Crystal Structure of the Plasmodium falciparum Thioredoxin Reductase–Thioredoxin Complex

Over the last decades, malaria parasites have been rapidly developing resistance against antimalarial drugs, which underlines the need for novel drug targets. Thioredoxin reductase (TrxR) is crucially involved in redox homeostasis and essential for Plasmodium falciparum. Here, we report the first crystal structure of P. falciparum TrxR bound to its substrate thioredoxin 1. Upon complex formation, the flexible C-terminal arm and an insertion loop of PfTrxR are rearranged, suggesting that the C-terminal arm changes its conformation during catalysis similar to human TrxR. Striking differences between P. falciparum and human TrxR are a Plasmodium-specific insertion and the conformation of the C-terminal arm, which lead to considerable differences in thioredoxin binding and disulfide reduction. Moreover, we functionally analyzed amino acid residues involved in substrate binding and in the architecture of the intersubunit cavity, which is a known binding site for disulfide reductase inhibitors. Cell biological experiments indicate that P. falciparum TrxR is indeed targeted in the parasite by specific inhibitors with antimalarial activity. Differences between P. falciparum and human TrxR and details on substrate reduction and inhibitor binding provide the first solid basis for structure-based drug development and lead optimization.     

Plasmodium falciparum RuvB2 translocates in 5′–3′ direction,relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA + family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5′–3′ direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.     

Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having a COOH-terminal orotate phosphoribosyltransferase (OPRT) and an NH₂-terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, were stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (k_cat/K_m). The k_cat was selectively enhanced up to three orders of magnitude, while the K_m was not much affected and remained at low μM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivities and metabolic functions on molecular evolution.     

An interplay between 2 signaling pathways: Melatonin-cAMP and IP3–Ca signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca²⁺) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca²⁺ imaging showed that LZ treatment completely abolished Ca²⁺ oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP₃–Ca²⁺ and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.     

Functional Importance of Bacteriophage ?29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx₂h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx₂hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.     

Plasmodium falciparum DDX55 is a nucleocytoplasmic protein and a 3′-5′ direction-specific DNA helicase

Malaria is one of the major causes of mortality as well as morbidity in many tropical and subtropical countries around the world. Although artemisinin combination therapies (ACTs) are contributing to substantial decline in the worldwide malaria burden, it is becoming vulnerable by the emergence of artemisinin resistance in Plasmodium falciparum leading to clinical failure of ACTs in Southeast Asia. Helicases play important role in nucleic acid metabolic processes and have been also identified as therapeutic drug target for different diseases. Previously, it has been reported that P. falciparum contains a group of DEAD-box family of helicases which are homologous to Has1 family of yeast. Here, we present the characterization of a member of Has1 family (PlasmoDB number PF3D7_1419100) named as PfDDX55. The biochemical characterization of PfDDX55C revealed that it contains both DNA- and RNA-dependent ATPase activity. PfDDX55C unwinds partially duplex DNA in 3′ to 5′ direction and utilizes mainly ATP or dATP for its activity. The immunofluorescence assay and q-RT PCR analysis show that PfDDX55 is a nucleocytoplasmic protein expressed in all the intraerythrocytic development of P. falciparum 3D7 strain with maximum expression level in trophozoite stage. The LC-MS/MS experiment results and STRING analysis show that PfDDX55 interacts with AAA-ATPase which has been shown to be involved in ribosomal biogenesis.

     

PICK1 is implicated in organelle motility in an Arp2/3 complex–independent manner

PICK1 is a modular scaffold implicated in synaptic receptor trafficking. It features a PDZ domain, a BAR domain, and an acidic C-terminal tail (ACT). Analysis by small- angle x-ray scattering suggests a structural model that places the receptor-binding site of the PDZ domain and membrane-binding surfaces of the BAR and PDZ domains adjacent to each other on the concave side of the banana-shaped PICK1 dimer. In the model, the ACT of one subunit of the dimer interacts with the PDZ and BAR domains of the other subunit, possibly accounting for autoinhibition. Consistently, full-length PICK1 shows diffuse cytoplasmic localization, but it clusters on vesicle-like structures that colocalize with the trans-Golgi network marker TGN38 upon deletion of either the ACT or PDZ domain. This localization is driven by the BAR domain. Live-cell imaging further reveals that PICK1-associated vesicles undergo fast, nondirectional motility in an F-actin–dependent manner, but deleting the ACT dramatically reduces vesicle speed. Thus the ACT links PICK1-associated vesicles to a motility factor, likely myosin, but, contrary to previous reports, PICK1 neither binds nor inhibits Arp2/3 complex.     

A transient α-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1

The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays show that the defects of the proline mutants are the result of impaired binding of Top3 and Rmi1 to Sgs1. Extending mutagenesis N-terminally revealed a second functionally critical region that spans residues 9–17. Depending on the position of the proline substitution in the helix functional impairment of Sgs1 function varied, gradually increasing from the C- to the N-terminus. The multiscale approach we used to interrogate structure/function relationships in the long disordered N-terminal segment of Sgs1 allowed us to precisely define a functionally critical region and should be generally applicable to other disordered proteins.     

Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5–SMG7 and SMG6

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7. To understand the molecular mechanisms, we recapitulated these steps of NMD in vitro using purified components. We find that a short C-terminal segment of phosphorylated UPF1 containing the last two Ser-Gln motifs is recognized by the heterodimer of SMG5 and SMG7 14–3–3-like proteins. In contrast, the SMG6 14–3–3-like domain is a monomer. The crystal structure indicates that the phosphoserine binding site of the SMG6 14–3–3-like domain is similar to that of SMG5 and can mediate a weak phospho-dependent interaction with UPF1. The dominant SMG6–UPF1 interaction is mediated by a low-complexity region bordering the 14–3–3-like domain of SMG6 and by the helicase domain and C-terminal tail of UPF1. This interaction is phosphorylation independent. Our study demonstrates that SMG5–SMG7 and SMG6 exhibit different and non-overlapping modes of UPF1 recognition, thus pointing at distinguished roles in integrating the complex NMD interaction network.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization,and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.     

An insertion/deletion polymorphism in the 3′ untranslated region of β-transducin repeat-containing protein (βTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese

Hepatocellular carcinoma (HCC) is an epithelial cancer which originates from hepatocytes or their progenitors. As a positive regulator of NFκB signaling pathway, β-transducin repeat-containing protein (βTrCP) is overexpressed and oncogenic in epithelial cancers, suggesting a potential role of βTrCP in HCC susceptibility. We carried out a case-control study in a Chinese population (256 cases and 367 controls) to estimate the susceptibility to HCC associated with a 9 bp insertion/deletion polymorphism (rs16405) in 3′ untranslated region of βTrCP. Using unconditional logistic regression, we found that 9N del/del and 9N ins/del genotypes were significantly associated with decreased HCC risk: OR = 0.44 (0.24-0.83) (p = 0.004) and OR = 0.56 (0.31-1.00) (p = 0.034), respectively. Furthermore, in vivo experiments showed that mRNA levels of βTrCP from HCC tumor tissues were correlated with rs16405 genotypes. HCC tumor tissues with homozygous for 9N ins/ins has the highest level of βTrCP, which are 3.99 and 7.04-fold higher than heterozygous 9N ins/del and homozygous 9N del/del, respectively. Based on bioinformatics prediction, we found that the risk allele for rs16405 disrupted a binding site for human microRNA-920 which would negatively regulate βTrCP. We propose a microRNA-920 mediated βTrCP regulation model depending on rs16405 genotype, which warrants further replication association studies and follow-up functional experiments.     

Cellular iron homeostasis mediated by the Mrs4–Ccc1–Smf3 pathway is essential for mitochondrial function,morphogenesis and virulence in Candida albicans

Iron bioavailability is crucial for mitochondrial metabolism and biosynthesis. Dysregulation of cellular iron homeostasis affects multiple aspects of mitochondrial physiology and cellular processes. However, the intracellular iron trafficking pathway in Candida albicans remains unclear. In this study, we characterized the Mrs4–Ccc1–Smf3 pathway, and demonstrated its important role in maintaining cellular iron levels. Double deletion of vacuolar iron exporter SMF3 and mitochondrial iron transporter MRS4 further elevated cellular iron levels in comparison with the single MRS4 deletion. However, deletion of vacuolar iron importer CCC1 in the mrs4?/? mutant restored cellular iron homeostasis to normal wild-type levels, and also normalized most of the defective phenotypes in response to various environmental stresses. Our results also suggested that both Mrs4 and Ccc1 contributed to the maintenance of mitochondrial function. The mrs4?/? and mrs4?/?smf3?/? mutants exhibited an obvious decrease in aconitase activities and mitochondrial membrane potential, whereas deletion of CCC1 in the mrs4?/? mutant effectively rescued these defects. Furthermore, we also found that the Mrs4–Ccc1–Smf3 pathway was indispensable for cell-wall stability, antifungal drug tolerance, filamentous growth and virulence, supporting the novel viewpoint that mitochondria might be the promising target for better antifungal therapies. Interestingly, the addition of exogenous iron failed to rescue the defects on non-fermentable carbon sources or hyphae-inducing medium, indicating that the defects in mitochondrial respiration and filamentous development might result from the disturbance of cellular iron homeostasis rather than environmental iron deprivation. Taken together, our results propose the Mrs4–Ccc1–Smf3 pathway as a potentially attractive target for antifungal drug development.     

The Crystal Structure of the Pseudomonas dacunhae Aspartate-β-Decarboxylase Dodecamer Reveals an Unknown Oligomeric Assembly for a Pyridoxal-5′-Phosphate-Dependent Enzyme

The Pseudomonas dacunhael-aspartate-β-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-decarboxylation of l-aspartate to produce l-alanine and CO₂. This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an l-Asp/l-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 Å resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arginine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.     

Isolation and structure elucidation of 5′-O-β-d-glucopyranosyl-dihydroascorbigen from Cardamine diphylla rhizome

From the methanol extract of Cardamine diphylla rhizome, 5′-O-β-d-glucopyranosyl-dihydroascorbigen (1) and 6-hydroxyindole-3-carboxylic acid 6-O-β-d-glucopyranoside (2) were isolated. The structures of the compounds were elucidated using spectroscopic methods. This is the second report on the presence of a glucosylated indole ascorbigen in plants.     

Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.