PROJECT SUMMARY There exists a fundamental gap at the molecular level in understanding how intermediate filament proteins as- semble into mature filaments. Continued existence of this gap is an important problem because, until this need is met, understanding of how intermediate filaments function in large macromolecular complexes and how path- ologic mutations alter filament assembly will remain elusive. The long-term goal is to intelligently manipulate and target intermediate filament systems for the prevention and treatment of human diseases. The objective in this application is to define the molecular mechanisms that enable intermediate filament proteins to assemble into mature filaments and to interact with epidermal proteins in large macromolecular complexes. The central hy- pothesis is that the knob-pocket tetramerization mechanism in intermediate filaments drives mature filament formation and facilitates the assembly of cutaneous protein complexes critical for establishing skin barrier integ- rity. This hypothesis is generated from preliminary data produced in the applicant’s lab. The rationale for the proposed research is that, once intermediate filament assembly and protein interaction mechanisms are molec- ularly characterized, intermediate filament networks can be specifically targeted and pharmacologically regu- lated for the clinical benefit of patients currently without adequate therapies. Supported by robust preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine how 1B knob-pocket interactions regulate assembly of mature intermediate filaments; and 2) Elucidate the molecular basis of keratin interactions with desmoplakin and filaggrin. Under the first aim, a combination of mutagenesis, light scattering, filament as- sembly assays coupled with negative stain electron microscopy, x-ray crystallography, and transient transfection cell biology will be used to establish the molecular functions of the “knob-pocket” tetramerization mechanism located in the 1B domain of intermediate filaments. Under the second aim, binding assays, mutagenesis, iso- thermal titration calorimetry, x-ray crystallography, electron microscopy, and cellular adhesion and migration assays will be used to characterize the molecular interfaces driving keratin intermediate filaments to form com- plexes with desmoplakin and filaggrin in human epidermis. The approach is innovative because it takes a new intermediate filament assembly mechanism discovered in the applicant’s lab and examines previously unex- plored questions about how this mechanism contributes to human skin barrier integrity through protein complex formation in the epidermis and through regulation of cell migration. It is also innovative because it utilizes novel peptides developed in the applicant’s lab to probe intermediate filament functions in cells. The proposed re- search is significant because it will advance the knob-pocket tetramerization mechanism as a nov...