Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.     

A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world''s major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.Parasites of the Plasmodium genus are the etiological agents responsible for malaria, an infectious disease mostly occurring in developing countries with up to 40% of the world''s population described as being at risk of the disease. Among the Plasmodium species that can affect humans, Plasmodium falciparum is responsible for the highest mortality, causing around one million deaths annually, mostly in children under the age of five (1). The clinical symptoms of malaria occur during the cyclic asexual blood stage of the parasite lifecycle when merozoites, that have invaded and replicated within host erythrocytes, are released into the bloodstream before invading new red blood cells (2). Despite intensive efforts from the research community there is currently no licensed vaccine for malaria. The leading candidate RTS,S/AS01, which targets the pre-erythrocytic stage of the disease and was tested in phase III trials, conferred 30 to 50% protection from clinical malaria, depending on the age group studied (3, 4). This limited efficacy has led to calls for a more effective vaccine and many have suggested that a combinatorial vaccine that additionally targets the blood stage may increase efficacy.A vaccine targeting the proteins expressed on the surface of the blood stage of the parasite is conceptually attractive because merozoites are repeatedly and directly exposed to the human humoral immune system and naturally acquired antibodies against these proteins have been shown to confer at least partial immunity (5–8). Despite this, only a few antigens discovered before the completion of the parasite genome sequence have been assessed in detail (9) and clinical vaccine trials using antigens that target the blood stage have so far shown limited efficacy, mostly caused by antigenic diversity (10). The sequencing of the parasite genome (11) has identified all possible targets but the systematic screening of these new candidates to assess their potential as a vaccine is hampered by the inability to systematically express recombinant Plasmodium proteins in their native conformation (12–15). Likely explanations might be the high (∼80%) A:T content of the P. falciparum genome resulting in low codon usage compatibility in heterologous expression systems, the large size (> 50 kDa) of many proteins, the presence of long stretches of highly repetitive amino acids, and the difficulty in identifying clear structural domains within these proteins using standard prediction computer programs (11). Extracellular proteins, in particular, present an additional challenge because they often have signal peptides and transmembrane regions that can negatively impact expression (16–18) and contain structurally important disulfide bonds. However, unlike most other eukaryotic extracellular proteins, Plasmodium cell surface and secreted proteins are not modified by N-linked glycans because of the absence of the necessary enzymes (19).To express Plasmodium proteins for basic research and vaccine development, a diverse range of expression systems have been tried (12) ranging from bacteria (17, 18), yeast (13), Dictyostelium (20), and plants (21) to mammalian cells (22) and cell-free systems (23–25). To circumvent the problem of codon usage, bacterial (26) and yeast (27) strains with modified tRNA pools have been developed, or sequences of the gene of interest synthesized and codon-optimized to match that of the expression host (28, 29). Although Escherichia coli has been the most popular expression system because of its relative simplicity and cost effectiveness, large-scale production of soluble functional Plasmodium falciparum recombinant proteins remains challenging with success rates ranging from just 6 to 21% (17, 18) and is often hindered by the need for complex refolding procedures. Similarly, attempts have been made to compile large panels of parasite proteins using in vitro translation systems (23, 25, 30, 31). These systems, however, require reducing conditions and are therefore not generally suitable for the systematic expression of extracellular proteins that occupy an oxidizing environment and critically require the formation of disulfide bonds for proper function. As a result, functional analyses of extracellular parasite proteins have often been restricted to smaller subfragments of the proteins that can be expressed in a soluble form rather than the entire extracellular region. Although eukaryotic expression systems are able to add disulfide bonds, they also often inappropriately glycosylate parasite proteins, adding further complication (32). A generic method that would overcome these technical challenges to express, in a systematic way, panels of recombinant Plasmodium proteins that have retained their native function and conformation would therefore be a valuable resource for the molecular investigations of erythrocyte invasion and the development of a blood stage vaccine.To generate a resource of correctly folded recombinant merozoite proteins, we used a mammalian expression system and established the parameters necessary for high-level expression. Using this method, we compiled a panel of 42 proteins that corresponds to the repertoire of abundant cell surface and secreted merozoite proteins of the 3D7 strain of Plasmodium falciparum. Biochemical activity of these proteins was demonstrated by recapitulating known protein interactions and by showing conformation-sensitive immunoreactivity of the recombinant proteins using immune sera.     

Evidence for Catalytic Roles for Plasmodium falciparum Aminopeptidase P in the Food Vacuole and Cytosol

The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 °C in the pH range 5.0–7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.Malaria remains one of the most deadly global infectious diseases with an estimated 500 million clinical cases and 2 million deaths annually (1, 2). Clinical manifestations of the disease arise as the protozoan malaria parasite replicates asexually within human erythrocytes. Five species of the genus Plasmodium infect humans. The cytoadherent properties of red blood cells infected with Plasmodium falciparum, coupled with the ability of the parasite to reach high parasitemia, make it the most virulent species. The emergence of strains of P. falciparum that are resistant to affordable anti-malarial drugs such as chloroquine has complicated efforts to manage malaria, and new drugs are urgently needed.Aminopeptidases catalyze the hydrolysis of amino acids from the amino termini of proteins and peptides. They participate in a wide range of biological processes, including peptide catabolism, protein maturation, antigen presentation on immune cells, and regulation of hormone activity. During the asexual erythrocytic replication cycle of the malaria parasite, aminopeptidases contribute to the catabolism of peptides generated by two major proteolytic pathways. One of these is initiated at the proteasome, a multifunctional protease that plays an important role in the turnover of ubiquitinated cellular proteins in the cytosol (3–5). In addition, the parasite transports host red blood cell cytosol (consisting primarily of hemoglobin) to an acidic degradative organelle, the food vacuole, where it is degraded in a proteasome-independent pathway (6, 7). As up to 75% of the host cell hemoglobin is catabolized during the intraerythrocytic cycle (8, 9), flux through the vacuolar pathway is substantial. Three aminopeptidases have been identified as key players in recycling amino acids from peptides generated by the proteasomal and vacuolar catabolic pathways: leucine aminopeptidase, aminopeptidase N (PfA-M1), and aminopeptidase P (10–14). The latter two enzymes have been found in the food vacuole and therefore may play a direct role in hemoglobin catabolism (11). An aspartyl aminopeptidase is also expressed in asexual stage parasites and hydrolyzes amino-terminal aspartyl and glutamyl substrates (15); however, disruption of its gene does not prevent efficient intraerythrocytic replication (11).Aminopeptidase P (APP)² homologs exhibit high specificity for proline in the second position of the substrate (the P1′ position in the nomenclature of Schechter and Berger (16)) and catalyze the hydrolysis of the X-Pro amide bond, where X is any aminoacyl residue (17). Because of the cyclic nature of the proline side chain, X-Pro-containing peptides are not easily accommodated in the active sites of broad specificity aminopeptidases (17). In mammals, three APP isozymes have been identified. APP1 is found in the cytosolic fraction of cell lysates and has been characterized from a variety of tissues (18–20). Although this enzyme has not, to our knowledge, been localized in intact cells, the apparent lack of specific targeting information is consistent with a role in cytosolic peptide turnover. Active cytosolic forms of APP have been reported in plants (21), fruit flies (22), the microsporidian parasite Encephalitozoon cuniculi (23), and in intestinal cells in Caenorhabditis elegans (24), where it is believed to play a role in the catabolism of peptides produced from ingested bacteria. Mammalian APP2 is a glycosylated ectoenzyme anchored into the membrane of endothelial and epithelial cells with a glycosylphosphatidylinositol attachment (25). The best characterized role of APP2 is the inactivation of the plasma hormone bradykinin, a nonapeptide, through cleavage of the Arg-Pro amino-terminal peptide bond (26, 27). Inhibition of APP2 potentiates the vasodilatory and cardioprotective properties of bradykinin, and APP2 has been considered a target for the development of cardiovascular drugs (28–30). A third isoform, APP3, has been identified in the human genome and may be a mitochondrial enzyme but has not yet been characterized (31). Prokaryotic APP homologs contribute to intracellular peptide turnover (32).P. falciparum aminopeptidase P (PfAPP) appears to be important for intraerythrocytic growth, as parasites with a disrupted PfAPP gene could not be isolated (11). We have previously localized a PfAPP-yellow fluorescent protein fusion to the food vacuole and the cytosol of the parasite (11). The cytosolic pool of PfAPP probably fulfills a role in peptide turnover and amino acid recycling that is orthologous to those of the cytosolic enzymes described above. In contrast, there is no report to our knowledge of an aminopeptidase P homolog functioning in an acidic environment akin to the malarial food vacuole. Moreover, characterization of mammalian aminopeptidase P homologs typically reveals a pH optimum of 7–8 with relatively little, if any, activity in the pH range 5.0–5.5 (18–20). Although we have previously detected PfAPP activity at acidic pH (11), the catalytic efficiency of the enzyme has not been characterized. Thus, at the outset of this study it was not clear whether PfAPP has a significant catalytic role in the food vacuole.Here we have localized untagged, native PfAPP in the parasite and have confirmed the dual cytosolic/vacuolar distribution of the enzyme. The domain organization, quaternary structure, and metal requirement of PfAPP were characterized. To evaluate the plausibility of a catalytic role for PfAPP at acidic and near-neutral pH, its stability in the pH range 5.0–7.5 was assessed and compared with that of human cytosolic APP1, an enzyme that does not, to our knowledge, have a physiological role in an acidic environment. The catalytic efficiency of PfAPP at a range of pH values was characterized with three X-Pro-containing peptides, two of which are found in the sequences of human α- and β-globin and therefore represent potentially physiological substrates.     

RTS,S Vaccination Is Associated With Serologic Evidence of Decreased Exposure to Plasmodium falciparum Liver- and Blood-Stage Parasites

The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator vaccine (n = 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together, these data provide insight into the mechanism by which RTS,S protects from malaria.The RTS,S malaria vaccine candidate provides partial protection against clinical malaria in African children, which has been repeatedly demonstrated in Phase IIb and Phase III clinical trials (1–5). The RTS,S target is the Plasmodium falciparum circumsporozoite protein (CSP), and it has been shown to generate high antibody titers that remain above levels acquired naturally for years (6). However, it remains unclear how the vaccine, which targets sporozoites, provides protection against disease caused by blood-stage parasites. A rational mechanism has been proposed, based on antibody and T cell responses to the CSP (7), but antibodies have not consistently correlated with protection when clinical disease was the trial end point (8). We and others hypothesized that partial blockage of pre-erythrocytic development would result in low-level blood-stage infections that go untreated in RTS,S vaccinees and that this would boost the blood-stage immune response, contributing to protection from malaria disease (8–10).We set out to address the question of how the vaccine works by investigating the response to malaria parasites in the context of RTS,S vaccination. However, until recently, the means of assessing the response to malaria parasites has been limited to a sparse selection of recombinant proteins or parasite lysates. The P. falciparum (Pf) proteome contains more than 5,300 proteins, and, until recently, less than 0.5% of them have been closely investigated (11). Similar to the approach taken with gene expression microarrays, protein arrays offer the opportunity to screen antibody responses to partial or complete proteomes (12). This approach was taken in this study to identify the breadth and magnitude of naturally acquired immune responses in Mozambican children vaccinated with RTS,S/AS02¹, the predecessor to the RTS,S/AS01 formulation used in the current Phase III trial, or comparator vaccine.In addition to characterizing the RTS,S mode of action, we aimed to identify biomarker correlates of protection against clinical malaria. Malaria vaccinology is lacking in surrogate markers of protection, and such biomarkers would be a highly useful measure for assessment of vaccine efficacy, especially when control or placebo vaccine groups are no longer available (13). This could mitigate the current inefficient means of measuring efficacy in clinical trials. In the post-genomic era, with systems approaches employed for questions to complex problems in biology and medicine, perhaps alternative thinking is required to tackle the question of how to assess vaccines (14, 15). In this study, we took steps in that direction in order to identify antibody signatures of protection that contribute toward a surrogate marker for the RTS,S and other vaccines.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

An Essential Role for the Plasmodium Nek-2 Nima-related Protein Kinase in the Sexual Development of Malaria Parasites

The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission.Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is a major public health problem in the developing world (1). The species responsible for the vast majority of lethal cases is Plasmodium falciparum. The life cycle of malaria parasites consists of a succession of developmental stages: asexual multiplication occurs in the human host (first in a single round of schizogony in a hepatocyte infected by a sporozoite injected by the mosquito vector, and then multiple rounds of schizogony in erythrocytes), whereas the sexual cycle is initiated by the formation of cell cycle-arrested gametocytes in infected erythrocytes and proceeds, in the midgut of the mosquito vector, to gametogenesis, fertilization, and formation of a motile ookinete. The ookinete crosses the midgut epithelium and establishes an oocyst at the basal lamina, in which sporogony occurs, generating sporozoites that render the vector infectious once they reach its salivary glands. The alternation of proliferative and non-proliferative phases implies that the control of cell cycle progression is of prime importance for completion of the life cycle of the parasite.The NIMA-related protein kinases (Neks)⁵ constitute an extended family of eukaryotic mitotic serine/threonine kinases. The best characterized members of the Nek family include NIMA (never in mitosis/Aspergillus), the founding member from the fungus Aspergillus nidulans (2), and its closest homologue in mammals, Nek2 (3, 4). Initially identified as a kinase essential for mitotic entry in Aspergillus, NIMA has been also shown to participate in nuclear membrane fission (5). Eleven members of the NIMA kinase family (Nek1–11) have now been identified in various human tissues, and together fulfill a number of cell cycle-related functions in centrosome separation, mitosis, meiosis, and checkpoint control (reviewed in Ref. 6). It has been proposed that expansion of the Nek family accompanied the evolution of a complex system for the coordination of progression through the cell cycle with the replication of cellular components such as cilia, basal bodies, and centrioles. Several human Neks have C-terminal extensions to their catalytic domain, which contain regulatory elements (e.g. PEST sequences that function as target for cell cycle-dependent proteolytic degradation, or coiled-coil domains mediating dimerization). Nek6 and Nek7 have no large extensions, but bind to the C-terminal non-catalytic tail of Nek9, an enzyme that becomes activated during mitosis and is likely to be responsible for the activation of Nek6 (7). This may represent a novel cascade of mitotic NIMA family protein kinases whose combined function is important for mitotic progression.The P. falciparum kinome includes four NIMA-related serine/threonine kinases (8). Pfnek-1 (PlasmoDB identifier PFL1370w) clusters within the Aspergillus NIMA/human Nek2 branch in phylogenetic trees, whereas clear orthology to mammalian or yeast Neks could not be assigned for the three other P. falciparum sequences (Pfnek-2, -3, and -4, PlasmoDB identifiers PFE1290w, PFL0080c, and MAL7P1.100, respectively) (9). Microarray data (10) available in the PlasmoDB data base (11) indicate that Pfnek-1 is expressed in asexual and sexual stages, whereas mRNA encoding the other three enzymes is predominantly or exclusively expressed in gametocytes, suggesting a possible role in the sexual development of the parasite. Consistent with this hypothesis, we previously showed that rodent malaria parasites Plasmodium berghei lacking the Nek-4 enzyme are unable to complete DNA replication to 4C in the zygote prior to meiosis (9). Pfnek-1, -3, and -4 have been characterized at the biochemical level and are active as recombinant enzymes (9, 12, 13). Pfnek-1 and Pfnek-3 have surprisingly been implicated as possible regulators of an atypical mitogen-activated protein kinase (MAPK), as both enzymes synergize with the Pfmap-2 MAPK in vitro (12, 13); the physiological relevance of these observations remains to be demonstrated.Here, we demonstrate that Pfnek-2, like the other three members of the P. falciparum Nek family, is a bona fide protein kinase. Analysis of the expression pattern demonstrates that low levels of Pfnek-2 mRNA are actually detectable in asexual parasites, even though transgenic parasites expressing a green fluorescent protein (GFP)-tagged Pfnek-2 under the control of its cognate promoter display female gametocyte-specific expression. To investigate the function of this kinase, parasite clones with a disrupted nek-2 gene were generated in P. falciparum and P. berghei; transmission experiments identified an important role for nek-2 in sexual development: nek-2⁻ parasites are able to differentiate into mature gametocytes and to undergo gametogenesis, but do not develop into ookinetes. Further investigations on the pbnek-2⁻ parasites showed that pre-meiotic DNA replication is dysregulated in the mutant clones.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]