Hsc70-Auxilin复合物的拉伸分子动力学模拟

Hsc70与auxilin蛋白组成的系统是Hsp70/Hsp40分子伴侣系统家族的一员,在热休克反应中发挥重要作用。本文为得出auxilin蛋白J结构域的关键氨基酸,首先采用由二硫键交联的Hsc70 R171C与auxilin D876C的复合物结晶结构作为初始模型,进行分子动力学模拟,通过比较平衡后的结合部位发现,将形成二硫键的氨基酸突变为原来的氨基酸结构在结合位点上与生化结果较为相近,之后利用此结构通过拉伸动力学模拟分析了auxilin蛋白J结构域与Hsc70的ATPase功能域的解离过程,并探讨了Hsc70与auxilin蛋白之间的相互作用力。结果表明位于HPD loop上的His874,Asp876,Thr879,螺旋Ⅲ上的Glu884,Asn895,Asp896,Ser899,Glu902,Asn903为关键氨基酸,这些数据符合之前核磁共振实验证实的T抗原J结构域的HPD基序和螺旋Ⅲ与Hsc70的ATPase功能域之间的相互作用。     

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.     

一种基于信息理论的距离系数

给出了一种基于信息理论的距离系数,这一新的信息系数是通过对信息论中的离散增量系数改进而得,并证明其满足距离系数的三个性质．将其应用于一组同源辅助蛋白的聚类分析,表明是可行的．与离散增量系数及经典的欧氏距离系数的聚类结果进行比较,应用相干系数对聚类结果进行评价,结果表明由新信息距离系数所确定的聚类结构与聚类数据问的拟合程度最好.     

Swa2p-dependent clathrin dynamics is critical for Flo11p processing and ‘Mat’ formation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to form complex multicellular structures called mats on low-density agar Petri plates. Mat formation strictly depends on Flo11p, a cell surface mannoprotein that mediates the adhesion of yeast cells to the agar surface. Here, we show that Swa2p, an auxilin ortholog required for clathrin-coated vesicle uncoating, is strictly required for biofilm formation. We show that the maturation and cellular levels of Flo11p are affected in Δswa2 cells, yet without compromising invasive growth. Both the TPR and J-domains of Swa2p, but not its clathrin-binding and ubiquitin-association motifs, are required for its function in Flo11p processing.