Molecular function of an intermediate filament assembly mechanism in epidermal protein complexes and cell migration

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 novel molecular
target for developing intermediate filament-specific therapies that prevent or treat human diseases.

Grant Number: 5R01AR079428-05
NIH Institute/Center: NIH
Principal Investigator: Christopher Bunick