Hydrophilic aromatic residue and in silico structure for carbohydrate binding module

Carbohydrate binding modules (CBMs) are found in polysaccharide-targeting enzymes and increase catalytic efficiency. Because only a relatively small number of CBM structures have been solved, computational modeling represents an alternative approach in conjunction with experimental assessment of CBM functionality and ligand-binding properties. An accurate target-template sequence alignment is the crucial step during homology modeling. However, low sequence identities between target/template sequences can be a major bottleneck. We therefore incorporated the predicted hydrophilic aromatic residues (HARs) and secondary structure elements into our feature-incorporated alignment (FIA) algorithm to increase CBM alignment accuracy. An alignment performance comparison for FIA and six others was made, and the greatest average sequence identities and similarities were achieved by FIA. In addition, structure models were built for 817 representative CBMs. Our models possessed the smallest average surface-potential z scores. Besides, a large true positive value for liagnd-binding aromatic residue prediction was obtained by HAR identification. Finally, the pre-simulated CBM structures have been deposited in the Database of Simulated CBM structures (DS-CBMs). The web service is publicly available at http://dscbm.life.nthu.edu.tw/ and http://dscbm.cs.ntou.edu.tw/.     

Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.     

Hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread

Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues.     

Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes

Introduction

Synovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress.

Methods

Human chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm²). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes.

Results

Stimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression.

Conclusions

These data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.     

All-trans-retinoid acid induces the differentiation of P19 cells into neurons involved in the PI3K/Akt/GSK3β signaling pathway

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III β-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3β (GSK3β), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3β, phosphorylated GSK3β, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3β signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.     

HCoV-NL63N蛋白不同片段的表达纯化及血清学检测应用分析

将HCoV-NL63核衣壳蛋白N端(Np1~154aa)、C端(Cp141~306aa)基因片段克隆到原核表达载体pET30a(+)上进行原核表达,制备相应的纯化蛋白Np、Cp蛋白,利用Np、Cp蛋白建立基于Western-Blot条带印迹的HCoV-NL63抗体检测法,并与基于全长N蛋白(Nf)的HCoV-NL63抗体检测法相平行筛查了50份成年体检血清。结果显示:50份成年体检血清中,采用Nf、Np、Cp分别检出25、27、36份HCoV-NL63抗体阳性血清,检出率分别为50%、54%、72%。不同N蛋白与血清反应抗体阳性谱存在差异,其中Np与Nf检出一致率为64%,Cp与Nf检出一致率为54%,而Np与Cp检出一致率为54%。本研究表明人冠状病毒NL63在我国人群中感染常见,N蛋白C端(Cp)检出率比全长N(Nf)及N端(Np)要高,Nf、Np、Cp在抗体检测上存在不一致性。这为HCoV-NL63血清学试剂研发及免疫学研究提供了依据与实验基础。