Date of Award

January 2021

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Christopher Bunick

Abstract

Intermediate filaments (IFs) are a group of proteins with diverse functions in the body including structural integrity, cell signaling, and motility. Furthermore, IFs are implicated in the pathogenesis of cancer metastasis. There is a significant gap in our knowledge of how IF protomers form mature filaments due to the limited availability of atomic-resolution structural data describing the higher-order assembly of IFs. Herein, we report the crystal structure of the 1B subdomain of the keratin 1-keratin 10 (K1/K10-1B) heterotetramer. K1/K10 are the most abundant keratin pair in differentiated epidermis. This structure captures the first physiologic keratin tetramer and reveals a conserved hydrophobic pocket and anchoring knob interaction that drives tetramerization. The knob-pocket interaction was characterized using site-directed alanine mutagenesis, light scattering, electron microscopy and cell transfection to show that the knob-pocket mechanism is necessary for IF assembly. Furthermore, we demonstrate that an edge-to-face aromatic-aromatic interaction is central to driving the knob-pocket interaction. Targeting of the knob-pocket was studied to explore the feasibility of finding a drug to treat cancer metastasis by disrupting IF assembly, and preliminary success was achieved using a peptide that was observed by electron microscopy to inhibit in vitro filament assembly and successfully inhibited filament assembly in cultured cells.Two approaches were implemented to use the K1/K10-1B structure to gain insight into the molecular mechanism of hereditary IF disease pathogenesis: i) proteins containing pathogenic mutations were crystallized and compared to the wild-type structure, and ii) the wild-type structure was used to computationally model the mutated protein structure. Mutations associated with palmoplantar keratoderma, epidermolysis bullosa, pachyonychia congenita, hair disease, liver disease, and cataracts were studied. The resulting crystal structures and models revealed two common molecular mechanisms of mutation pathogenesis: changes in hydrophobic interactions leading to disrupted dimer or tetramer interfaces, and changes in surface charge leading to disrupted higher order filament assembly. The biochemical and structural basis behind dermatology drug mechanisms was also investigated. Existing structural data describing the binding of drugs to their targets was used to correlate the structures of biologic drugs with their clinical use in the treatment of psoriasis. The target epitopes of inhibitors of tumor necrosis factor alpha (TNFα), interleukin-17 (IL-17), and interleukin-23 (IL-23) were analyzed for binding surface hydrophobicity, charge, and location. Differences in the binding locations among different TNFα inhibitors and IL-23 inhibitors were found to correlate with clinical efficacy and side effects. The epitope charges significantly differed between the acidic TNFα inhibitor epitopes and the basic IL-17 inhibitor epitopes. Unanswered questions about the specific epitope locations of IL-17A and IL-23 inhibitors also highlighted the need for more complete structural identification of biologic drugs and their targets. As a whole, the work in this thesis advances our understanding of the structural and biochemical basis of how IFs contribute to skin function and disease, how IFs can be targeted for novel drug development, and how dermatologic drugs function.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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