Structural Features Of Keratin And Related Intermediate

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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.