'DEAD-box' helicase from Plasmodium falciparum is active at wide pH and is Schizont stage-specific

Background & objectives: DNA helicases catalyse unwinding of duplex DNA in an ATP-dependent manner and are involved in all the basic genetic processes. In order to study these important enzymes in the human malaria parasite we have recently cloned the first full-length 'DEAD-box' helicase gene from Plasmodium falciparum (3D7). In the present study, we report some of the important activities of the encoded protein. Methods: We have expressed the P. falciparum helicase in Escherichia coli and characterised the encoded biochemically active helicase protein. The characterisation of the protein was carried out using radioactively labeled substrate and the standard strand displacement assay. The localisation of the enzyme was studied using immunofluorescence assay. Results & conclusion: P. falciparum helicase gene is 1551 bp in length and encodes for a protein consisting of 516 amino acid residues with a predicted molecular mass of 59.8 kDa. The protein is designated as Plasmodium falciparum DEAD-box helicase 60 kDa in size (PfDH60). Purified PfDH60 showed ATP and Mg2+ dependent DNA unwinding, ssDNA-dependent ATPase and ATP-binding activities. Interestingly, this is a unique helicase because it works at a wide pH range (from 5.0-10.0). The peak expression of PfDH60 is mainly in schizont stages of the development of P. falciparum, where DNA replication is active. The cell-cycle dependent expression suggests that PfDH60 may be involved in the process of DNA replication and distinct cellular processes in the parasite and this study should make an important contribution in our better understanding of DNA metabolic pathways in the parasite

Uptake of proteins and degradation of human serum albumin by Plasmodium falciparum – infected human erythrocytes

BACKGROUND: Intraerythrocytic malaria parasites actively import obligate nutrients from serum and export proteins and lipids to erythrocyte cytoplasm and membrane. The import of macromolecules in the malaria parasite has been the subject of many debates. To understand the import of macromolecules by the parasite, we studied the uptake of proteins by Plasmodium falciparum infected human erythrocyte. METHODS: Proteins were biotin labelled individually, purified on a gel filtration column and added to uninfected and infected asynchronized culture. The uptake of these proteins by malaria parasites was determined by western blot analysis of parasite pellet and their different fractions using streptavidin-horseradish conjugate. To further confirm this import, we studied the uptake of (125)I-labelled proteins by western blot analysis as well as used direct immunofluorescence method. RESULTS: Here we show that biotin labelled and radio-iodinated polypeptides of molecular sizes in the range of 45 to 206 kDa, when added in the culture medium, get direct access to the parasite membrane through a membrane network by by-passing the erythrocyte cytosol. The import of these polypeptides is ATP-dependent as sodium azide treatment blocks this uptake. We also show that malaria parasites have the ability to take up and degrade biotin labelled human serum albumin, which has been shown to be essential for the parasite growth. CONCLUSIONS: These results can be used, as a basis to explore the role of human serum albumin in the intraerythrocytic development of parasites, and this in turn can be an important adjunct to the development of novel antimalarial drugs

Identification and characterization of a novel Plasmodium falciparum merozoite apical protein involved in erythrocyte binding and invasion

Proteins that coat Plasmodium falciparum merozoite surface and those secreted from its apical secretory organelles are considered promising candidates for the vaccine against malaria. In the present study, we have identified an asparagine rich parasite protein (PfAARP; Gene ID PFD1105w), that harbors a predicted signal sequence, a C-terminal transmembrane region and whose transcription and translation patterns are similar to some well characterized merozoite surface/apical proteins. PfAARP was localized to the apical end of the merozoites by GFP-targeting approach using an inducible, schizont-stage expression system, by immunofluorescence assays using anti-PfAARP antibodies. Immuno-electron microsopic studies showed that PfAARP is localized in the apical ends of the rhoptries in the merozoites. RBC binding assays with PfAARP expressed on COS cells surface showed that it binds to RBCs through its N-terminal region with a receptor on the RBC surface that is sensitive to trypsin and neuraminidase treatments. Sequencing of PfAARP from different P. falciparum strains as well as field isolates showed that the N-terminal region is highly conserved. Recombinant protein corresponding to the N-terminal region of PfAARP (PfAARP-N) was produced in its functional form in E. coli. PfAARP-N showed reactivity with immune sera from individuals residing in P. falciparum endemic area. The anti-PfAARP-N rabbit antibodies significantly inhibited parasite invasion in vitro. Our data on localization, functional assays and invasion inhibition, suggest a role of PfAARP in erythrocyte binding and invasion by the merozoite

Helix packing motif common to the crystal structures of two undecapeptides containing dehydrophenylalanine residues: implications for the de novo design of helical bundle super secondary structural modules

De novo designed peptide based super secondary structures are expected to provide scaffolds for the incorporation of functional sites as in proteins. Self-association of peptide helices of similar screw sense, mediated by weak interactions, has been probed by the crystal structure determination of two closely related peptides: Ac-Gly1-Ala2-ΔPhe3-Leu4-Val5-ΔPhe6-Leu7-Val8-ΔPhe9-Ala10-Gly11-NH2 (I) and Ac-Gly1-Ala2-ΔPhe3-Leu4-Ala5-ΔPhe6-Leu7-Ala8-ΔPhe9-Ala10-Gly11-NH2 (II). The crystal structures determined to atomic resolution and refined to R factors 8.12 and 4.01%, respectively, reveal right-handed 310-helical conformations for both peptides. CD has also revealed the preferential formation of right-handed 310-helical conformations for both molecules. Our aim was to critically analyze the packing of the helices in the solid state with a view to elicit clues for the design of super secondary structural motifs such as two, three, and four helical bundles based on helix-helix interactions. An important finding is that a packing motif could be identified common to both the structures, in which a given peptide helix is surrounded by six other helices reminiscent of transmembrane seven helical bundles. The outer helices are oriented either parallel or antiparallel to the central helix. The helices interact laterally through a combination of N-H ... O, C-H ... O, and C-H ... π hydrogen bonds. Layers of interacting leucine residues are seen in both peptide crystal structures. The packing of the peptide helices in the solid state appears to provide valuable leads for the design of super secondary structural modules such as two, three, or four helix bundles by connecting adjacent antiparallel helices through suitable linkers such as tetraglycine segment

Isolation and Characterization of Type I Signal Peptidase of Different Malaria Parasites

Type I signal peptidases are important membrane-bound serine proteases responsible for the cleavage of the signal peptide of the proteins. These enzymes are unique serine proteases that carry out catalysis using a serine/lysine catalytic dyad. In the present study, we report the isolation of type I signal peptidase from the malaria parasites Plasmodium falciparum, Plasmodium knowlesi, and Plasmodium yoelii and some characterization of type I signal peptidase of Plasmodium falciparum. We show that these enzymes are homologous to signal peptidases from various sources and also contain the conserved boxes present in other type I signal peptidases. The type I signal peptidase from P falciparum is an intron-less and a single-copy gene. The results also show that the enzyme from Plasmodium falciparum is subject to self-cleavage and it has been demonstrated to possess type I signal peptidase activity in E coli preprotein processing in vivo by complementation assay. This study will be helpful in understanding one of the important metabolic pathways “the secretory pathway” in the parasite and should make an important contribution in understanding the complex process of protein targeting in the parasite

A systematic classification of Plasmodium falciparum P-loop NTPases: structural and functional correlation

Background: The P-loop NTPases constitute one of the largest groups of globular protein domains that play highly diverse functional roles in most of the organisms. Even with the availability of nearly 300 different Hidden Markov Models representing the P-loop NTPase superfamily, not many P-loop NTPases are known in Plasmodium falciparum. A number of characteristic attributes of the genome have resulted into the lack of knowledge about this functionally diverse, but important class of proteins. Method: In the study, protein sequences with characteristic motifs of NTPase domain (Walker A and Walker B) are computationally extracted from the P. falciparum database. A detailed secondary structure analysis, functional classification, phylogenetic and orthology studies of the NTPase domain of repertoire of 97 P. falciparum P-loop NTPases is carried out. Results: Based upon distinct sequence features and secondary structure profile of the P-loop domain of obtained sequences, a cladistic classification is also conceded: nucleotide kinases and GTPases, ABC and SMC family, SF½ helicases, AAA+ and AAA protein families. Attempts are made to identify any ortholog(s) for each of these proteins in other Plasmodium sp. as well as its vertebrate host, Homo sapiens. A number of P. falciparum P-loop NTPases that have no homologue in the host, as well as those annotated as hypothetical proteins and lack any characteristic functional domain are identified. Conclusion: The study suggests a strong correlation between sequence and secondary structure profile of P-loop domains and functional roles of these proteins and thus provides an opportunity to speculate the role of many hypothetical proteins. The study provides a methodical framework for the characterization of biologically diverse NTPases in the P. falciparum genome. The efforts made in the analysis are first of its kind; and the results augment to explore the functional role of many of these proteins from the parasite that could provide leads to identify novel drug targets against malaria

ProtRepeatsDB: a database of amino acid repeats in genomes

BACKGROUND: Genome wide and cross species comparisons of amino acid repeats is an intriguing problem in biology mainly due to the highly polymorphic nature and diverse functions of amino acid repeats. Innate protein repeats constitute vital functional and structural regions in proteins. Repeats are of great consequence in evolution of proteins, as evident from analysis of repeats in different organisms. In the post genomic era, availability of protein sequences encoded in different genomes provides a unique opportunity to perform large scale comparative studies of amino acid repeats. ProtRepeatsDB is a relational database of perfect and mismatch repeats, access to which is designed as a resource and collection of tools for detection and cross species comparisons of different types of amino acid repeats. DESCRIPTION: ProtRepeatsDB (v1.2) consists of perfect as well as mismatch amino acid repeats in the protein sequences of 141 organisms, the genomes of which are now available. The web interface of ProtRepeatsDB consists of different tools to perform repeat s; based on protein IDs, organism name, repeat sequences, and keywords as in FASTA headers, size, frequency, gene ontology (GO) annotation IDs and regular expressions (REGEXP) describing repeats. These tools also allow formulation of a variety of simple, complex and logical queries to facilitate mining and large-scale cross-species comparisons of amino acid repeats. In addition to this, the database also contains sequence analysis tools to determine repeats in user input sequences. CONCLUSION: ProtRepeatsDB is a multi-organism database of different types of amino acid repeats present in proteins. It integrates useful tools to perform genome wide queries for rapid screening and identification of amino acid repeats and facilitates comparative and evolutionary studies of the repeats. The database is useful for identification of species or organism specific repeat markers, interspecies variations and polymorphism

Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide

<p>Abstract</p> <p>Background</p> <p>The <it>de novo </it>design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α, β-dehydrophenylalanine (ΔPhe) comes in use for spawning well-defined structural motifs. Introduction of ΔPhe induces β-bends in small and 3₁₀-helices in longer peptide sequences.</p> <p>Results</p> <p>The present report is an investigation of the effect of incorporating two glycines in the middle of a ΔPhe containing undecapeptide. A de novo designed undecapeptide, Ac-Gly¹-Ala²-ΔPhe³-Leu⁴-Gly⁵-ΔPhe⁶-Leu⁷-Gly⁸-ΔPhe⁹-Ala¹⁰-Gly¹¹-NH₂, was synthesized and characterized using X-ray diffraction and Circular Dichroism spectroscopic methods. Crystallographic studies suggest that, despite the presence of L-amino acid (L-Ala and L-Leu) residues in the middle of the sequence, the peptide adopts a 3₁₀-helical conformation of ambidextrous screw sense, one of them a left-handed (A) and the other a right-handed (B) 3₁₀-helix with A and B being antiparallel to each other. However, CD studies reveal that the undecapeptide exclusively adopts a right-handed 3₁₀-helical conformation. In the crystal packing, three different interhelical interfaces, Leu-Leu, Gly-Gly and ΔPhe-ΔPhe are observed between the helices A and B. A network of C-H...O hydrogen bonds are observed at ΔPhe-ΔPhe and Gly-Gly interhelical interfaces. An important feature observed is the occurrence of glycine zipper motif at Gly-Gly interface. At this interface, the geometric pattern of interhelical interactions seems to resemble those observed between helices in transmembrane (TM) proteins.</p> <p>Conclusion</p> <p>The present design strategy can thus be exploited in future work on de novo design of helical bundles of higher order and compaction utilizing ΔPhe residues along with GXXG motif.</p

Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, Anopheles culicifacies

<p>Abstract</p> <p>Background</p> <p>The main vector for transmission of malaria in India is the <it>Anopheles culicifacies </it>mosquito species, a naturally selected subgroup of which is completely refractory (R) to transmission of the malaria parasite, <it>Plasmodium vivax</it>;</p> <p>Results</p> <p>Here, we report the molecular characterization of a serine protease (<it>acsp30</it>)-encoding gene from <it>A. culicifacies</it>, which was expressed in high abundance in the refractory strain compared to the susceptible (S) strain. The transcriptional upregulation of <it>acsp30 </it>upon <it>Plasmodium </it>challenge in the refractory strain coincided with ookinete invasion of mosquito midgut. Gene organization and primary sequence of <it>acsp30 </it>were identical in the R and S strains suggesting a divergent regulatory status of <it>acsp30 </it>in these strains. To examine this further, the upstream regulatory sequences of <it>acsp30 </it>were isolated, cloned and evaluated for the presence of promoter activity. The 702 bp upstream region of <it>acsp30 </it>from the two strains revealed sequence divergence. The promoter activity measured by luciferase-based reporter assay was shown to be 1.5-fold higher in the R strain than in the S. Gel shift experiments demonstrated a differential recruitment of nuclear proteins to upstream sequences of <it>acsp30 </it>as well as a difference in the composition of nuclear proteins in the two strains, both of which might contribute to the relative abundance of <it>acsp30 </it>in the R strain;</p> <p>Conclusion</p> <p>The specific upregulation of <it>acsp30 </it>in the R strain only in response to <it>Plasmodium </it>infection is suggestive of its role in contributing the refractory phenotype to the <it>A. culicifacies </it>mosquito population.</p