https://scholar.dgist.ac.kr/handle/20.500.11750/11739 2026-04-04T13:10:21Z 2026-04-04T13:10:21Z Yeom, Jiwoo Kim, Suji Lee, Chang-Hun https://scholar.dgist.ac.kr/handle/20.500.11750/57688 2025-07-25T04:24:55Z 2024-03-23T15:00:00Z

Title: Keratin 17 tail domain has a role in keratin 6/17 filament formation Author(s): Yeom, Jiwoo; Kim, Suji; Lee, Chang-Hun Abstract: Keratins are the largest subgroup of intermediate filament (IF) proteins, which form 10-nm filaments with type I/II heterodimers in epithelial cells. The keratin 6 (K6) and keratin 17 (K17) pairs (K6/K17) are stress-induced keratins specifically expressed when wounds occur on the skin and play a key role in driving wound healing. To perform these functions, keratins require the assembly and bundling of filaments. It has been known that the rod domain plays a major role in forming this filament structure, and tail domains are not required for filament formation. For example, a tail deletion mutant of the K14, the basal layer-specific keratin, can form 10-nm filaments indistinguishable from wild-type keratins. In this study, however, we suggest that the K17 tail domain (K17T) plays a significant role in filament structure formation in the case of K6/K17. In this report, we used recombinant human proteins, including wildtype K6, wildtype K17, K17 rod domain (K17R), and tail-less K17 (K17ΔT), which were purified and reconstituted as described in previous works of literature. We conducted in vitro filament assembly studies on K6/K17, K6/K17R, and K6/K17ΔT pairs. To verify the abilities for filament structure formation among the groups, a high-speed sedimentation assay with Air-driven ultracentrifuge (Airfuge) was used. Using Transmission Electron Microscope (TEM), we directly observed the filament formation patterns of each group. Also, to elucidate the mechanism of how K17T influences K6/17 filament formation, we conducted a binding assay for K17T using a cross-linker, disuccinimidyl suberate (DSS). Furthermore, for structural analysis of K17T, NMR experiments with 13C- & 15N-labeled K17T were conducted. In the high-speed sedimentation assay using Airfuge, the K6/K17 group showed that most of the proteins were retrieved from the pellet. However, the K6/K17ΔT group displayed only about half of the proteins in the pellet. The K6/K17R group also revealed that most of the proteins were found in the supernatant. Using TEM, we observed that the K6/K17 group exhibited an elongated filament structure, while the K6/K17ΔT group displayed truncated filaments with the presence of spherical particles. In the case of the K6/K17R group, it did not exhibit any filamentous structures, only spherical particles. In the binding assay for K17T using the cross-linker DSS, K17T formed self-dimerization. In the structural analysis using NMR experiments, we discovered that K17T exhibited a high level of disorder. In the High-speed sedimentation assay, the majority of K6/K17 proteins were localized in the pellet, whereas the K6/K17ΔT group exhibited about half of the proteins in the pellet. This suggests that although the keratin 17 tail is not indispensable, it significantly influences keratin filament formation, as also confirmed by TEM imaging. TEM observations further confirmed that the K6/K17R pair failed to generate filaments and only formed particles, highlighting the insufficiency of the K17 rod alone for K6/K17 filament assembly. Additionally, with the presence of shorter filaments and spherical particles in the K6/K17ΔT group, we can infer the contributory roles of the K17T in both filament formation and stabilization. As the mechanism of how the K17T stabilizes the K6/K17 filament structures, we suggest interactions between the K17T, as confirmed by the binding assay with DSS. We also conducted a structural analysis for the K17T, and we have confirmed that K17T exhibits intrinsically disordered protein (IDP) characteristics.

2024-03-23T15:00:00Z Jeong, Jinju Kwon, Mi Kyung Nam, Yongho Lee, Chang-Hun Lee, Young-Sam https://scholar.dgist.ac.kr/handle/20.500.11750/57687 2025-07-25T02:42:52Z 2024-03-24T15:00:00Z

Title: The In vitro reconstituted the lens-specific intermediate filament with filensin and phakinin replicates the genotype-phenotype correlation for cataracts Author(s): Jeong, Jinju; Kwon, Mi Kyung; Nam, Yongho; Lee, Chang-Hun; Lee, Young-Sam Abstract: The intermediate filaments (IFs) family is one of the cyto-skeletons that regulate cell shape, size, stiffness, and movement. Unlike other cytoskeletons such as actin and tubulin, alpha-helical linear proteins of the IFs family constitute filament formation, which can be classified in six types depending on their assembly mechanisms. Filensin and phakinin, which are classified into the type-VI IFs, are only expressed in lens fiber cells and are composed of the lens-specific IFs. However, it is unclear how filensin and phakinin constitute filaments and have an impact on lens properties. Here, we studied the in vitro molecular assembly of human filensin and phakinin to identify the structural and functional relationships. We reconstituted the co-assembled filaments with human recombinant filensin and phakinin and determined its stoichiometry as the ratio of one-to-one. Filensin and phakinin filaments interacted with alpha-crystallin and assembled to make a beeded structure detected by the sedimentation assay and TEM. Moreover, the cataract disease mutant phakinin E233del caused short filaments and reduced resistance against heat and shear stress. We further showed that the alpha-helical rod domains in each protein are involved in the interaction between two proteins, and the intrinsically disordered head and tail domains regulates filament extension. Overall, we determined the molecular interaction in the lens-IFs, which confers the crystalline lens with stability against physical stress. These results suggest that the impaired integrity of the IF can lead to age-related diseases like cataracts and presbyopia.

2024-03-24T15:00:00Z