Nanobodies against Pfs230 block Plasmodium falciparum transmission.

Transmission blocking interventions can stop malaria parasite transmission from mosquito to human by inhibiting parasite infection in mosquitos. One of the most advanced candidates for a malaria transmission blocking vaccine is Pfs230. Pfs230 is the largest member of the 6-cysteine protein family with 14 consecutive 6-cysteine domains and is expressed on the surface of gametocytes and gametes. Here, we present the crystal structure of the first two 6-cysteine domains of Pfs230. We identified high affinity Pfs230-specific nanobodies that recognized gametocytes and bind to distinct sites on Pfs230, which were isolated from immunized alpacas. Using two non-overlapping Pfs230 nanobodies, we show that these nanobodies significantly blocked P. falciparum transmission and reduced the formation of exflagellation centers. Crystal structures of the transmission blocking nanobodies with the first 6-cysteine domain of Pfs230 confirm that they bind to different epitopes. In addition, these nanobodies bind to Pfs230 in the absence of the prodomain, in contrast with the binding of known Pfs230 transmission blocking antibodies. These results provide additional structural insight into Pfs230 domains and elucidate a mechanism of action of transmission blocking Pfs230 nanobodies.

Plasmodium 6-Cysteine Proteins: Functional Diversity, Transmission-Blocking Antibodies and Structural Scaffolds.

The 6-cysteine protein family is one of the most abundant surface antigens that are expressed throughout the Plasmodium falciparum life cycle. Many members of the 6-cysteine family have critical roles in parasite development across the life cycle in parasite transmission, evasion of the host immune response and host cell invasion. The common feature of the family is the 6-cysteine domain, also referred to as s48/45 domain, which is conserved across Aconoidasida. This review summarizes the current approaches for recombinant expression for 6-cysteine proteins, monoclonal antibodies against 6-cysteine proteins that block transmission and the growing collection of crystal structures that provide insights into the functional domains of this protein family.

Characterization of Plasmodium falciparum macrophage migration inhibitory factor homologue and its cysteine deficient mutants.

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) is a homologue of the multifunctional human host cytokine MIF (HsMIF). Upon schizont rupture it is released into the human blood stream where it acts as a virulence factor, modulating the host immune system. Whereas for HsMIF a tautomerase, an oxidoreductase, and a nuclease activity have been identified, the latter has not yet been studied for PfMIF. Furthermore, previous studies identified PfMIF as a target for several redox post-translational modifications. Therefore, we analysed the impact of S-glutathionylation and S-nitrosation on the protein's functions. To determine the impact of the four cysteines of PfMIF we produced His-tagged cysteine to alanine mutants of PfMIF via site-directed mutagenesis. Recombinant proteins were analysed via mass spectrometry, and enzymatic assays. Here we show for the first time that PfMIF acts as a DNase of human genomic DNA and that this activity is greater than that shown by HsMIF. Moreover, we observed a significant decrease in the maximum velocity of the DCME tautomerase activity of PfMIF upon alanine replacement of Cys3, and Cys3/Cys4 double mutant. Lastly, using a yeast reporter system, we were able to verify binding of PfMIF to the human chemokine receptors CXCR4, and demonstrate a so-far overlooked binding to CXCR2, both of which function as non-cognate receptors for HsMIF. While S-glutathionylation and S-nitrosation of PfMIF did not impair the tautomerase activity of PfMIF, activation of these receptors was significantly decreased.

Development of a new peptide-bead coupling method for an all peptide-based Luminex multiplexing assay for detection of Plasmodium falciparum antibody responses.

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas.

A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity.

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.

Structures of the Plasmodium falciparum heat-shock protein 70-x ATPase domain in complex with chemical fragments identify conserved and unique binding sites.

Plasmodium falciparum invades erythrocytes and extensively modifies them in a manner that increases the virulence of this malaria parasite. A single heat-shock 70 kDa-type chaperone, PfHsp70-x, is among the parasite proteins exported to the host cell. PfHsp70-x assists in the formation of a key protein complex that underpins parasite virulence and supports parasite growth during febrile episodes. Previous work resolved the crystallographic structures of the PfHsp70-x ATPase and substrate-binding domains, and showed them to be highly similar to those of their human counterparts. Here, 233 chemical fragments were screened for binding to the PfHsp70-x ATPase domain, resulting in three crystallographic structures of this domain in complex with ligands. Two binding sites were identified, with most ligands binding proximal to the ATPase nucleotide-binding pocket. Although amino acids participating in direct ligand interactions are conserved between the parasite and human erythrocytic chaperones, one nonconserved residue is also present near the ligand. This work suggests that PfHsp70-x features binding sites that may be exploitable by small-molecule ligands towards the specific inhibition of the parasite chaperone.

Structural basis of malaria RIFIN binding by LILRB1-containing antibodies.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to ß2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.

Structural basis for placental malaria mediated by Plasmodium falciparum VAR2CSA.

Plasmodium falciparum VAR2CSA binds to chondroitin sulfate A (CSA) on the surface of the syncytiotrophoblast during placental malaria. This interaction facilitates placental sequestration of malaria parasites resulting in severe health outcomes for both the mother and her offspring. Furthermore, CSA is presented by diverse cancer cells and specific targeting of cells by VAR2CSA may become a viable approach for cancer treatment. In the present study, we determined the cryo-electron microscopy structures of the full-length ectodomain of VAR2CSA from P. falciparum strain NF54 in complex with CSA, and VAR2CSA from a second P. falciparum strain FCR3. The architecture of VAR2CSA is composed of a stable core flanked by a flexible arm. CSA traverses the core domain by binding within two channels and CSA binding does not induce major conformational changes in VAR2CSA. The CSA-binding elements are conserved across VAR2CSA variants and are flanked by polymorphic segments, suggesting immune selection outside the CSA-binding sites. This work provides paths for developing interventions against placental malaria and cancer.

The nucleotide specificity of succinyl-CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone.

Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP  = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.

Enhanced Antimalarial Efficacy Obtained by Targeted Delivery of Artemisinin in Heparin-Coated Magnetic Hollow Mesoporous Nanoparticles.

Malaria is one of the deadliest infectious diseases threatening half of the world population. With the deterioration of the parasiticidal effect of the current antimalarials, novel approaches such as screening of more specific inhibitors and targeted delivery of drugs have been under intensive research. Herein, we prepare hollow mesoporous ferrite nanoparticles (HMFNs) of 200 nm with ferromagnetic properties using a one-pot hydrothermal reaction. A magnetically targeted drug-delivery system coloaded with artemisinin in the inner magnetite shell and heparin on the outer mesoporous shell (HMFN@ART@HEP) is developed. Specific targeting of the magnetic nanoparticles to the parasite-infected erythrocytes is achieved by the attraction between the HMFNs and hemozoin (paramagnetic), a vital metabolite of plasmodium in the erythrocytic stage. With the hemozoin production reaching the maximum during the schizont period of the parasite, HMFN@ART@HEPs are adsorbed to the infected red blood cells (iRBCs), which not only interferes with the release of merozoites but also significantly enhances the inhibitory efficacy due to the increased local concentration of artemisinin. Subsequently, the heparin coated on the surface of the nanoparticles can efficiently interfere with the invasion of freshly released merozoites to new RBCs through the specific interaction between the parasite-derived ligands and heparin, which further increases the inhibitory effect on malaria. As a cluster of heparin, heparin-coated nanoparticles provide stronger blocking capability than free heparin, resulting from multivalent interactions with surface receptors on merozoite. Thus, we have developed a HMFN-based delivery system with considerable antimalarial efficacy, which is a promising platform for treatment against malaria.

Conus venom fractions inhibit the adhesion of Plasmodium falciparum erythrocyte membrane protein 1 domains to the host vascular receptors.

Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2ß3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment. BIOLOGICAL SIGNIFICANCE: Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems. We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies. This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1).

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site.

Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5.IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it-and the other parasite proteins it interacts with-promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

Structure of the Plasmodium falciparum PfSERA5 pseudo-zymogen.

PfSERA5, a significantly abundant protein present within the parasitophorous vacuole (PV) and essential for normal growth during the blood-stage life cycle of the malaria parasite Plasmodium falciparum, displays structural similarity to many other cysteine proteases. However, PfSERA5 does not exhibit any detectable protease activity and therefore the role of the PfSERA5 papain-like domain (PfSERA5E), thought to remain bound to its cognate prodomain, remains unknown. In this study, we present a revised structure of the central PfSERA5E domain at a resolution of 1.2 Å, and the first structure of the "zymogen" of this papain-like domain including its cognate prodomain (PfSERA5PE) to 2.2 Å resolution. PfSERA5PE is somewhat structurally similar to that of other known proenzymes, retaining the conserved overall folding and orientation of the prodomain through, and occluding, the archetypal papain-like catalytic triad "active-site" cleft, in the same reverse direction as conventional prodomains. Our findings are congruent with previously identified structures of PfSERA5E and of similar "zymogens" and provide a foundation for further investigation into the function of PfSERA5.

Antimalarial activity of tetrahydro-ß-carbolines targeting the ATP binding pocket of the Plasmodium falciparum heat shock 90 protein.

A series of tetrahydro-ß-carboline derivatives of a lead compound known to target the heat shock 90 protein of Plasmodium falciparum were synthesized and assayed for both potency against the parasite and toxicity against a human cell line. Using a rationalized structure based design strategy, a new lead compound with a potency two orders of magnitude greater than the original lead compound was found. Additional modeling of this new lead compound suggests multiple avenues to further increase potency against this target, potentially paving the path for a therapeutic with a mode of action different than any current clinical treatment.

Solution structure of Plasmodium falciparum Hsp90 indicates a high flexible dimer.

Hsp90 is a ubiquitous, homodimer and modular molecular chaperone. Each Hsp90 protomer has three different domains, named the N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD). The Hsp90 molecular cycle involves ATP binding and hydrolysis, which drive conformational changes. Hsp90 is critical for the viability of eukaryotic organisms, including the protozoan that causes the severe form of malaria, Plasmodium falciparum, the growth and differentiation of which are compromised when Hsp90 is inhibited. Here, we characterize the structure of a recombinant P. falciparum Hsp90 (PfHsp90) protein, as well as its MD (PfHsp90MD) and NTD plus MD (PfHsp90NMD) constructs. All the proteins were obtained with high purity and in the folded state. PfHsp90 and PfHsp90NMD interacted with adenosine nucleotides via the NTD, and Mg2+ was critical for strong binding. PfHsp90 behaved mostly as elongated and flexible dimers in solution, which dissociate with a sub-micromolar dissociation constant. The PfHsp90MD and PfHsp90NMD constructs behaved as globular and elongated monomers, respectively, confirming the importance of the CTD for dimerization. Small angle X-ray scattering data were obtained for all the constructs, and ab initio models were constructed, revealing PfHsp90 in an open conformation and as a greatly elongated and flexible protein.

Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone.

Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

Yield improvement and enzymatic dissection of Plasmodium falciparum plasmepsin V.

To survive within a red blood cell (RBC), malaria parasites establish striking modifications to the permeability, rigidity and cytoadherence properties of the host cell. This is mediated by the export of hundreds of proteins from the parasite into the erythrocyte. Plasmodium falciparum plasmepsin V (PfPMV), is an ER resident aspartic protease that processes proteins for export into the host erythrocyte, plays a crucial role in parasite virulence and survival and is considered a potential malaria drug target. Most attempts at its heterologous expression in Escherichia coli have resulted in mainly the production of insoluble proteins. In this study, we employed a multipurpose fusion tag to improve the production of PfPMV in E. coli. Recombinant PfPMVm, comprising residues 84-521, was substantially obtained in soluble form and could be purified in a single step, yielding a 3.7-fold increase in purified PfPMVm compared to previous reports. Additionally, we have mutated the catalytic residues (D118N and D365N), individually and together, and the unpaired cysteine residue C178 to evaluate the effects on catalytic efficiency. Mutation of D365 had more pronounced effects on the catalytic efficiency than that of D118, suggesting that the D365 may act as a catalytic nucleophile to activate the water molecule. The importance of C178 was also confirmed by the inhibition by metal ions, indicating that C178 is partially involved in the substrate recognition. Collectively, our results describe an improved system to produce recombinant PfPMVm in E. coli and dissect the amino acids involved in catalysis and substrate recognition.

Biochemical and physiological investigations on adenosine 5' monophosphate deaminase from Plasmodium spp.

The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter-conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in-turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over-expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti-parasitic agents.

Bias in False Discovery Rate Estimation in Mass-Spectrometry-Based Peptide Identification.

Accurate target-decoy-based false discovery rate (FDR) control of peptide identification from tandem mass-spectrometry data relies on an important but often neglected assumption that incorrect spectrum annotations are equally likely to receive either target or decoy peptides. Here we argue that this assumption is often violated in practice, even by popular methods. Preference can be given to target peptides by biased scoring functions, which result in liberal FDR estimations, or to decoy peptides by correlated spectra, which result in conservative estimations.