Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase

The OspF family of phosphothreonine lyase, including SpvC from Salmonella, irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination. We determined crystal structures of SpvC and its complex with a phosphopeptide substrate. SpvC adopts a unique fold of alpha/beta type. The disordered N terminus harbors a canonical D motif for MAPK substrate docking. The enzyme-substrate complex structure indicates that recognition of the phosphotyrosine followed by insertion of the threonine phosphate into an arginine pocket places the phosphothreonine into the enzyme active site. This requires the conformational flexibility of pT-X-pY, which suggests that p38 (pT-G-pY) is likely the preferred physiological substrate. Structure-based biochemical and enzymatic analysis allows us to propose a general acid/base mechanism for beta elimination reaction catalyzed by the phosphothreonine lyase. The mechanism described here provides a structural understanding of MAPK inactivation by a family of pathogenic effectors conserved in plant and animal systems and may also open a new route for biological catalysis.     

Dithiothreitol decreases in vitro activity of ADP-glucose pyrophosphorylase from leaves of apple (Malus domestica Borkh.) and many other plant species

Inclusion of dithiothreitol (DTT) in the extraction buffer and pre-incubation of apple leaf ADP-glucose pyrophosphorylase (AGPase) with DTT resulted in a decrease in AGPase activity whether the assay was performed in the presence or absence of 3-phosphoglycerate (PGA). When PGA was included in the pre-incubation mixture or when pre-incubation of AGPase with PGA was followed by DTT, the latter did not cause any decrease in AGPase activity. However, once AGPase was decreased by DTT, subsequent incubation of the enzyme with PGA did not reverse the decrease. Pre-incubation of AGPase from leaves of Arabidopsis thaliana, sorghum, soybean, tobacco, spinach, wheat, barley, tomato and potato, and tubers of potato with DTT, generally caused a decrease in AGPase activity when assayed in the presence of PGA. When assayed in the absence of PGA, however, a diverse response of AGPase was observed among species to pre-incubation with DTT. The activity of AGPase from potato tubers was increased by DTT; the activity of AGPase from both potato and tomato leaves was not affected by DTT; the activity of AGPase from leaves of other species was decreased by DTT. It is concluded that DTT decreases in vitro activity of AGPase from leaves of apple and many other plant species such that DTT should not be routinely included in the extraction or assay mixture of leaf AGPase.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

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紧密连接蛋白claudins应用于肿瘤治疗的进展

Claudins蛋白家族是组成紧密连接(Tight junctions,TJs)必不可少的骨架蛋白,在维持上皮和内皮细胞中的细胞极性、细胞间的粘附固定、细胞旁路的离子运输等发挥重要作用。近年来大量的研究结果证明,claudins在许多人类恶性肿瘤中异常表达。因此,claudins也被作为癌症治疗的潜在靶标。文中就claudin蛋白家族在肿瘤中的表达情况及其相关药物的研究进展进行阐述。     

Epidemiological Characteristics of Influenza A and B in Macau, 2010–2018

Virologica Sinica - Influenza is one of the major respiratory diseases in humans. Macau is a tourist city with high density of population and special population mobility. The study on the...     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

Effect of surgical castration on expression of TRPM8 in urogenital tract of male rats

Trpm8 (melastatin-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily, encoding a cation channel named TRPM8, has been shown to be a primary androgen-responsive gene and play an important role in prostate physiology. To investigate the expression feature of TRPM8 in urogenital tract of male rats, and whether TRPM8 was also regulated by androgen receptor in these organs, male Sprague–Dawley rats were divided into three groups of 35 animals as follows: sham-operated (SHAM), orchidectomized (ORX), orchidectomized plus DHT treatment (O + D). Organs in urogenital tract, including kidney, prostate, seminal vesicle (SV), testis, epididymis and penis, were collected at different post-castration periods. RT-PCR, real-time PCR and Western blotting were used to detect the expression of androgen receptor (AR) and trpm8 in these tissue. As a result, AR and trpm8 can be detected in all these organs at mRNA or/and protein level. The mRNA expression of trpm8 in kidney, prostate, SV and penis decreased 24 or 72 h after castration and kept decreasing in a time-dependant manner. However, treatment of dihydrotestosterone (DHT) could reverse the effect of surgical castration. Collectively, our data provide evidence that TRPM8 and AR were expressed generally in urogenital tract of male rats, and in these organs, expression of trpm8 was regulated by serum androgen.     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells

Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.