Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum.

Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

Thin film Gallium nitride (GaN) based acoustofluidic Tweezer: Modelling and microparticle manipulation.

Gallium nitride (GaN) is a compound semiconductor which shows advantages in new functionalities and applications due to its piezoelectric, optoelectronic, and piezo-resistive properties. This study develops a thin film GaN-based acoustic tweezer (GaNAT) using surface acoustic waves (SAWs) and demonstrates its acoustofluidic ability to pattern and manipulate microparticles. Although the piezoelectric performance of the GaNAT is compromised compared with conventional lithium niobate-based SAW devices, the inherited properties of GaN allow higher input powers and superior thermal stability. This study shows for the first time that thin film GaN is suitable for the fabrication of the acoustofluidic devices to manipulate microparticles with excellent performance. Numerical modelling of the acoustic pressure fields and the trajectories of mixtures of microparticles driven by the GaNAT was performed and the results were verified from the experimental studies using samples of polystyrene microspheres. The work has proved the robustness of thin film GaN as a candidate material to develop high-power acoustic tweezers, with the potential of monolithical integration with electronics to offer diverse microsystem applications.

Dose Effects of Orally Administered Spirulina Suspension on Colonic Microbiota in Healthy Mice.

Oral supplemented nutraceuticals derived from food sources are surmised to improve the human health through interaction with the gastrointestinal bacteria. However, the lack of fundamental quality control and authoritative consensus (e.g., formulation, route of administration, dose, and dosage regimen) of these non-medical yet bioactive compounds are one of the main practical issues resulting in inconsistent individual responsiveness and confounded clinical outcomes of consuming nutraceuticals. Herein, we studied the dose effects of widely used food supplement, microalgae spirulina (Arthrospira platensis), on the colonic microbiota and physiological responses in healthy male Balb/c mice. Based on the analysis of 16s rDNA sequencing, compared to the saline-treated group, oral administration of spirulina once daily for 24 consecutive days altered the diversity, structure, and composition of colonic microbial community at the genus level. More importantly, the abundance of microbial taxa was markedly differentiated at the low (1.5 g/kg) and high (3.0 g/kg) dose of spirulina, among which the relative abundance of Clostridium XIVa, Desulfovibrio, Eubacterium, Barnesiella, Bacteroides, and Flavonifractor were modulated at various degrees. Evaluation of serum biomarkers in mice at the end of spirulina intervention showed reduced the oxidative stress and the blood lipid levels and increased the level of appetite controlling hormone leptin in a dose-response manner, which exhibited the significant correlation with differentially abundant microbiota taxa in the cecum. These findings provide direct evidences of dose-related modulation of gut microbiota and physiological states by spirulina, engendering its future mechanistic investigation of spirulina as potential sources of prebiotics for beneficial health effects via the interaction with gut microbiota.

Crosstalk between cancer and immune cells: Role of tumor-associated macrophages in the tumor microenvironment.

Tumor microenvironment is a complex system that contains multiple cells and cytokines. Among the multiple immune cells, macrophage is particularly abundant and plays an important role throughout the tumor progression process, namely, tumor-associated macrophage (TAM) in this special tumor microenvironment. Many kinds of cytokines from TAMs and other immune cells in tumor niche are involved in the linkage of inflammation, immunity and tumorigenesis. Inflammatory responses induced by TAMs are crucial to tumor development of different stages. This review highlights the critical role of TAMs in the linkage of inflammation, immunity, and cancer. It outlines the molecules of inflammatory cytokines, chemokines, and growth factors mainly from TAMs in tumor microenvironment and their functions in tumor development during the major issues of angiogenesis, chronic inflammation, and immune suppression. Additionally, the signaling pathways involved in tumor progression and the crosstalk between them are also summarized.

Progress of Antimicrobial Discovery Against the Major Cariogenic Pathogen Streptococcus mutans.

Dental caries, also known as tooth decay, or cavities, remains a global public health issue. Streptococcus mutans is considered the most significant contributor to dental caries. Its cariogenicity typically depends on its unique metabolic activity and lifestyle, including acid production, acid tolerance and biofilm formation. Currently used anti-caries therapies, such as fluoride and chlorhexidine, are characterized by side-effects and drug resistance. Therefore, the development of alternative inhibitors against S. mutans growth is urgently needed. In the last decade, a larger number of natural products and their derivatives from plants, marine organisms and microorganisms were studied to evaluate their antibacterial activity against S. mutans. In addition, drug-repositioning base screening and target based high-throughput screening were employed, resulting in inspiring progresses in recent years. In this review, we summarized the available evidences regarding the inhibition of S. mutans growth. We focus on the sources, structures and potential mechanism of action of these inhibitors. Beside small molecular compounds, we also considered antibacterial peptides and protein inhibitors developed in this field.

Cyclic Dimeric Guanosine Monophosphate: Activation and Inhibition of Innate Immune Response.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a universally conserved second messenger that contributes to the pathogenicity of numerous bacterial species. In recent years, growing evidence has shown that bacterial extracellular c-di-GMP can interact with the innate immune system and regulate host immune responses. This review summarizes our current understanding on the dual roles of bacterial c-di-GMP in pathogen-host interaction: activation of the antibacterial innate immune response through the cytosolic surveillance pathway and inhibition of innate immune defense for iron restriction.

The Small Regulatory Antisense RNA PilR Affects Pilus Formation and Cell Motility by Negatively Regulating pilA11 in Synechocystis sp. PCC 6803.

Pili are found on the surface of many bacteria and play important roles in cell motility, pathogenesis, biofilm formation, and sensing and reacting to environmental changes. Cell motility in the model cyanobacterium Synechocystis sp. PCC 6803 relies on expression of the putative pilA9-pilA10-pilA11-slr2018 operon. In this study, we identified the antisense RNA PilR encoded in the noncoding strand of the prepilin-encoding gene pilA11. Analysis of overexpressor [PilR(+)] and suppressor [PilR(-)] mutant strains revealed that PilR is a direct negative regulator of PilA11 protein. Although overexpression of PilR did not affect cell growth, it greatly reduced levels of pilA11 mRNA and protein and decreased both the thickness and number of pili, resulting in limited cell motility and small, distinct colonies. Suppression of PilR had the opposite effect. A hypothetical model on the regulation of pilA9-pilA10-pilA11-slr2018 operon expression by PilR was proposed. These results add a layer of complexity to the mechanisms controlling pilA11 gene expression and cell motility, and provide novel insights into how sRNA and the intergenic region secondary structures can work together to discoordinatly regulate target gene in an operon in cyanobacterium.

Uncovering Drug Mechanism of Action by Proteome Wide- Identification of Drug-Binding Proteins.

Small molecule drugs obtained from synthetic compounds or natural products show their therapeutic effects by specifically binding to one or a few target proteins and modulating their functions. In contrast, undesirable drug-protein interactions may provoke harmful side effects. Furthermore, drug-protein interactions also play roles in drug activation, transport, metabolism and regulation of drug resistance. Therefore, systematically identifying binding proteins of a drug molecule is critical for understanding the mechanism of action of the drug at molecular level. In this review, we summarize current widely-used experimental approaches for proteome-wide target identification of small molecule drugs, including affinity purification, chemical proteomics and protein thermal stability based methods. The advance of these methods will quicken the pace of target deconvolution of small molecule drugs and hold promise for drug repositioning research. Moreover, these approaches also provide a powerful arsenal for exploring the signaling pathways of small molecule second messengers, signaling lipids and other regulatory metabolites.

Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections.

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.

High resolution structures of Plasmodium falciparum GST complexes provide novel insights into the dimer-tetramer transition and a novel ligand-binding site.

Protection from oxidative stress and efficient redox regulation are essential for malarial parasites which have to grow and multiply rapidly in pro-oxidant rich environments. Therefore, redox active proteins currently belong to the most attractive antimalarial drug targets. The glutathione S-transferase from Plasmodium falciparum (PfGST) is a redox active protein displaying a peculiar dimer-tetramer transition that causes full enzyme-inactivation. This distinct structural feature is absent in mammalian GST isoenzyme counterparts. A flexible loop between residues 113-119 has been reported to be necessary for this tetramerization process. However, here we present structural data of a modified PfGST lacking loop 113-119 at 1.9 Å resolution. Our results clearly show that this loop is not essential for the formation of stable tetramers. Moreover we present for the first time the structures of both, the inactive and tetrameric state at 1.7 Å and the active dimeric state in complex with reduced glutathione at 2.4 Å resolution. Surprisingly, the structure of the inactive tetrameric state reveals a novel non-substrate binding-site occupied by a 2-(N-morpholino) ethane sulfonic acid (MES) molecule in each monomer. Although it is known that the PfGST has the ability to bind lipophilic anionic ligands, the location of the PfGST ligand-binding site remained unclear up to now.

Purification, crystallization and preliminary crystallographic analysis of a ribosome-recycling factor from Thermoanaerobacter tengcongensis (TteRRF).

Ribosome-recycling factor (RRF) plays an essential role in the fourth step of protein synthesis in prokaryotes. RRF combined with elongation factor G (EF-G) disassembles the post-termination ribosome complex and recycles the protein synthesis machine for the next round of translation. A reductive-methylation-modified RRF from Thermoanaerobacter tengcongensis (TteRRF) has been crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.1 M citric acid pH 3.5, 3.0 M NaCl and 50 mg ml(-1) methylated protein solution at 289 K. A complete data set was collected from a crystal to 2.80 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space group P6122/P6522 with unit-cell parameters a = b = 103.26, c = 89.17 Å. The asymmetric unit was estimated to contain one molecule of TteRRF.

Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex.

Protein preparation, crystallization and preliminary X-ray analysis of Polygonum cuspidatum bifunctional chalcone synthase/benzalacetone synthase.

The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution.

Expression, purification and preliminary crystallographic analysis of the T6SS effector protein Tse3 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to inject effector proteins into rival cells in niche competition. Tse3, one of the effectors of T6SS, is delivered into the periplasm of recipient cells. Tse3 functions as a muramidase that degrades the ß-1,4-linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan, thus leading to lysis of the recipient cells and providing a competitive advantage to the donor cells. Here, the preliminary crystallographic study of Tse3 is reported. A crystal of Tse3 diffracted to 1.5 Šresolution. It belonged to space group C121, with unit-cell parameters a = 166.99, b = 70.13, c = 41.94 Å, α = 90.00, ß = 90.52, γ  = 90.00° and one molecule per asymmetric unit.

Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function.

Tse1 (Tse is type VI secretion exported), an effector protein produced by Pseudomonas aeruginosa, is an amidase that hydrolyses the γ-D-glutamyl-DAP (γ-D-glutamyl-L-meso-diaminopimelic acid) linkage of the peptide bridge of peptidoglycan. P. aeruginosa injects Tse1 into the periplasm of recipient cells, degrading their peptidoglycan, thereby helping itself to compete with other bacteria. Meanwhile, to protect itself from injury by Tse1, P. aeruginosa expresses the cognate immunity protein Tsi1 (Tsi is type VI secretion immunity) in its own periplasm to inactivate Tse1. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6-148)-Tsi1-(20-end) complex at 1.4 Å and 1.6 Å (1 Å=0.1 nm) resolutions respectively. The Tse1 structure adopts a classical papain-like α+ß fold. A cysteine-histidine catalytic diad is identified in the reaction centre of Tse1 by structural comparison and mutagenesis studies. Tsi1 binds Tse1 tightly. The HI loop (middle finger tip) from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1, and CD, EF, JK and LM loops (thumb, index finger, ring finger and little finger tips) interact with Tse1, thus blocking the binding of enzyme to peptidoglycan. The catalytic and inhibition mechanisms provide new insights into how P. aeruginosa competes with others and protects itself.

Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant from Ammopiptanthus mongolicus.

Calcineurin B-like protein 1 (CBL1) is a calcium sensor in plants. It transmits the calcium signal through the downstream protein CBL-interacting protein kinase (CIPK). CBL1 and CIPK play crucial roles in the response to environmental stresses such as low K+, osmotic shock, high salt, cold and drought. Recombinant CBL1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. However, the crystal did not diffract well. A mutant prepared using the surface-entropy method and crystallized using the hanging-drop method at 298 K with PEG 2000 MME as a precipitant diffracted to 2.90 Šresolution. The crystal belonged to space group P2(1)2(1)2, with unit-cell parameters a=99.87, b=114.42, c=63.80 Å, α=ß=γ=90.00° and three molecules per asymmetric unit.

Crystal structure of the complex between Pseudomonas effector AvrPtoB and the tomato Pto kinase reveals both a shared and a unique interface compared with AvrPto-Pto.

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9A and of the AvrPtoB(121-205)-Pto complex at a resolution of 3.3 A. AvrPtoB(121-205) exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.

Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione.

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.

Cloning, expression, purification, characterization, crystallization and x-ray diffraction of bifunctional pyrimidine deaminase/reductase from Shigella flexneri 2a.

Bifunctional pyrimidine deaminase/reductase (RibD) plays an important role during riboflavin biosynthesis in many microorganisms. The 40.4 kDa RibD from Shigella flexneri 2a has been cloned, expressed, purified and characterized. Three Crystals of RibD have been obtained by the hanging-drop technique at 291 K using PEG 20k or NaCl as precipitant. The RibD crystal using PEG 20k as precipitant diffracted to 2.5A.

Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2.

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.