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1. chinaXiv:201605.01801 [pdf]

Expanding the genetic code for site-specific labelling of tobacco mosaic virus coat protein and building biotin-functionalized virus-like particles

Wu, F. C.; Zhou, Q.; Wu, M.; Tian, Y.; Niu, Z. W.; Huang, Y.; Wu, F. C.; Zhou, Q.; Wu, F. C.; Ballard, Z.; Zhang, H.; Wang, J. Y.
Subjects: Biology >> Biophysics

A method for site-specific and high yield modification of tobacco mosaic virus coat protein (TMVCP) utilizing a genetic code expanding technology and copper free cycloaddition reaction has been established, and biotin-functionalized virus-like particles were built by the self-assembly of the protein monomers.

submitted time 2016-05-18 Hits3172Downloads1328 Comment 0

2. chinaXiv:201605.01493 [pdf]

Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay

Men, Dong; Zhou, Juan; Zhang, Zhi-Ping; Cui, Zong-Qiang; Deng, Jiao-Yu; Wang, Dian-Bing; Zhang, Xian-En; Men, Dong; Zhang, Ting-Ting; Hou, Li-Wei; Shi, Yuan-Yuan; Zhang, Jin-Li
Subjects: Biology >> Biophysics

The self-assembly of nanoparticles into larger superstructures is a powerful strategy to develop novel functional nanomaterials, as these superstructures display collective properties that are different to those displayed by individual nanoparticles or bulk samples. However, there are increasing bottlenecks in terms of size control and multifunctionalization of nanoparticle assemblies. In this study, we developed a self-assembly strategy for construction of multifunctional nanoparticle assemblies of tunable size, through rational regulation of the number of self-assembling interaction sites on each nanoparticle. As proof-of-principle, a size-controlled enzyme nanocomposite (ENC) was constructed by self-assembly of streptavidin-labeled horseradish peroxidase (SA-HRP) and autobiotinylated ferritin nanoparticles (bFNP). Our ENC integrates a large number of enzyme molecules, together with a streptavidin-coated surface, allowing for a drastic increase in enzymatic signal when the SA is bound to a biotinylated target molecule. As result, a 10000-fold increase in sensitivity over conventional enzyme-linked immunosorbent assays (ELISA) methods was achieved in a cardiac troponin immunoassay. Our method presented here should provide a feasible approach for constructing elaborate multifunctional superstructures of tunable size useful for a broad range of biomedical applications.

submitted time 2016-05-12 Hits1758Downloads1068 Comment 0

3. chinaXiv:201605.01443 [pdf]

Insights into the interaction between Cren7 and DNA: the role of loop beta 3-beta 4

Zhang, Zhenfeng; Li, Hongbin; Huang, Li; Gong, Yong; Chen, Yuanyuan
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Sulfolobus synthesizes large amounts of small chromatin proteins Cren7 and Sul7d. The two proteins share overall structural similarity, but differ distinctly in the DNA-binding region between beta 3- and beta 4-strands. While Sul7d possesses a hinge of two amino acid residues, Cren7 contains a flexible seven-residue loop (loop beta 3-beta 4) in the region. Here, we report the role of loop beta 3-beta 4 in the interaction of Cren7 with duplex DNA. We show that all residues with a large side chain on the loop, i.e., Pro30, Lys31, Arg33 and Lys34, contributed significantly to the binding of Cren7 to DNA. The three basic amino acids affected the ability of Cren7 to constrain negative DNA supercoils in a residue number-dependent manner. The crystal structure of a complex between a mutant Cren7 protein (GR) with loop beta 3-beta 4 replaced by two residues (Gly and Arg) to mimic the hinge at the corresponding position in Sul7d and an 8-bp dsDNA has been determined. Structural comparison between the GR-DNA and Cren7-DNA complexes shows that GR resembles Sul7d more than Cren7 in DNA-binding size and in the effect on the width of the major groove of DNA and the pattern of DNA bending. However, GR induces smaller DNA curvature than Sul7d. Our results suggest that Cren7 and Sul7d package chromosomal DNA in a slightly different fashion, presumably permitting different chromosomal accessibility by proteins functioning in DNA transactions.

submitted time 2016-05-12 Hits7076Downloads984 Comment 0

4. chinaXiv:201605.01352 [pdf]

Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling

Yuan, Tianyi; Liu, Lin; Wei, Lisi; Zhao, Shiqun; Zheng, Xiaolu; Huang, Xiaoshuai; Xie, Lihan; Zong, Weijian; Yang, Lu; Liu, Yanmei; Chen, Liangyi; Yuan, Tianyi; Liu, Lin; Wei, Lisi; Zhao, Shiqun; Zheng, Xiaolu; Huang, Xiaoshuai; Xie, Lihan; Zong, Weijian; Yang, Lu
Subjects: Biology >> Biophysics >> Cell Biology

Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (similar to 10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of preformed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5 beta/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations.

submitted time 2016-05-11 Hits1505Downloads885 Comment 0

5. chinaXiv:201605.01195 [pdf]

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

Zhang, Hai-zhen; Chen, Hua-yan; Nan, Fa-jun; Xu, Xiao-lan; Ali, Sher; Wang, Dan; Yu, Jun-wei; Xu, Tao
Subjects: Biology >> Biophysics

Aim: Ca2+-release-activated Ca2+ (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors. Methods: HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca2+ influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP. Results: A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl) urea inhibited Ca2+ influx with IC50 of 3.25 +/- 0.17 mu mol/L. SAR study on its derivatives showed that the alkyl substituent on the a-position of the left-side benzylic amine (R1) was essential for Ca2+ influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N-H of urea was not necessary to maintain the high potency of Ca2+ influx inhibition. The N, N'-disubstituted or N'-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity. Conclusion: 1-Phenyl-3-(1-phenylethyl) urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca2+ influx inhibition, immune inhibition and low cytotoxicity.

submitted time 2016-05-11 Hits1388Downloads776 Comment 0

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