Structural Features Of Keratin And Related Intermediate

中间丝是真核生物细胞骨架的重要组成部分。它们由五种不同的类型组成，其中数量最多和最复杂的是广泛表达于上皮中的I型和II型角蛋白。我们感兴趣的不仅是人皮肤角蛋白IF的结构、功能和表达及其在角蛋白病疾病中的作用，而且还对其他细胞类型的相关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已得到充分了解，但仍有必要进行进一步的结构研究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，通过使用X射线晶体学技术来解决波形蛋白IF的三维结构，从而启动了一项重大研究。选择这些IF是因为：（a）它们是均聚的，因此可能更容易求解;和（B）它们与角蛋白IF具有非常高的序列同源性，因此为波形蛋白提出的许多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杆结构域片段的最后35个残基的结构，其中包括螺旋终止基序，以及成功的IF组装所需的可能的触发基序。目前正在进行的工作是许多其他的构建体已累积覆盖整个杆域部分的波形蛋白。我们先前已经通过详细的交联实验证明了表皮角蛋白分子对以三种模式排列，称为A11，A22和A12。当同化到IF中时，在同一轴向行中的分子对采用称为ACN的第四模式，其中一个分子的末端与相邻分子的开始重叠约1 nm。有趣的是，几乎所有已知的角蛋白病突变/置换都存在于该重叠窗口中。我们还表明，III型IF分子采用相同的基本四个模式，但前三个的排列略有偏移。这充分解释了为什么III型和I/II型角蛋白链不能在体内或体外共组装。在过去一年中，取得了以下进展： 1.完成并发表了一项关于角化过程中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。许多缺失/突变构建体的化学测序已经揭示了N-末端100个氨基酸与分子的卷曲螺旋区的相互作用。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，预计不久将对结构进行进一步可能的细化，从而完成本研究。 3.一个类似的项目的头发角蛋白的头部结构域的头发角蛋白中间丝的组装中的作用开始。构建了32个突变体和缺失构建体，并纯化了蛋白质。通过化学测序，用两个构建体进行的细丝组装的初步实验揭示了头部和杆结构域之间的特定氨基酸相互作用。将继续与德国和新西兰的合作者一起进一步完善数据。 4.已经完成并发表了一项关于trychohyalin与毛囊中角蛋白丝相互作用的研究。它涉及接触点的测定和从毛囊内根鞘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.