Plasmodium Falciparum Biology - Molecular Biology and Biochemistry - Molecular Biology

Molecular Biology

The messenger RNA capping system appears to be more similar to that of fungi than to vertebrates. It encodes RNA guanylyltransferase (Pgt1) and RNA triphosphatase (Prt1) enzymes. The triphosphatase enzyme is a member of the fungal/viral family of metal dependent phosphohydrolases. These are structurally and mechanistically unrelated to the cysteine phosphatase type RNA triphosphatases found in metazoans and plants.

There are two translation elongation factor G proteins encoded in the genome. One is located in the mitochondrion and the second in the plastid. Both appear to be inhibitable with fusidic acid

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. A P. falciparum gene encoding a protein with 24% homology to human PNKP has been cloned. This enzyme dephosphorylates single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks.

Sir2A is a member of the sirtuin family of nicotinamide adenine dinucleotide dependent deacetylases. In P. falciparum it has been has been shown to regulate the expression of surface antigens to evade the detection by host immune surveillance. While it is a poor deacetylator of histones it also catalyzes the hydrolysis of medium and long chain fatty acyl groups from lysine residues. Proteins are present in P. falciparum with these modifications and these can be removed by can be removed by PfSir2A in vitro. This suggests that this may be its role rather than the deacetylation of histones.

The telomerase (tert) is a large protein (2518 codons) and has a predicted molecular weight of ~280 kiloDaltons. It has the usual telomerase specific motifs within the N-terminal half of the protein (GQ/N, CP, QFP and T) and reverse transcriptase (RT) specific motifs in the C-terminal half. The N-terminal half is required for efficient binding of the RNA template, defining the 5′ RNA template boundary, multimerization and interactions with associated proteins. The RT domain is essential for the catalytic activity. The protein contains several nuclear localization signals and is found in the nucleolus.

A histone deacetylase (HDAC1) has been cloned. The protein has 449 amino acid residues and localises to the nucleus. Its molecular weight is 50 kiloDaltons and it is predominantly expressed in mature asexual blood stages and in gametocytes.

A novel DNA/RNA binding protein PfAlba has been described. This protein is related to the archaeal protein Alba (Acetylation lowers binding affinity). There are at least four paralogs of the PfAlba gene and these proteins form a complex with the P. falciparum specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric regions. Also associated with the TARE6 regions are PfSir2 a histone deacetylase. In the early blood stages the PfAlba proteins are enriched at the nuclear periphery and associate with the PfSir2 proteins. When the parasite switches from trophozoite to the schizont stage the PfAlba proteins move to the cytoplasm. These proteins will also bind single stranded RNA but the reason for this binding is not known.

A number of novel DNA binding sites have been identified along the genome. Their function - if any - remains to be determined.

Aminoacyl-tRNA synthetases are required for protein synthesis. Alanine tRNA synthetase, glycine tRNA synthetase and threonine tRNA synthetase are dually localised to the cytosol and the apicoplast. These enzymes do not appear to be present in the mitochondrion.

Tyrosyl tRNA synthetase is secreted by the parasite into the cytoplasm of the infected erythrocyte. On lysis of the erythrocyte it is released into the blood stream where it is pro inflammatory. It is specifically bound by and taken up by host macrophages and leads to enhanced secretion of the cytokines tumor necrosis factor-alpha and interleukin 6. This interaction also increases the adherence linked host endothelial receptors ICAM-1 and VCAM-1.

The cytoplasmic lysyl-tRNA synthetase is dimeric unlike the human version which may be dimeric or tetrameric. It is capable of synthesizing the signalling molecule diadenosine tetraphosphate using ATP as a substrate.

The eukaryotic translation initiation factor 2α has a regulatory serine at position 51. This can be phosphorylated by several kinases. Three are known in P falciparum: IK1, IK2 and PK4. IK1 regulates stress response to amino acid starvation; IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands; and PK4 is essential for the completion of the parasite's erythrocytic cycle.

The centromeres occupy a 4-4.5 kilobase region in each chromosome. The centromeres cluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion.

The single stranded DNA binding protein (SSB) plays an important role in all known organisms. A SSB protein is encoded in the genome and localises to the apicoplast. It forms a homo-tetramer alone and when bound to single stranded DNA. The protein binds 52-65 nucleotides/tetramer. While similar in its overall structure to that of the SSB of E. coli it differs at the carboxy terminal region. Although it binds single stranded DNA in a similar fashion to the SSB of E. coli it does so with the opposite polarity. There are a number of other functional differences between this protein and that of E. coli. The basis for these differences has yet to be determined.

A protein - RPA1L - is the homologue of the bacterial single-stranded binding protein (SSB) and acts in initiating homologous pairing and strand exchange activity. It is negatively regulated in a dose dependent manner by RPA1S.

The eukaryotic homologue of the bacterial RecA protein is Rad51. The Plasmodium falciparum Rad51 protein exhibits ATPase activity and promotes DNA strand exchange. This protein interacts with Rad54 and replication protein A.

The RuvB protein belongs to AAA+ family of enzymes which are involved in diverse cellular activities. There are at least 3 copies of this protein in the genome. RuvB3 possesses the Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. It has single stranded DNA dependent ATPase activity. The protein is mainly expressed during intraerythrocytic schizont stages and localizes to the nuclear region. In the merozoite the protein relocalizes to the sub nuclear region.

A helicase - PfH45 - of 398 amino acid residues (molecular weight 45 kiloDaltons) is a unique bipolar helicase with both the 3' to 5' and 5' to 3' directional helicase activities. It is expressed in all the intraerythrocytic developmental stages and has a role in translation.

The transcription factor NF-YB is localised in the nucleus during the erythrocytic stages of the life cycle. Melatonin and cyclic adenosine monophosphate modulate the expression of NF-YB. NF-YB is also more ubiquitinated in the presence of melatonin.

SET is a conserved nuclear protein involved in chromatin dynamics. In P falciparum it is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. In P falciparum there are two distinct promoters upstream. he one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter.

A protein (PfSR1) involved in alternative splicing has been described. It appears that regulation of this gene is essential for the parasite's normal physiology.

A number of the DExD/DExH-box containing pre-mRNA processing proteins (Prps) - PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p - are present in the genome. PfPrp16p a helicase and a member of DEAH-box protein family with nine collinear sequence motifs has been cloned. It binds to RNA, hydrolyses ATP and appears to be involved in splicing.

A putative tyrosine site specific recombinase has been isolated. The N-terminus has the typical alpha helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. The C-terminal domain has the putative tyrosine recombinase conserved active site residues Lysine-Histadine-Lysine-(Histadine/Tryptophan)-Tyrosine. The gene is expressed differentially during the erythrocytic stages being maximal in the schizont stage. The open reading frame encodes a ∼57 kiloDalton protein. Knockout mutants are viable and appear normal. DNA binding studies suggest a number of targets include the subtelomeric regions.

Apetala 2 (AP2) family proteins are transcription factors that have DNA-binding domains of ~60 amino acids called AP2 domains. 27 AP2-family genes have been identified in the Plasmodium falciparum genome. One of these proteins appears to play a critical role in the liver stage development of the parasite.

Several nucleic acid repair pathways are known. These include the nucleotide excision repair, the mismatch repair, the base excision repair, the double strand break repair and the cross link repair pathways. DNA replication errors - base substitution mismatches and insertion-deletion loops - are primarily corrected by the mismatch repair system.

The DNA helicase II (uvrD) is a superfamily 1A helicase which plays an essential role in the mismatch repair pathway. Homologues of UvrD include the proteins PcrA and Rep. These proteins have a two domain (1 and 2) structure with each domain made of two sub-domains (1A, 1B, 2A and 2B) and a C-terminal extension. They are DNA-dependent ATPases with 3′ to 5′ helicase activity. The helicase activity is located in the N terminal domain.

The UvrD protein of P. falciparum has been cloned. This gene (PFE0705c) is located on chromosome 5 and contains no introns. It is 4326 bases in length, encodes a protein of 1441 amino acids and has a predicted molecular weight of ~170 kiloDaltons. The two domains and their subdomains are present: The 1A domain is from amino acid 1–722; the 1B domain is from amino acid 150–464; the 2A domain is from amino acid 723–1441; and the 2B domain is from amino acid 896–1359. There is no C-terminal extension. The ATPase and helicase activty are confined to domain 1A and 1B (the N-terminal and first half of the C terminal). It is expressed in the schizont stages of intraerythrocytic development and it colocalizes with PfMLH, a protein involved in mismatch repair. Both PfDH60 - another helicase - and PfMLH are also expressed in schizont stages.

The MutL homolog (MLH) - part of the DNA mismatch repair system - has been cloned. MLH possess ATPase and endonuclease activities. Its expression is maximal in the schizont stage.

A 3'-5' DNA helicase has been identified in the parasite. The apparent molecular weight is 90 kiloDaltons. Its activity is dependent on the presence of magnesium and ATP.

Posttranslational covalent modifications of the histones (acetylation, methylation and phosphorylation) are known to occur. How these influence gene transcription is poorly understood. The 14-3-3 protein appears to act as a phosphorlated histone 3 reader.

A number of mini chromosome maintenance proteins are present in the genome. These are large proteins and members of the AAA ATPase family with a conserved region of ~200 amino acids responsible for nucleotide binding. They are responsible for unwinding DNA at the replication forks and are involved in other chromosome transactions such as transcription, chromatin remodeling and genome stability.

A SIP2 gene, a member of the ApiAP2 family of putative transcription factors, has been cloned. It appears to be involved in maintaince of the chromosome ends rather than in the regulation of particular genes.

A homolog of UAP56 (U2AF65 associated protein) - a member of the DEAD box helicase family - has been cloned. This homolog - PfU52 - contains the RNA dependent ATPase, RNA helicase and RNA binding activities. This protein is expressed in all the intraerythrocytic developmental stages of the parasite. Residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this binding activity is required for its enzymatic activities.

Another DEAD box helicase - a homologue of Has1p from yeast which has DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities - has also been cloned.

An unusual helicase - a homologue of Dbp5 and DDX19 from yeast and human respectively - has been cloned. It possesses DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. A Q motif is required for its activity.

DDX6/DOZI (development of zygote inhibited) is a member of the DEAD box family and is involved in the sexual development of the protozoan parasite. The gene is known as PfDZ50 in P. falciparum. It binds DNA and RNA and has nucleic acid-dependent ATPase and RNA unwinding activities. It interacts with eIF4E mainly through domain 1 and inhibits translation. It is localized mainly in the granular bodies found throughout the cytoplasm during the asexual intraerythrocytic developmental stages.

The DNA polymerase is unusual. They share a large amino-terminal domains with putative helicase/primase elements features that are known only in the thermophilic viruses and Aquificae. A horizont transfer seems the most likely explanation for these findings.

Two ribosome recycling factors (RRF1 and RR2) are present in the genome. Both proteins are targeted to both the apicoplast and the mitochondrion. RRF2 is also present in the cytoplasm. Unusually it forms dimers. RRF1 has a 108 amino acid insert compared with that of other organisms. The function of this insert - if any - is currently unknown.

The long adenosine/thymidine tracts that are scattered throught the genome may play a role in gene duplication.