New insight on porcine carboxylesterases expression and activity in lung tissues.

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase.

Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2's cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.

Cytosolic UDP-L-arabinose synthesis by bifunctional UDP-glucose 4-epimerases in Arabidopsis.

L-Arabinose (L-Ara) is a plant-specific sugar found in cell wall polysaccharides, proteoglycans, glycoproteins, and small glycoconjugates, which play physiologically important roles in cell proliferation and other essential cellular processes. L-Ara is synthesized as UDP-L-arabinose (UDP-L-Ara) from UDP-xylose (UDP-Xyl) by UDP-Xyl 4-epimerases (UXEs), a type of de novo synthesis of L-Ara unique to plants. In Arabidopsis, the Golgi-localized UXE AtMUR4 is the main contributor to UDP-L-Ara synthesis. However, cytosolic bifunctional UDP-glucose 4-epimerases (UGEs) with UXE activity, AtUGE1, and AtUGE3 also catalyze this reaction. For the present study, we first examined the physiological importance of bifunctional UGEs in Arabidopsis. The uge1 and uge3 mutants enhanced the dwarf phenotype of mur4 and further reduced the L-Ara content in cell walls, suggesting that bifunctional UGEs contribute to UDP-L-Ara synthesis. Through the introduction of point mutations exchanging corresponding amino acid residues between AtUGE1 with high UXE activity and AtUGE2 with low UXE activity, two mutations that increase relative UXE activity of AtUGE2 were identified. The crystal structures of AtUGE2 in complex forms with NAD+ and NAD+/UDP revealed that the UDP-binding domain of AtUGE2 has a more closed conformation and smaller sugar-binding site than bacterial and mammalian UGEs, suggesting that plant UGEs have the appropriate size and shape for binding UDP-Xyl and UDP-L-Ara to exhibit UXE activity. The presented results suggest that the capacity for cytosolic synthesis of UDP-L-Ara was acquired by the small sugar-binding site and several mutations of UGEs, enabling diversified utilization of L-Ara in seed plants.

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana.

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

Acyl-Coa Thioesterases: A Rheostat That Controls Activated Fatty Acids Modulates Dengue Virus Serotype 2 Replication.

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. The loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that functional differences and substrate specificities due to the location (cytosol and mitochondria, respectively) of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell, highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

METTL16 exerts an m6A-independent function to facilitate translation and tumorigenesis.

METTL16 has recently been identified as an RNA methyltransferase responsible for the deposition of N6-methyladenosine (m6A) in a few transcripts. Whether METTL16 methylates a large set of transcripts, similar to METTL3 and METTL14, remains unclear. Here we show that METTL16 exerts both methyltransferase activity-dependent and -independent functions in gene regulation. In the cell nucleus, METTL16 functions as an m6A writer to deposit m6A into hundreds of its specific messenger RNA targets. In the cytosol, METTL16 promotes translation in an m6A-independent manner. More specifically, METTL16 directly interacts with the eukaryotic initiation factors 3a and -b as well as ribosomal RNA through its Mtase domain, thereby facilitating the assembly of the translation-initiation complex and promoting the translation of over 4,000 mRNA transcripts. Moreover, we demonstrate that METTL16 is critical for the tumorigenesis of hepatocellular carcinoma. Collectively, our studies reveal previously unappreciated dual functions of METTL16 as an m6A writer and a translation-initiation facilitator, which together contribute to its essential function in tumorigenesis.

Cytosolic phosphofructokinases are important for sugar homeostasis in leaves of Arabidopsis thaliana.

BACKGROUND AND AIMS: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focuses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. METHODS: We isolated homozygous knockout individual mutants (pfk1, pfk3, pfk6 and pfk7) and two double mutants (pfk1/7 and pfk3/6), and characterized their growth and metabolic phenotypes. KEY RESULTS: In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and showed a clear visual and metabolic phenotype with reduced shoot growth, early flowering and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. CONCLUSIONS: We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently in source-sink relationships in A. thaliana, while PFK3 and PFK6 only play a minor role under normal growth conditions.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

The Iron Maiden. Cytosolic Aconitase/IRP1 Conformational Transition in the Regulation of Ferritin Translation and Iron Hemostasis.

Maintaining iron homeostasis is fundamental for almost all living beings, and its deregulation correlates with severe and debilitating pathologies. The process is made more complicated by the omnipresence of iron and by its role as a fundamental component of a number of crucial metallo proteins. The response to modifications in the amount of the free-iron pool is performed via the inhibition of ferritin translation by sequestering consensus messenger RNA (mRNA) sequences. In turn, this is regulated by the iron-sensitive conformational equilibrium between cytosolic aconitase and IRP1, mediated by the presence of an iron-sulfur cluster. In this contribution, we analyze by full-atom molecular dynamics simulation, the factors leading to both the interaction with mRNA and the conformational transition. Furthermore, the role of the iron-sulfur cluster in driving the conformational transition is assessed by obtaining the related free energy profile via enhanced sampling molecular dynamics simulations.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.

Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.

Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.

Effect of Salt Stress on the Expression and Promoter Methylation of the Genes Encoding the Mitochondrial and Cytosolic Forms of Aconitase and Fumarase in Maize.

The influence of salt stress on gene expression, promoter methylation, and enzymatic activity of the mitochondrial and cytosolic forms of aconitase and fumarase has been investigated in maize (Zea mays L.) seedlings. The incubation of maize seedlings in 150-mM NaCl solution resulted in a several-fold increase of the mitochondrial activities of aconitase and fumarase that peaked at 6 h of NaCl treatment, while the cytosolic activity of aconitase and fumarase decreased. This corresponded to the decrease in promoter methylation of the genes Aco1 and Fum1 encoding the mitochondrial forms of these enzymes and the increase in promoter methylation of the genes Aco2 and Fum2 encoding the cytosolic forms. The pattern of expression of the genes encoding the mitochondrial forms of aconitase and fumarase corresponded to the profile of the increase of the stress marker gene ZmCOI6.1. It is concluded that the mitochondrial and cytosolic forms of aconitase and fumarase are regulated via the epigenetic mechanism of promoter methylation of their genes in the opposite ways in response to salt stress. The role of the mitochondrial isoforms of aconitase and fumarase in the elevation of respiration under salt stress is discussed.

Comprehensive characterization of mRNAs associated with yeast cytosolic aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are a conserved family of enzymes with an essential role in protein synthesis: ligating amino acids to cognate tRNA molecules for translation. In addition to their role in tRNA charging, aaRSs have acquired non-canonical functions, including post-transcriptional regulation of mRNA expression. Yet, the extent and mechanisms of these post-transcriptional functions are largely unknown. Herein, we performed a comprehensive transcriptome analysis to define the mRNAs that are associated with almost all aaRSs present in S. cerevisiae cytosol. Nineteen (out of twenty) isogenic strains of GFP-tagged cytosolic aaRSs were subjected to immunoprecipitation with anti-GFP beads along with an untagged control. mRNAs associated with each aaRS were then identified by RNA-seq. The extent of mRNA association varied significantly between aaRSs, from MetRS in which none appeared to be statistically significant, to PheRS that binds hundreds of different mRNAs. Interestingly, many target mRNAs are bound by multiple aaRSs, suggesting co-regulation by this family of enzymes. Gene Ontology analyses for aaRSs with a considerable number of target mRNAs discovered an enrichment for pathways of amino acid metabolism and of ribosome biosynthesis. Furthermore, sequence and structure motif analysis revealed for some aaRSs an enrichment for motifs that resemble the anticodon stem loop of cognate tRNAs. These data suggest that aaRSs coordinate mRNA expression in response to amino acid availability and may utilize RNA elements that mimic their canonical tRNA binding partners.

Characterization of human sulfotransferases catalyzing the formation of p-cresol sulfate and identification of mefenamic acid as a potent metabolism inhibitor and potential therapeutic agent for detoxification.

p-Cresol sulfate, the primary metabolite of p-cresol, is a uremic toxin that has been associated with toxicities and mortalities. The study objectives were to i) characterize the contributions of human sulfotransferases (SULT) catalyzing p-cresol sulfate formation using multiple recombinant SULT enzymes (including the polymorphic variant SULT1A1*2), pooled human liver cytosols, and pooled human kidney cytosols; and ii) determine the potencies and mechanisms of therapeutic inhibitors capable of attenuating the production of p-cresol sulfate. Human recombinant SULT1A1 was the primary enzyme responsible for the formation of p-cresol sulfate (Km = 0.19 ±â€¯0.02 µM [with atypical kinetic behavior at lower substrate concentrations; see text discussion], Vmax = 789.5 ±â€¯101.7 nmol/mg/min, Ksi = 2458.0 ±â€¯332.8 µM, mean ±â€¯standard deviation, n = 3), while SULT1A3, SULT1B1, SULT1E1, and SULT2A1 contributed negligible or minor roles at toxic p-cresol concentrations. Moreover, human recombinant SULT1A1*2 exhibited reduced enzyme activities (Km = 81.5 ±â€¯31.4 µM, Vmax = 230.6 ±â€¯17.7 nmol/mg/min, Ksi = 986.0 ±â€¯434.4 µM) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (Km = 14.8 ±â€¯3.4 µM, Vmax = 1.5 ±â€¯0.2 nmol/mg/min) and substrate inhibition in kidney cytosols (Km = 0.29 ±â€¯0.02 µM, Vmax = 0.19 ±â€¯0.05 nmol/mg/min, Ksi = 911.7 ±â€¯278.4 µM). Of the 14 investigated therapeutic inhibitors, mefenamic acid (Ki = 2.4 ±â€¯0.1 nM [liver], Ki = 1.2 ±â€¯0.3 nM [kidney]) was the most potent in reducing the formation of p-cresol sulfate, exhibiting noncompetitive inhibition in human liver cytosols and recombinant SULT1A1, and mixed inhibition in human kidney cytosols. Our novel findings indicated that SULT1A1 contributed an important role in p-cresol sulfonation (hence it can be considered a probe reaction) in liver and kidneys, and mefenamic acid may be utilized as a potential therapeutic agent to attenuate the generation of p-cresol sulfate as an approach to detoxification.

Direct evidence of cytosolic PNGase activity in Arabidopsis thaliana: in vitro assay system for plant cPNGase activity.

Cytosolic peptide:N-glycanase (cPNGase), which occurs ubiquitously in eukaryotic cells, is involved in the de-N-glycosylation of misfolded glycoproteins in the protein quality control system. In this study, we aimed to provide direct evidence of plant cPNGase activity against a denatured glycoprotein using a crude extract prepared from a mutant line of Arabidopsis thaliana lacking 2 acidic PNGase genes.

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP).

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0-8.0), although PfM1AAP also possesses some activity (< 20%) at the lower pH range of 5.0-5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.

Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases.

Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

Cloning, purification and characterisation of cytosolic fructose-1,6-bisphosphatase from mung bean (Vigna radiata).

To improve the crop yield and quality, the cytosolic fructose-1,6-bisphosphatase (cFBPase) from mung bean (Vigna radiata), a rate-limiting enzyme in gluconeogenesis, was cloned, purified, and structurally characterised. To function it required Mg2+ and Mn2+ at 0.01-10 mM. The Michaelis-Menton constant and adenosine monophosphate (AMP) inhibitory constant (Ki) were 7.96 and 111.09 µM, respectively. The functional site residues of AMP binding (Arg30, Asp32, and Phe33) and the active site residues (Asn218 and Met251) were tested via site-directed mutagenesis and molecular docking. Asn218 and Met251 were replaced by Tyr and Leu, respectively. The M251L mutant showed enhanced substrate affinity and activity, resulting from decreased binding energy (-2.58 kcal·mol-1) and molecular distance (4.2 Å). AMP binding site mutations changed the enzyme activities, indicating a connection between the binding and active sites. Furthermore, Ki and docking analysis revealed that Asp32 plays a key role in maintaining the AMP binding conformation.

Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines.

The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.