Enhancing the water-resistance of MOF-199 film through incorporation of microporous organic networks for solid-phase microextraction of BTEX in aqueous environments with improved efficiency.

BACKGROUND: The practical application of moisture sensitive metal organic frameworks (MOFs) in extraction technology faces challenges related to competitive adsorption and water stability. The target analytes cannot be effectively extracted under humid conditions due to the competitive moisture adsorption and/or framework structure collapse of MOFs. In this study, the microporous organic networks (MONs) were synthesized through Sonogashira coupling reaction to use for hydrophobic modification on the surface of MOF-199. RESULTS: The MOF-199@MON as coating was deposited on stainless steel wires for solid-phase microextraction (SPME) of benzene series (BTEX) in aqueous environments. Under the optimal extraction conditions, the MOF-199@MON coated fiber for SPME coupled with GC-MS for the determination of BTEX gave the linear range of 0.5-500 µg L-1, the limit of detections (LODs, S/N = 3) of 0.01-0.04 µg L-1, the limit of quantifications (LOQs, S/N = 10) of 0.04-0.12 µg L-1, the enhancement factors of 3567-4878, and the intra-day, inter-day and fiber-to-fiber precisions (relative standard deviations, RSDs) of 1.0-9.8, 1.9-7.9 and 4.5-9.5 %, respectively. The developed method was successfully applied to the analysis of BTEX in water samples with the recoveries of 71.0 %-113 %. SIGNIFICANCE: This work reveals the home-made SPME fibers have a long service life (the extraction efficiency of fiber decreased by only 7.26 %-13.14 % after 100 cycles). The potential of MON functionalized MOFs as effective adsorbents for the SPME of pollutants in the water environment.

The UbL-UBA Ubiquilin4 protein functions as a tumor suppressor in gastric cancer by p53-dependent and p53-independent regulation of p21.

Ubiquilin4 (Ubqln4), a member of the UbL-UBA protein family, serves as an adaptor in the degradation of specific substrates via the proteasomal pathway. However, the biological function of Ubqln4 remains largely unknown, especially in cancer. Here, we reported that Ubqln4 was downregulated in gastric cancer tissues and functioned as a tumor suppressor by inhibiting gastric cancer cell proliferation in vivo and in vitro. Overexpression of Ubqln4-induced cellular senescence and G1-S cell cycle arrest in gastric cancer cells and activated the p53/p21 axis. Moreover, Ubqln4 regulated p21 through both p53-dependent and p53-independent manners. Ubqln4 interacted with RNF114, an E3 ubiquitin ligase of p21, and negatively regulated its expression level, which in turn stabilized p21 by attenuating proteasomal degradation of p21. These effects of Ubqln4 were partly abrogated in gastric cancer cells upon silencing of p21. Our findings not only establish the anti-tumor potential of Ubqln4 in gastric cancer but also reveal a role for Ubqln4 in regulation of the cell cycle and cellular senescence via stabilizing p21.

Oxidation-resistant and thermostable forms of alpha-1 antitrypsin from Escherichia coli inclusion bodies.

Native α1-antitrypsin (AAT) is a 52-kDa glycoprotein that acts as an antiprotease and is the physiological inhibitor of neutrophil serine proteases. The main function of AAT is to protect the lung from proteolytic damage induced by inflammation. AAT deficiency (AATD) is a codominant autosomal disorder caused by pathogenic mutations in SERPINA1 gene, leading to reduced levels of serum AAT. The deficiency is known to increase the risk of pulmonary emphysema and chronic obstructive pulmonary disease as a consequence of proteolytic imbalance induced by inflammation, associated in many instances with cigarette smoking and other environmental hazards. Currently, the available therapy for lung disease associated with AATD is serum purified human AAT injected into patients on a weekly basis. It would be advantageous to replace serum-derived AAT with a recombinant version which is stable and resistant to oxidation. We have expressed AAT in Escherichia coli as inclusion bodies and developed a highly efficient refolding and purification process. We engineered a series of mutant forms of AAT to achieve enhance thermostability and oxidation resistance. Moreover, we synthesized an active form of AAT via cysteine-pegylation to achieve a markedly extended half-life in vivo. The resulting molecule, which retains comparable activity to the wild-type form, is expected to be an improved therapeutic agent for treating hereditary emphysema. In addition, the molecule may also be used to treat other types of emphysema caused by smoking, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease.

Crystal Structure of Tetrameric Arabidopsis MYC2 Reveals the Mechanism of Enhanced Interaction with DNA.

Jasmonates (JAs) are essential plant hormones that play important roles in the regulation of plant growth and the response to environmental stress. In the JA signaling pathway, the core transcription factors are a class of basic helix-loop-helix (bHLH) proteins, including MYC2, MYC3, and MYC4, that have different regulatory capacities. Here, we report the 2.7 Å crystal structure of the MYC2 bHLH domain complexed with G-box DNA, showing a cis-tetrameric structure. Biochemical assays confirmed that full-length MYC2 forms a stable homo-tetramer both in solution and in DNA-bound states, whereas MYC3 forms only a homodimer. Isothermal titration calorimetry (ITC) assays demonstrated that tetramerization enhanced DNA binding affinity, and fluorescence resonance energy transfer (FRET) assay indicated DNA looping potential of tetrameric MYC2. Luciferase assay further confirmed the importance of tetramerization in transcriptional regulation. Our studies provide a mechanistic explanation for the regulatory differences of MYC transcription factors.

Convenient and controllable preparation of a novel uniformly nitrogen doped porous graphene/Pt nanoflower material and its highly-efficient electrochemical biosensing.

By employing dopamine as a nitrogen source and reducing agent, the block copolymer P123 as a pore forming agent, and graphene oxide as a carbon precursor, we present, for the first time, a convenient and controllable approach to the preparation of a novel uniformly nitrogen doped porous graphene (N-PGR) material. Using the prepared N-PGR as the supporting material, a novel nitrogen doped porous graphene/Pt nanoflower material (Pt/N-PGR) was obtained by a green and simple method. The characterization results of scanning electron microscopy (SEM), element mapping, transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) demonstrate that Pt nanoflowers are uniformly dispersed on nitrogen doped porous graphene. Electrochemical measurements show that Pt/N-PGR-900/GCE exhibits improved electrocatalytic activity towards H2O2 reduction and glucose oxidation. Linear responses are found for H2O2 and glucose in the range of 0.5-40 326 µM and 0.5-133.5 mM with the detection limit (S/N = 3) of 0.2 µM and 0.05 mM, respectively. In addition, Pt/N-PGR-900/GCE exhibits high sensitivity and good anti-interference ability. The superior catalytic activity and selectivity make Pt/N-PGR a promising nanomaterial for application in electrochemical biosensing studies.

Rat and human STINGs profile similarly towards anticancer/antiviral compounds.

Cyclic dinucleotides (CDNs) and antitumor/antiviral agents (DMXAA and CMA) trigger STING-dependent innate immunity activation. Accumulative evidences have showed that DMXAA and CMA selectively activate mouse, but not human STING signaling. The mechanism underlying this species selectivity remains poorly understood. In this report, we have shown that human and rat STINGs display more similar signaling profiles toward DMXAA and CMA than that of human and mouse STINGs, suggesting that rat is more suitable for preclinical testing of STING-targeted drugs. We have also determined the crystal structures of both apo rat STING and its complex with cyclic GMP-AMP with 2'5' and 3'5' phosphodiester linkage (2'3'-cGAMP), a human endogenous CDN. Structure-guided biochemical analysis also revealed the functional importance of the connecting loop (A140-N152) between membrane and cytosolic domains in STING activation. Taken together, these findings reveal that rat STING is more closely related to human STING in terms of substrate preference, serving as a foundation for the development of STING-targeted drugs.

Structural and biochemical characterization of MdaB from cariogenic Streptococcus mutans reveals an NADPH-specific quinone oxidoreductase.

The smu.1420 gene from the cariogenic pathogen Streptococcus mutans encodes a putative protein which has sequence homology to NQO [ NAD(P)H: quinone oxidoreductase] family members, including mammalian NQO and bacterial MdaB (modulator of drug activity B). NQO can detoxify quinones by converting them to hydroquinones and prevent the generation of reactive oxygen species. Thus, comprehensive studies on Smu.1420 will be important for uncovering the antioxidation and antidrug mechanisms of S. mutans. Here, the catalytic properties of Smu.1420 have been characterized, and its structure was determined in complexes with NADP(+) and menadione, respectively. Smu.1420 binds menadione directly and exhibits a pronounced preference for NADPH over NADH as a substrate, demonstrating that it is an NADPH-specific quinone oxidoreductase. The structure of Smu.1420 shows a compact homodimer with two substrate pockets located in the cleft of the dimer interface. The nicotinamide moiety of NADP(+) is bound on top of the isoalloxazine moiety of the FAD cofactor and overlaps with the binding site of menadione, suggesting a hydride-transfer process from NADPH to FAD and then to menadione. Two strongly basic patches near the substrate pocket are expected to confer the preference for NADPH over NADH. These studies shed light on future drug development against the cariogenic pathogen S. mutans.

Structural genomics studies of human caries pathogen Streptococcus mutans.

Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.

The regulatory mechanism of the caspase 6 pro-domain revealed by crystal structure and biochemical assays.

Caspase 6 (CASP6) is a neuron degeneration-related protease and is widely considered to be a potential drug-design target against neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. The N-terminal pro-peptide of CASP6, also referred to as the pro-domain, contains 23 residues and its functional role remains elusive. In this study, the crystal structure of a full-length CASP6 zymogen mutant, proCASP6H121A, was solved. Although the pro-domain was flexible in the crystal, without visible electron density, structural analyses combined with biochemical assays revealed that the pro-domain inhibited CASP6 auto-activation by inhibiting intramolecular cleavage at the intersubunit cleavage site TEVD(193) and also by preventing this site from intermolecular cleavage at low protein concentration through a so-called `suicide-protection' mechanism. Further experiments showed that the length of the pro-domain and the side chain of Asn18 played critical roles in suicide protection. These results disclosed a new inhibitory mechanism of CASP6 and shed light on the pathogenesis and therapeutically relevant study of CASP6-related neurodegenerative diseases.

Structural and functional characterization of a novel α/ß hydrolase from cariogenic pathogen Streptococcus mutans.

The protein Smu.1393c from Streptococcus mutans is annotated as a putative α/ß hydrolase, but it has low sequence identity to the structure-known α/ß hydrolases. Here we present the crystal structure of Smu.1393c at 2.0 Å resolution. Smu.1393c has a fully open alkaline substrate pocket, whose conformation is unique among other similar hydrolase structures. Three residues, Ser101, His251, and Glu125, were identified as the active center of Smu.1393c. By screening a series of artificial hydrolase substrates, we demonstrated Smu.1393c had low carboxylesterase activity towards short-chain carboxyl esters, which provided a clue for exploring the in vivo function of Smu.1393c.

Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA.

CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7' base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.

Ellman's reagent in promoting crystallization and structure determination of Anabaena CcbP.

Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca2+-binding protein (CcbP). In this report, the promoting effect of Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid); DTNB] on the crystallization of CcbP is described. CcbP contains one free cysteine. A quick and simple oxidation reaction with DTNB blocked the free cysteine in purified CcbP and generated a homogenous monomeric protein for crystallization. The crystal structure of DTNB-modified CcbP was determined by the single-wavelength anomalous diffraction method. Structure analysis indicated that DTNB modification facilitated crystallization of CcbP by inducing polar interactions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.

Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans.

Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni(2+)-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Šresolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = ß = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Šresolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, ß = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing.

The crystal structure of the MPN domain from the COP9 signalosome subunit CSN6.

The COP9 signalosome (CSN) is a multiprotein complex containing eight subunits and is highly conserved from fungi to human. CSN is proposed to widely participate in many physiological processes, including protein degradation, DNA damage response and signal transduction. Among those subunits, only CSN5 and CSN6 belong to JAMM family. CSN5 possesses isopeptidase activity, but CSN6 lacks this ability. Here we report the 2.5Å crystal structure of MPN domain from Drosophila melanogaster CSN6. Structural comparison with other MPN domains, along with bioinformation analysis, suggests that MPN domain from CSN6 may serve as a scaffold instead of a metalloprotease.

Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays.

The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.

Structure of the bifunctional methyltransferase YcbY (RlmKL) that adds the m7G2069 and m2G2445 modifications in Escherichia coli 23S rRNA.

The 23S rRNA nucleotide m(2)G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass spectrometric analyses of 23S rRNAs showed that the N-terminal region of YcbY and Smu472 are functionally equivalent and add the m(2)G2445 modification, while the C-terminal region of YcbY is responsible for the m(7)G2069 methylation on the opposite side of the same helix (H74). Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs. The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL. Our structural and functional data provide insights into how this bifunctional enzyme evolved.

Get phases from arsenic anomalous scattering: de novo SAD phasing of two protein structures crystallized in cacodylate buffer.

The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA) and the human caspase-6 (Casp6), were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD) phasing method. Arsenic (As), an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V)). This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III)), in the presence of the reducing agent, dithiothreitol (DTT), and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography.

Crystal structures of cobalamin-independent methionine synthase (MetE) from Streptococcus mutans: a dynamic zinc-inversion model.

Cobalamin-independent methionine synthase (MetE) catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to l-homocysteine to form methionine. Previous studies have shown that the MetE active site coordinates a zinc atom, which is thought to act as a Lewis acid and plays a role in the activation of thiol. Extended X-ray absorption fine structure studies and mutagenesis experiments identified the zinc-binding site in MetE from Escherichia coli. Further structural investigations of MetE from Thermotoga maritima lead to the proposition of two models: "induced fit" and "dynamic equilibrium", to account for the catalytic mechanisms of MetE. Here, we present crystal structures of oxidized and zinc-replete MetE from Streptococcus mutans at the physiological pH. The structures reveal that zinc is mobile in the active center and has the possibility to invert even in the absence of homocysteine. These structures provide evidence for the dynamic equilibrium model.

High-resolution structure of a new crystal form of BamA POTRA4-5 from Escherichia coli.

In Escherichia coli, the BAM complex is employed to mediate correct folding of the outer membrane (OM) proteins into ß-barrels and their insertion into the OM. BamA, which is an essential component of the complex, consists of a C-terminal transmembrane region and five N-terminal polypeptide transport-associated (POTRA) domains. Although deletion studies have shown that each of the POTRA domains plays an important role in the process of BAM complex formation, only POTRA5 is essential for cell viability. Here, the crystal structure of POTRA4-5 has been determined to 1.50 Šresolution with an R factor of 14.7% and an Rfree of 18.9%.

Crystallization and preliminary X-ray analysis of argininosuccinate lyase from Streptococcus mutans.

Argininosuccinate lyase (ASL) is an important enzyme in arginine synthesis and the urea cycle, which are highly conserved from bacteria to eukaryotes. The gene encoding Streptococcus mutans ASL (smASL) was amplified and cloned into expression vector pET28a. The recombinant smASL protein was expressed in a soluble form in Escherichia coli strain BL21 (DE3) and purified to homogeneity by two-step column chromatography. Crystals suitable for X-ray analysis were obtained and X-ray diffraction data were collected to a resolution of 2.5 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 254.5, c = 78.3 Å.