Bioinformatics-based approaches to engineer the transmembrane Δ6 desaturase from Micromonas pusilla

The increased awareness of the health benefits of ω3-long chain polyunsaturated fatty acids (ω3-LCPUFAs) has led to a drastic increase in the consumption of fish-oil supplements. This has resulted in environmental concerns and the identification of key membrane-bound desaturases involved in the biosynthesis of ω3-LCPUFAs in order to generate a sustainable source of ω3-LCPUFAs. The Micromonas pusilla Δ6 desaturase (MpΔ6des) is a membrane-bound desaturase that is specific for ω3-LCPUFA precursors and acyl-Coenzyme A substrates (acyl-CoAs). The incorporation of MpΔ6des into the ω3-LCPUFA biosynthesis pathway allows efficient ω3-LCPUFA production in transgenic plants. However, little is known of the molecular basis underlying its ω3-specificity, stability and acyl-CoAs specificity. MpΔ6des is relatively challenging in terms of protein engineering targets in that there is no molecular structure available, it cannot be expressed in easily manipulated prokaryotic systems such as Escherichia coli, and the activity cannot be rapidly screened via the conventional techniques. Thus, computational, structure-based, protein design and high-throughput directed evolution could not be used. To overcome the technical hurdles, we have applied bioinformatics-based techniques (consensus mutagenesis, ancestral protein reconstruction and sequence similarity networks) to engineer MpΔ6des and to better define the sequence-structure-function relationship of proteins within the desaturase superfamily. Consensus mutagenesis of MpΔ6des (Chapter 2) demonstrated that it is possible to modulate the ω3/ω6-specificity of MpΔ6des semi-independently. The geometry of the substrate-binding pocket of MpΔ6des was not only influenced by the residues located in the substrate-binding cavity, but also by distal residues, possibly through intramolecular interaction networks. An ancestral algal front-end Δ6 desaturase (ANC175) was inferred (Chapter 3), which resembles the properties of the progenitor of the algal Δ6 desaturases. The comparison between ANC175 and contemporary desaturases indicated that the divergence of the ω3/ω6-specificity of algal Δ6 desaturases is associated with the environmental differences seen in the habitats of the different algal species. Chapter 4 describes a bioinformatics analysis of the desaturase superfamily, showing that the four major desaturase subfamilies (the first desaturases, methyl-end desaturases, front-end desaturases and Δ4 sphingolipid desaturases) are structurally and functionally distinct. Conserved motif analysis of the front-end desaturases suggested that two cytosolic regions (a loop between AH1 and H2, and the cytosolic side of TM3) play crucial roles in determining the substrate head-group specificity of the front-end desaturase. Altogether, this thesis promotes a more detailed structural and functional understanding of the front-end desaturases, especially MpΔ6des. It validates the use of bioinformatics-based approaches such as consensus mutagenesis and ancestral protein reconstruction, showing that small libraries that are relatively “rich” in valuable mutations can be produced, even in the absence of detailed structural information or a high-throughput screen. We have successfully created novel variants of MpΔ6des with significantly enhanced ω3-specificity and with enhanced expression. These results also shed new light on the evolution of ω3/ω6-specificity in the front-end desaturase subfamily. Finally, the use of sequence similarity networks allowed us to propose a more detailed classification of the desaturase superfamily and identify specific sequence motifs that can be used to predict the substrate “head-group” specificity of these enzymes

A unified multichannel far-field speech recognition system: combining neural beamforming with attention based end-to-end model

Far-field speech recognition is a challenging task that conventionally uses signal processing beamforming to attack noise and interference problem. But the performance has been found usually limited due to heavy reliance on environmental assumption. In this paper, we propose a unified multichannel far-field speech recognition system that combines the neural beamforming and transformer-based Listen, Spell, Attend (LAS) speech recognition system, which extends the end-to-end speech recognition system further to include speech enhancement. Such framework is then jointly trained to optimize the final objective of interest. Specifically, factored complex linear projection (fCLP) has been adopted to form the neural beamforming. Several pooling strategies to combine look directions are then compared in order to find the optimal approach. Moreover, information of the source direction is also integrated in the beamforming to explore the usefulness of source direction as a prior, which is usually available especially in multi-modality scenario. Experiments on different microphone array geometry are conducted to evaluate the robustness against spacing variance of microphone array. Large in-house databases are used to evaluate the effectiveness of the proposed framework and the proposed method achieve 19.26\% improvement when compared with a strong baseline

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.This work was supported by National Health and Medical Research Council Grant 1042272 (to K. K.) and Australian Research Council Discovery Project Grant DP150102883 (to K. K. and G. G. v. D.), Linkage Project Grant LP150101226 (to K. K.), Discovery Early Career Researcher Award DE160101035 (to A. M. L.), QEII Fellowship DP110103144 (to G. G. v. D.), and Future Fellowship FT120100164 (to C. J. T.). C. J. T. is grateful for institutional support from the Victorian State Government Operational Infrastructure Support Program and the National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme

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 growt

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 PfATP4(G358S) 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 PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

The effect of grain size on deformation modes and deformation heterogeneity in a rolled Mg–Zn–Ca alloy

Grain refinement could promote the activation of non-basal slip and enhance the strength and ductility of Mg alloy simultaneously. In this work, the grain size effect on deformation modes and deformation heterogeneity and its relations to crystallographic orientation in a rolled Mg–1.58Zn–0.1Ca alloy sheet with a weakened transverse direction (TD) spread texture were investigated via intragranular misorientation axis (IGMA) and high resolution-digital image correlation (HR-DIC) measurement. The results indicate that a decrease of the grain size from 55 to 13 μm increased the yield strength (YS) by 56.9%, from 72 MPa to 113 MPa, for the TD samples, but only by 8% for the rolling direction (RD) samples, from 125.7 to 135.7 MPa. Moreover, grain refinement enhanced the elongation by 76.4% for the TD samples, from 11% to 19.1%, and merely by 26.5% for the RD samples, from 16.6% to 21%. The transition in dominant deformation modes due to grain size decreasing can be summarized as: for the RD samples: basal slip → prismatic slip; for the TD samples: basal slip + tension twinning → prismatic slip. The enhanced activity of prismatic slip in the fine-grained (FG)-TD and FG-RD samples can be ascribed to the reduced ratio of critical resolved shear stress (CRSS)prismatic/CRSSbasal. For both the RD and TD samples, grain refinement facilitated deformation homogeneity, which could be related to the enhanced activity of basal slip and non-basal slip, as well as the resulting improved deformation compatibility across grain boundary

Effects of ECAP and Annealing Treatment on the Microstructure and Mechanical Properties of Mg-1Y (wt. %) Binary Alloy

Microstructure and mechanical properties development of extruded Mg-1Y (wt. %) binary alloy during equal channel angular pressing (ECAP) with route Bc at 400 °C, and subsequent annealing treatment between 300–400 °C at different holding time of 5–120 min were investigated using an optical and scanning electron microscope (SEM), electron back scattered diffraction (EBSD), tensile test, and hardness test. The grain size of as-extruded material (~10.9 μm) was refined significantly by 1-pass ECAP (~5.8 μm), and resulted in a remarkably enhanced elongation to failure (EL) (~+62%) with a slightly decreased ultimate tensile strength (UTS) (~−3%) comparing to the as-extruded condition (EL = 11.3%, UTS = 200 MPa). The EL was further increased to 27.3% (~+142%) after four passes of ECAP comparing to the as-extruded condition, which was mainly caused by the much more homogenized microstructure. The split basal poles with about 60° rotations to the extruded direction (ED), the relatively coarsened grain size by static recrystallization (SRX) and post-dynamic recrystallization (PDRX) after four passes of ECAP might be responsible for the decreased strength with increasing ECAP pass. During the annealing treatment, recovery dominantly occurred at 300 °C, SRX and grain growth emerged at 350 °C and 400 °C, respectively. Meanwhile, the grain grew and hardness decreased rapidly even within 5 min for 1-pass ECAPed material at 400 °C, indicating a larger grain boundary mobility of ECAPed materials induced by higher deformation energy than the as-extruded ones

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

Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End 6-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 biotechnologyThis work was funded by Commonwealth Science and Industrial Research Organisation Agriculture Flagship. D.L. was funded by an ANU Ph.D. Scholarshi

High-ductility fine-grained Mg-1.92Zn-0.34Y alloy fabricated by semisolid and then hot extrusion

The combination of semisolid and hot extrusion processing was applied to refine the icosahedral quasicrystalline phase (I-phase) in an extruded Mg-1.92Zn-0.34Y(wt.%) alloy for the first time. The semisolid isothermal heat treatment transformed the micron-sized I-phase particles into nano lamellar eutectic (α-Mg + I-phase) with a lamellar spacing of ~86 nm. After subsequent hot extrusion at 250 °C, the nano lamellar eutectic phases were broken into uniformly dispersed nanoscale I-phase particles. What's more, the matrix microstructure was significantly refined with an equiaxed average grain size of 2.59 ± 0.81 µm, and an unusual texture component (most of the grains’ c-axis is parallel to the extrusion direction) was observed. The processed alloy exhibited a high tensile elongation to failure (EL) of 44 ± 2.6% with an ultimate tensile strength (UTS) of 258 ± 2.0 MPa and a tensile yield strength (TYS) of 176 ± 1.6 MPa at room temperature. The high ductility from the combined effects of the grain refinement, dispersion of nanoscale I-phase particles, and the unusual texture. The uniform dispersion of nanoscale I-phase particles could promote grain refinement by particle stimulated nucleation mechanism, and thus bring the unusual texture (where the c-axis is aligned parallel to the extrusion direction during dynamic recrystallization, which contributed to ductility)