are unable to form a functional channel or pore through which ions can be transported (Nose et al., 2006). It was therefore proposed, and subsequently verified, that copper transport protein monomers create a channel by associating in a homotrimeric complex (Aller and Unger, 2006; De Feo et al., 2009). This copper-permeable pore consequently forms a pathway that permits the controlled movement of copper across the membrane and into the cell. Given the importance of trimer formation to protein function (Aller et al., 2004), a similar formation is likely to exist for the Plasmodium copper transporters.
Supporting this likelihood, was the recent finding that the first transmembrane domain of the copper transporter serves as an adaptor that allows evolutionarily distant copper transport proteins to adopt a similar, but not identical, overall structure (De Feo et al., 2010).
The presence of a putative Plasmodium copper transport protein sequence suggests a dedicated system for copper acquisition in the parasite. This is supported by the presence of sequences for orthologs of cytochrome-c oxidase and its associated metallochaperones Cox11, Cox17 and Sco1 (Table 3.1). Cox17 is of particular interest since this protein localises to the cell cytoplasm and inner mitrochondrial space and is thought to be involved in shuttling copper from the copper transporter to the mitochondrion (Beers et al., 1997). This mechanism has been re-evaluated (Cobine et al., 2006a; Lutsenko, 2010), however, the importance of Cox17 to cell survival (Takahashi et al., 2002) has not been disputed. To better understand parasite copper pathways and homoeostasis, the Cox17 sequence was studied. Eight species of the Plasmodium parasite contained Cox17 metallochaperone sequences, suggesting a conserved presence and role for this protein. ClustalWTM alignment of the Plasmodium sequences, with other Cox17 sequences, highlighted essential and conserved motifs that are important to protein function (Figure 3.4). These include the CysCysXaaCys motif, which coordinates the cuprous ion to Cox17 (Abajian et al., 2004), plus additional cysteine residues required for structural disulfide bond formation (Figure 3.5). From the homology model for PfCox17 it appears that these conserved cysteine residues adopt a similar conformational orientation to the corresponding residues in human Cox17, resulting in PfCox17 forming a coiled coil-helix- coiled coil helix structural motif (Figure 3.5b and c). This is a common feature of related mitochondrial proteins (Barros et al., 2004; Nobrega et al., 2002), which suggests that Plasmodium Cox17 is likely to be involved in copper delivery to the parasite mitochondrion.
This modelling project was, however, prepared on a rudimentary level and thus requires further analysis to confirm the observed and inferred result.
Each Plasmodium Cox17 sequence was further examined for the presence of potential signal sequences and transmembrane regions. Characterisation of S. cerevisiae Cox17 found the protein to be localised to the cell cytosol as well as the inner mitochondrial space (Beers et al., 1997). A signal sequence directing the protein to the inner mitochondrial space is, however, absent raising the question as to how Cox17 is directed to the mitochondrion. Only recently was a possible mechanism behind this targeting action identified. Through a unified electrospray ionization mass spectrometry (ESI-MS) based strategy it was demonstrated that Cox17, amongst other copper chaperones, is driven toward its partner proteins by exploiting gradients of increasing copper binding affinity (Banci et al., 2010). Thus an absence of a signal peptide in the retrieved Plasmodium sequences would, to some degree, support the likelihood of these proteins being representative of Cox17 metallochaperones. Additionally, Cox17 is not known to anchor to cell or mitochondrial membranes, therefore the absence of long hydrophobic stretches of amino acids was expected. Each sequence was systematically analysed by SignalP, PlasmoAP, PlasMit (Bender et al., 2003), TMHMM and HMMTOP. As predicted, none of the retrieved sequences returned a positive result for any of the above evaluations. Overall, the presence of both the Cox17 copper chaperone and copper transport protein supports a physiological need for copper by the malaria parasite.