分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
Zhang, Jingjing
Yang, Zhenguo
Chen, Huapu
Wang, Sijie
Fan, Yiming
Zhang, Jingjing
Piontek, Anna
Qin, Zhihai
Piontek, Joerg
Ni, Chen
摘要： Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE(194-319)) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE(194-319) removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE(194-319). Incubation of zebrafish larvae with cCPE(194-319) removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE(194-319) incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE(194-319) could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an invivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.
分类： 生物学 >> 生物物理学 >> 细胞生物学 提交时间： 2016-05-12
Jia, Chenjun
Li, Mei
Zhang, Hongmei
Cao, Peng
Pan, Xiaowei
Chang, Wenrui
Li, Jianjun
Zhang, Jingjing
Lu, Xuefeng
Jia, Chenjun
Zhang, Jingjing
摘要： The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of C-n fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.
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