Neil D. Goldman

Molecular cloning and nuclear localization of a histone deacetylase homologue in Plasmodium falciparum

Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression and developmental events. The acetylation state of histones is controlled by a dynamic equilibrium between activities of histone acetylase and deacetylase enzymes. Histone deacetylase (HDAC) was recently suggested to be the target of a fungus-derived antiprotozoal agent exhibiting structural similarity to known HDAC inhibitors. We have initiated a study of HDAC of human malaria parasite, Plasmodium falciparum, to evaluate its potential as the target for novel antimalarials and its role in parasite development. We have isolated HDAC1 gene from the P. falciparum genomic and cDNA libraries. The nucleotide sequence contains no intervening sequence and its open reading frame (ORF) codes for a protein of 449 amino acid residues. We have named the protein, PfHDAC1, as the sequence shows significant homology to yeast, human and other eukaryotic HDACs. Northern blot analysis of the total RNA from different asexual and sexual stages of the parasite reveals the presence of single mRNA transcript, which is predominantly expressed in mature asexual blood stages and in gametocytes. Antiserum raised against a carboxyl terminal peptide immunoprecipitated an in vitro translated P. falciparum HDAC gene product and recognized an approximately 50 kDa protein in the Triton X-100 insoluble fraction of parasites. Immunoelectron microscopy analysis showed majority of the protein localized in the nucleus of P. falciparum. To our knowledge, this is the first HDAC gene isolated from the malaria parasite.

Stage-specific expression of a Plasmodium falciparum protein related to the eukaryotic mitogen-activated protein kinases.

We have identified a putative protein kinase gene from both Plasmodium falciparum cDNA and genomic DNA libraries. The nucleotide sequence contains an open-reading frame of 2646 bp, which codes for a predicted protein of 882 amino acid residues. Comparison of the predicted amino acid sequence with those in GenBank suggests that this gene codes for a protein similar to the mitogen-activated protein (MAP) kinase of other organisms. This MAP kinase-related protein, named PfMRP, contains the TDY dual phosphorylation site upstream of the highly conserved VATRWYRAPE sequence in subdomain VIII. PfMRP contains an unusually large and highly charged domain within its carboxyl-terminal segment, which includes two repetitive sequences of either a tetrapeptide or octapeptide motif. PfMRP gene is located on chromosome 14. Northern blot analysis of total RNA reveals the presence of a single mRNA transcript approximately 4.2 kb in length, which is predominantly expressed in gametocytes and gametes/zygotes.

Cloning of a Plasmodium falciparum gene related to the human 60-kDa heat shock protein

We have identified a gene encoding the 60 kDa heat shock protein (hsp60) from a Plasmodium falciparum blood stage cDNA library. The deduced protein sequence encodes for a polypeptide of 577 amino acids with a calculated molecular weight of 62158 Da. The primary structure of P. falciparum hsp60 contains a putative mitochondrial targeting peptide at its amino-terminus and a GGM motif at its carboxyl-terminus. The overall structure exhibits strong conservation (approximately 50%) to the hsp60 from human and other eukaryotes, but only low homology (< 30%) to a recently reported P. falciparum chaperonin 60 gene. The P. falciparum hsp60 gene is located on chromosome 10. During heat shock, the level of hsp60 transcript in blood stage parasites increases significantly and its accumulation correlates with the duration of the induction.

MOLECULAR CHARACTERIZATION AND ULTRASTRUCTURAL LOCALIZATION OF PLASMODIUM FALCIPARUM HSP 60

Heat shock proteins (Hsp) are a group of highly conserved proteins which are widely represented phylogenetically. Genes for members of the Hsp 70, 90 and 60 families have been cloned from the human malaria parasite Plasmodium falciparum. In this study, we have cloned and expressed the P. falciparum Hsp 60 (PfHsp60) in E. coli. The sequence analysis identified a previously unknown intron of 257 bp beginning after the nucleotide 142 in the coding sequence. Antisera raised against the recombinant PfHsp60 was employed in immunoprecipitation studies with biosynthetically labeled parasite extracts to investigate regulation of expression of PfHsp60 at various temperatures. In contrast to the three to four fold accumulation of PfHsp60 transcripts in heat shocked parasites (37-40 degrees C), the expression of PfHsp60 was not induced in the blood stages of P. falciparum. On the other hand, the effect of heat induction on PfHsp70 was seen both at the level of specific mRNA and protein. In these studies we also observed co-immunoprecipitation of a number of other cellular proteins suggesting possible interaction with PfHsp60. Immunofluorescence analysis indicated the presence of PfHsp60 in the cytoplasm of all the various stages of the parasite. In addition, immunoelectron microscopic analysis distinctly localized PfHsp60 in the mitochondrion of P. falciparum. This study suggests that different mechanisms are involved in the regulation of expression of various members of the heat shock proteins in the parasite.

Comparison of protein structure and genomic structure of human, rabbit, and limulus C-reactive proteins: Possible implications for function and evolution

The primary structures of human, rabbit, and Limulus C-reactive proteins (CRPs) have been compared by a computer program. Based on these data, a PAMs matrix (accepted point mutation per 100 residues) was constructed to generate topologies for the three proteins. Five trees with the shortest absolute length were generated, but only one positive tree was found. Using the relatively well-established distance between human and rabbit of 150 million years, we calculate that human and Limulus CRPs diverged at least 500 million years ago. The data indicate that the amino acid sequence indentity between Limulus CRPs and their mammalian counterparts is about 25%, strongly suggesting that human CRP, rabbit CRP, and Limulus CRPs share common ancestral genes. There are two highly conserved regions in the primary structures among the CRPs. Residues 52–67 in Limulus CRP and residues 51–66 in human CRP show identity in 10 of 16 positions, with 3 additional conservative replacements. This region of the molecule is thought to be involved in the binding of phosphorylcholine ligand. Residues 139–153 in Limulus CRP and residues 133–147 in human CRP show identity in 9 of 15 positions, with 5 additional conservative replacements. The biological function of this stretch of amino acid sequence is thought to be associated with the CA2+ binding of the CRPs.

Biochemical and biological analysis of human interleukin 6 expressed in rodent and primate cells

The cDNA for human interleukin 6 (IL 6) was stably expressed at high levels in the three mammalian cell lines COS-7, PA317, and GH3 to yield IL 6 proteins of 25 to 27, 26, 22 to 24, and 23 kDa molecular mass. Both size and relative amounts of the recombinant IL 6 (rIL 6) species produced correspond to those of natural IL 6 secreted by LPS-stimulated monocytes. Oligosaccharide analysis of recombinant IL 6 utilizing tunicamycin and endoglycosidases revealed O- and N-linked glycosylation that is comparable to that of natural IL 6 derived from human monocytes and fibroblasts. IL 6 expressed in each of the three cell lines was phosphorylated similarly to the IL 6 produced in human monocytes and fibroblasts. IL 6 secreted by the three different cell lines have marked differences in specific biological activities. COS-7 IL 6 appeared to be 12-fold more active in its hybridoma growth factor activity than that made in PA317 or GH3 cells. In contrast, PA317 and GH3 IL 6 were 230 and 6.7 times more effective than COS-7 IL 6 in inducing Ig production in CESS cells. Also, PA317 and GH3 IL 6 were more effective than COS-7 IL 6 in inducing the acute-phase protein fibrinogen in human hepatocytes. The rIL 6 species exhibited no antiviral activity.

Expression and characterisation of a Plasmodium falciparum protein containing domains homologous to sarcalumenin and a tyrosine kinase substrate, eps15.

We have identified in Plasmodium falciparum a novel gene encoding a putative bi-functional protein, termed PfPast-1, from genomic and cDNA libraries. Analysis indicated that the sequence encodes a 62 kDa protein of 529 amino acid residues with two distinctive domains: a sarcalumenin-like domain of approximately 320 amino acids at the amino half of the molecule, which shares homology to a major sarcoplasmic reticulum lumenal protein, sarcalumenin, and an eps15 homology domain of about 90 amino acids located at the carboxyl terminus. The eps15 homology domain, first identified in a tyrosine kinase substrate, eps15, and found in increasing numbers of mammalian proteins, has recently been suggested as a protein-protein interaction domain involved in intracellular sorting. Genomic sequences encoding similar proteins containing both the sarcalumenin-like and eps15 homology domains have been identified in humans and Drosophila. RNA blot analysis revealed the presence of a single messenger RNA transcript approximately 3.7 kb in size, which is expressed in all the developmental stages examined with the highest level in extracellular gametes followed by erythrocytic asexual stages, and the lowest in the gametocytes. In the attempt to define its biological function, we have expressed a full-length recombinant PfPast-1 protein in Escherichia coli. Specific immune serum directed against the recombinant protein recognised a approximately 55 kDa protein in the parasite lysate. Further characterisation of PfPast-1 may help in elucidation of its functions in P. falciparum.

HSF Activity of a Mononuclear Cell‐derived B‐Cell Growth Factor

A number of biological response modifiers (e.g., IL-I, TNF, and HSF) have been found to be directly responsible for the induction of many of the acute phase reactants (al-acid glycoprotein, al-antichymotrypsin, hemopexin, etc.). As a model for the acute phase induction, we have studied the prototype acute phase reactant, C-reactive protein (CRP), whose synthesis has been observed during infection to increase over