Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Δ6-Desaturase Family.

Marine algae are a major source of ω-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Δ6-desaturases, and enzymes from different species display a range of specificities toward ω3- and ω6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This data set provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

Characterization of the ATP4 ion pump in Toxoplasma gondii.

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+ Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.

A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii.

Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of "plant-like" Ca2+-dependent protein kinases (CDPKs) transduces cytosolic Ca2+ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca2+ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during Toxoplasma lytic growth.

A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

Simultaneous determination of trace Cd(II), Pb(II) and Cu(II) by differential pulse anodic stripping voltammetry using a reduced graphene oxide-chitosan/poly-l-lysine nanocomposite modified glassy carbon electrode.

The reduced graphene oxide (RGO) and Chitosan (CS) hybrid matrix RGO-CS were coated onto the glassy carbon electrode (GCE) surface, then, poly-l-lysine films (PLL) were prepared by electropolymerization with cyclic voltammetry (CV) method to prepare RGO-CS/PLL modified glassy carbon electrode (RGO-CS/PLL/GCE) for the simultaneous electrochemical determination of heavy metal ions Cd(II), Pb(II) and Cu(II). Combining the advantageous features of RGO and CS, RGO and CS are used together because the positively charged CS can interact with the negatively changed RGO to prevent their aggregation. Furthermore, CS has many amino groups along its macromolecular chains and possessed strongly reactive with metal ions. Moreover, PLL modified electrodes have good stability, excellent permselectivity, more active sites and strong adherence to electrode surface, which enhanced electrocatalytic activity. The RGO-CS/PLL/GCE was characterized voltammetrically using redox couples (Fe(CN)63-/4-), complemented with electrochemical impedance spectroscopy (EIS). Differential pulse anodic stripping voltammetry (DPASV) has been used for the detection of Cd(II), Pb(II) and Cu(II). The detection limit of RGO-CS/PLL/GCE toward Cd(II), Pb(II) and Cu(II) is 0.01µgL-1, 0.02µgL-1 and 0.02µgL-1, respectively. The electrochemical parameters that exert influence on deposition and stripping of metal ions, such as supporting electrolytes, pH value, deposition potential, and deposition time, were carefully studied.

Classification and substrate head-group specificity of membrane fatty acid desaturases.

Membrane fatty acid desaturases are a diverse superfamily of enzymes that catalyze the introduction of double bonds into fatty acids. They are essential in a range of metabolic processes, such as the production of omega-3 fatty acids. However, our structure-function understanding of this superfamily is still developing and their range of activities and substrate specificities are broad, and often overlapping, which has made their systematic characterization challenging. A central issue with characterizing these proteins has been the lack of a structural model, which has been overcome with the recent publication of the crystal structures of two mammalian fatty acid desaturases. In this work, we have used sequence similarity networks to investigate the similarity among over 5000 related membrane fatty acid desaturase sequences, leading to a detailed classification of the superfamily, families and subfamilies with regard to their function and substrate head-group specificity. This work will facilitate rapid prediction of the function and specificity of new and existing sequences, as well as forming a basis for future efforts to manipulate the substrate specificity of these proteins for biotechnology applications.

The intracellular HBV DNAs as novel and sensitive biomarkers for the clinical diagnosis of occult HBV infection in HBeAg negative hepatocellular carcinoma in China.

This study aimed to investigate the virological status in liver (both tumor and adjacent non-tumor tissue), the clinical features and the contribution of occult HBV infection (OBI) to postoperative prognosis in HBeAg-negative(-) hepatocellular carcinoma (HCC) patients in China. Using quantitative TaqMan fluorescent real-time PCR assays, HBV covalently closed circular DNA (cccDNA) and total DNA (tDNA) were both quantified in 11 (HBsAg(-)) and 57 (HBsAg-positive(+)) pairs of tumor tissue (TT) and adjacent non-tumor tissue (ANTT) obtained from HBeAg(-) HCC patients who received no antiviral treatment and were negative for anti-HCV before surgical treatment. Of 11 HBsAg(-) patients, 36% were with HBsAb(+) HBeAb(+) HBcAb(+). However, only 9% of the HBsAg(-) patients were HBsAb(-) HBeAb(+) HBcAb(+), which accounted for the majority (93%) in the HBsAg(+) group. TT and ANTT HBV tDNAs in 11 HCC patients with HBsAg (-) and HBeAg (-) were all detectable. HBV cccDNA and tDNA were all lower in the HBsAg(-) group than those in the HBsAg(+) group. By Kaplan-Meier analysis, patients with OBI were associated with a lower risk of cirrhosis and better overall survival (OS). The intracellular HBV DNAs, such as HBV cccDNA and tDNA are valuable biological markers for the diagnosis of occult HBV infection in HCC patients. This would assist the clinical implementation of a more personalized therapy for viral re-activation control and improve the survival rate of OBI patients.