Title

Structure-Function Relationship of Keratins in the Context of Epidermolysis Bullosa Simplex and Cancer

Alternative Title

단순 수포성 표피박리증과 암에서 케라틴의 구조-기능 상관관계 연구

DGIST Authors

Jewoo Kim ; Chang-Hun Lee ; Jin Hae Kim

Advisor

이창훈

Co-Advisor(s)

Jin Hae Kim

Issued Date

2025

Awarded Date

2025-02-01

Citation

Jewoo Kim. (2025). Structure-Function Relationship of Keratins in the Context of Epidermolysis Bullosa Simplex and Cancer. doi: 10.22677/THESIS.200000846149

Type

Thesis

Description

Keratin 5/14 (K5/K14), Keratin 8/19 (K8/K19), Epidermolysis Bullosa Simplex (EBS), Cancer, GSK3β, 14-3-3, Phosphorylation, Filament assembly, Protein interactions

Table Of Contents

Ⅰ. Introduction 1
1.1 Keratin5/Keratin14 and Epidermolysis Bullosa Simplex (EBS) 2
1.2 Keratin8/Keratin19 and Cancer 4
II. Methods and Materials 6
2.1 EBS mutant scoring and correlation analysis 6
2.1.1 Homology modeling by MODELLER 6
2.1.2 Aggregation scoring and correlation analysis 8
2.2 Plasmids and Protein expression 8
2.2.1 Mutagenesis and cloning 8
2.2.2 Transformation and protein expression 10
2.3 Protein purification 11
2.3.1 Purification of keratin 11
2.3.2 Purification of GSK3β and GST 12
2.3.3 Purification of 14-3-3 13
2.4 Assembly of Keratin filament 13
2.5 Assessing keratin filament assembly and bundling 14
2.5.1 Sedimentation assay 14
2.5.2 Transmission Electron Microscopy (TEM) 14
2.6 Measurement of material properties of K5/K14 15
2.7 Binding assay of K8/K19 filament with other protein 16
2.7.1 Co-sedimentation assay 16
2.7.2 Nuclear Magnetic Resonance (NMR) 16
III. Results 17
3.1 K5/K14 filaments show altered material properties in EBS mutants 17
3.1.1 Homology modeling 17
3.1.2 Correlation analysis between aggregation score and severity 21
3.1.3 Mutagenesis results 35
3.1.4 Protein purification 36
3.1.5 Sedimentation assay 38
3.1.6 TEM 39
3.1.7 Rheology Analysis 41
3.2 Functional differences between K8/K19 and K8/K19(S35E) 43
3.2.1 Protein purification 43
3.2.2 Sedimentation Assay 47
3.2.3 Comparison of wild-type and mutants (TEM, NMR) 49
3.2.4 Interaction with 14-3-3 (TEM, NMR, co-Sedimentation) 51
3.2.5 Interaction with GSK3β (TEM, NMR, co-Sedimentation) 56
IV. Discussion and Conclusion 61
V. Acknowledgement 65
VI. References 66
Abstract in Korean 70

URI

http://hdl.handle.net/20.500.11750/58014
http://dgist.dcollection.net/common/orgView/200000846149

DOI

10.22677/THESIS.200000846149

Degree

Master

Department

Department of New Biology

Publisher

DGIST

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