New functions for the Snail family of transcription factors: Two-faced proteins

Comment on: Yang D, et al. Cell Cycle 2010; 9:2789-802.     

Method to identify hexahistidine fusion proteins in crude cellular lysate

Summary The strong interaction of chelated nickel ions with a hexahistidine peptide was utilized to identify hexahistidine fusion proteins in crude cellular lysates. The technique involves a Ni²⁺-nitrilotriacetic acid derivative labelled with alkaline phosphatase. The new reagent was used to probe for hexahistidine fusion proteins after SDS-PAGE electrophoresis and blotting onto nitrocellulose. The minimal amount of fusion protein which could be detected with this method was 0.1 g.     

植物HD-Zip转录因子的生物学功能

HD-Zip转录因子属于Homeobox蛋白家族, 是植物特异转录因子, 由高度保守的HD(Homeodomain)结构域和Leu zipper(Zip)元件组成, 前者与DNA特异结合, 后者介导蛋白二聚体的形成。HD-Zip转录因子家族包括4个亚家族(HD-Zip Ⅰ-Ⅳ), 其成员通过与其他蛋白互作、参与激素介导的信号途径, 从而调控植物生长发育、光形态建成、花发育、果实发育和植物对逆境应答等生物学过程。文章对近几年关于植物HD-Zip转录因子参与上述生物学功能方面的研究进行了综述, 以期对新功能基因的挖掘和应用研究以及HD-Zip调控机制的阐明奠定基础。     

Imaging the impact of chemically inducible proteins on cellular dynamics in vivo

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters.     

Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins

SNARE proteins function at the center of membrane fusion reactions by forming complexes with each other via their coiled-coil domains. Several SNAREs have N-terminal domains (NTDs) that precede the coiled-coil domain and have critical functions in regulating the fusion cascade. This review will highlight recent findings on NTDs of syntaxins, the longin domain of VAMP proteins and SNAP-23/25 homologues in yeast. Biochemical and genetic experiments as well as the resolution of several NMR and crystal structures of SNARE NTDs shed light on their diverse function.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.