Structural Features Of Keratin And Related Intermediate

中间丝是真核细胞细胞骨架中普遍存在的成分。它们由五种不同的类型组成，其中数量最多、结构最复杂的是在上皮细胞中广泛表达的I型和II型角蛋白。我们不仅对人类皮肤角蛋白IF的结构、功能、表达及其在角化病疾病中的作用感兴趣，而且还对其他类型细胞的相关IF感兴趣，以了解它们在生物学中的作用。正在进行的结构研究虽然角蛋白在许多遗传性疾病中的作用现在已经被很好地理解，但进一步的结构研究是必要的，以开发合理的治疗方法。我们与瑞士、德国和新西兰的其他研究人员合作，开始了一项重大研究，以利用X射线结晶学技术解决波形蛋白IF的三维结构。选择这些IF是因为：(A)它们是均聚的，因此可能在某种程度上更容易解决；以及(B)它们与角蛋白IF具有非常高的序列同源性，因此所引用的许多针对Vimentin的结构原理应该适用于角蛋白IF。到目前为止，我们已经解决了2B杆状结构域片段最后35个残基的结构，它包含了螺旋末端基序，以及成功组装IF所需的可能触发基序。目前正在进行许多其他构建物的研究，这些构建物累积覆盖了vientin的整个杆状结构域部分。三细胞角蛋白分子的组织如果我们之前已经通过详细的交联实验证明，成对的表皮角蛋白分子以三种方式排列，分别为A11，A22和A12。当被同化为IF时，同一轴线上的分子对采用第四个模式，称为ACN，其中一个分子的末端与相邻分子的起点重叠约1 nm。有趣的是，几乎所有已知的角膜病变突变/替换都位于这个重叠窗口中。我们还表明，III型分子采用相同的基本四模，但前三种分子的排列略有偏移。这充分解释了为什么III型和I/II型角蛋白链不能也不能在体内或体外共同组装。一年来，取得了以下进展： 1.完成并发表了角化过程中三细胞角蛋白中间丝结构变化的研究。 2.研究了波形蛋白头部结构域在波形蛋白中间丝组装中的作用。对大量缺失/突变结构的化学测序表明，N-末端100个氨基酸与分子的卷曲区域相互作用。预计不久将通过从合作实验室(加州大学戴维斯分校)获得光谱(电子顺磁共振)数据来进一步完善该结构，从而完成这项研究。 3.启动了一个关于发角蛋白头部结构域在毛发角蛋白中间丝组装中的作用的类似项目。构建了32个突变型和缺失型重组蛋白，并对其进行了纯化。通过化学测序，用其中两个结构物进行细丝组装的初步实验表明，头部和杆状结构域之间存在特定的氨基酸相互作用。将继续与德国和新西兰的合作者一起进一步完善数据。 4.已完成并发表了一项关于酪蛋白与毛囊中角蛋白细丝相互作用的研究。它包括接触点的确定和从毛囊内根鞘中分离的分子间交联肽的化学测序。

Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryote cells. They consist of five different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested in not only the structure, function and expression of keratin IF of human skin and their roles in keratinopathy diseases, but also of the related IF of other cell types in order to understand their roles in biology. Ongoing structural studies While the roles of the keratins in many genetic diseases are now well understood, further structural studies are necessary to develop rational approaches to therapy. We have initiated a major study in collaboration with other investigators in Switzerland, Germany and New Zealand to solve the three-dimensional structure of vimentin IF by use of Xray crystallographic techniques. These IF have been chosen because: (a) they are homopolymeric, and therefore likely to be somewhat simpler to solve; and (b) they have a very high sequence homology with keratin IF, and thus many of the structural principles adduced for vimentin should be applicable to keratin IF. To date, we have solved the structure of the last 35 residues of the 2B rod domain segment, which encompasses the helix-termination motif, and the likely trigger motif required for successful IF assembly. Work is now in progress on numerous other constructs have been made which cumulatively cover the entire rod domain portion of vimentin. The organization of molecules in trichocyte keratin IF We have previously demonstrated by detailed cross-linking experiments that pairs of epidermal keratin molecules are aligned in three modes termed A11, A22 and A12. When assimilated into IF, pairs of molecules in the same axial row adopt a fourth mode termed ACN, in which the end of one molecule overlaps the beginning of the adjacent molecule by about 1 nm. Interestingly, almost all known keratinopathy mutations/substitutions reside in this overlap window. We also have shown that the molecules of type III IF adopt the same basic four modes, but the alignments of the former three are slightly offset. This adequately explains why type III and types I/II keratin chains cannot and do not coassemble in vivo or in vitro. Over the past year, the following progress was made: 1. A study on structural changes in trichocyte keratin intermediate filaments during keratinization was completed and published. 2. The role of the head domain of vimentin in the assembly of vimentin intermediate filaments was investigated. Chemical sequencing of numerous deletion / mutation constructs have revealed interactions of the N-terminal 100 amino acids with the coiled-coiled region of the molecule. Further possible refinement of the structure is expected shortly by the acquisition of spectroscopic (electron paramagnetic resonance) data from a collaborating laboratory (University of California at Davis) leading to completion of this study. 3. A similar project on the role of the head domain of hair keratins in the assembly of hair keratin intermediate filaments was initiated. 32 mutant and deletion constructs were created and the proteins have been purified. Initial experiments of filament assembly with two of the constructs revealed, by chemical sequencing, specific amino acid interactions between the head and rod domains. Work will continue to further refine the data with collaborators in Germany and New Zealand. 4. A study on the interaction of trychohyalin with the keratin filaments in the hair follicle has been completed and published. It involved determination of contact points and chemical sequencing of isolated intermolecular cross-linked peptides from the inner root sheath of the hair follicle.