Antigen presentation by MHC class II molecules represents a key step in the development of T cell mediated autoimmune diseases. The peptide repertoire presented to CD4 T cells is shaped by the action of HLA-DM, a protein with an MHC-like fold that acts as an enzyme which accelerates peptide exchange. The major goals of the project are to define the mechanism by which DM catalyzes peptide exchange and to examine whether low affinity peptides involved in autoimmune processes can be selectively removed by increasing the editing function of DM. We have identified four small molecules that accelerate DM-catalyzed peptide exchange and increase the activity of this enzyme. These small molecules represent unique tools to examine relevant structural and biological properties of DM. The focus of Aim 1 is to determine the structural basis of DM-catalyzed peptide exchange.
We aim to determine the structure of DM with one of the small molecule accelerators in order to define the small molecule binding site and the conformational change that increases the activity of the enzyme. This structural information will be used to design functional experiments on the catalytic mechanism. We also aim to crystallize the complex of DM with DR/peptide and will examine whether the small molecule accelerators promote the formation of crystals suitable for structure determination by shifting the equilibrium to an active conformation of DM. The focus of Aim 2 is to determine whether low affinity peptides can be selectively removed from MHC class II molecules by increasing the editing function of DM with small molecule accelerators. This question is relevant because T cells specific for low affinity self-peptides can escape tolerance induction and cause autoimmune diseases. For a systematic assessment of the peptide repertoire, we will analyze peptides eluted from MHC class II molecules by mass spectrometry and determine the binding properties of peptides whose abundance is significantly affected by the small molecules. In the B10.PL mouse model of multiple sclerosis, disease is mediated by CD4 T cells specific for the low affinity Ac(1-11 ) peptide of myelin basic protein (MBP) since T cells specific for high affinity determinants are subjected to central and peripheral tolerance mechanisms. T cell experiments will assess whether small molecule accelerators of DM prevent presentation of this self peptide to CD4 T cells. ? ?
