Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. While the parasite has multiple life cycle stages, it is in the blood stage where clinical symptoms of the disease are manifested. Following erythrocyte entry, the parasite resides in the parasitophorous vacuole and actively transports its own proteins to the erythrocyte cytosol. This host-parasite "cross-talk" results in tremendous modifications of the infected erythrocyte imparting properties that allow it to adhere to the endothelium preventing splenic clearance. The Hsp70-J protein (DnaJ/Hsp40) molecular chaperone machinery, involved in cellular protein homeostasis, is being investigated as a novel drug target in various cellular systems including malaria. In Plasmodium the diverse chaperone complement is intimately involved in infected erythrocyte remodelling associated with the development and pathogenesis of malaria. In this review, we provide an overview of the Hsp70-J protein chaperone complement in Plasmodium falciparum and compare it with other Plasmodium species including the ones that serve as experimental study models for malaria. We propose that the unique traits possessed by this machinery not only provide avenues for drug targeting but also inform the evolutionary fitness of this parasite to its environment.
The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.
Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I–IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.
Cytidine diphosphate (CDP)-diacylglycerol synthase (cytidine triph0sphate:phosphatidate cytidyltransferase, EC 2.7.7.41) catalyzes the formation of CDP-diacylglycerol, which is the precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We report the first cloning, to our knowledge, of two plant cDNAs, StCDSl and AtCDS1, coding for CDP-diacylglycerol synthases from potato (Solanum tuberosum) and Arabidopsis thaliana, respectively. The two proteins belong to the eukaryotic type of CDP-diacylglycerol synthases and contain eight predicted transmembrane-spanning domains. We analyzed gene expression in shoot and root tissues of potato plants and demonstrated enzyme activity by expression of N-terminally truncated, recombinant StCDSl in Escherichia coli.CDS (cytidine triph0sphate:phosphatidate cytidyltransferase, EC 2.7.7.41) catalyzes the synthesis of CDP-DG and PPi from PA and CTP. CDP-DG is the precursor of the minor lipids PI, PG (via PG phosphate), cardiolipin, and possibly phosphatidylserine (Moore, 1982). CDS activity is present in most plant membrane systems. Located at the inner envelope membrane of chloroplasts, CDS may be mainly engaged in PG synthesis (Andrews and Mudd, 1985). In mitochondria, CDP-DG is the substrate for PG phosphate synthase (EC 2.7.8.5) and cardiolipin synthase. CDS and other enzymes involved in cardiolipin biosynthesis are enriched at the inner mitochondrial membrane (Frentzen and Griebau, 1994). Microsomes isolated from castor bean endosperm contain approximately 75% of total extractable CDS activity (Kleppinger-Sparace and Moore, 1985). Microsomal membranes might account for most of the PI synthesis in plant cells (Moore, 1982). Studies on microsomes isolated from spinach leaves also suggest the presence of phosphatidylserine synthase (EC 2.7.8.8), which utilizes CDP-DG (Marshall and Kates, 1974). Moreover, CDS and other enzymes necessary for PI resynthesis from phospholipase C and D reaction products are present in plant plasma membranes (Wissing et al., 1992). As demonstrated by the recent discovery of an eye-specific CDS from Drosophila melanogaster, CDS activity is essential for IP,-mediated light perception in insect eyes (Wu et al., To investigate the role of CDS in plants we isolated and characterized the first plant cDNA clones of this enzyme. We present the predicted primary structures and properties of CDS from potato (Solanum tuberosum) (StCDSl) and Arabidopsis tkaliana (AtCDSl), a preliminary analysis of gene expression in potato plants, and enzyme activity of recombinant StCDSl. MATERIALS A N D METHODS Cloning and Sequencing of cDNAsA TBLASTN search (Altschul et al., 1990) for CDS homologous plant sequences was performed in the EST database at the National Center of Biotechnology Information (Bethesda, MD). Two overlapping ESTs from Arabidopsis tkaliana, T45653 and N97146, were found with a "bait" amino acid sequence of CDS from Esckerickia coli. The ESTs contained a continuous open reading frame that encoded a partia1 protein with highest sequence homol...
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