PROJECT SUMMARY/ABSTRACT Project 2 will verify and then study the molecular mechanisms of phenotypes created and genetically solved in Core B and Core C. We expect to find scores of mutations with modifier effects over the term of funding, but we will not study all of them mechanistically. Herein we describe the key criteria used in selecting mutations to study and propose to begin with four mutations identified as T1D modifiers in our preliminary work (affecting Dusp10, Paqr8, Xpnpep1, and Rapgef1). We will proceed by re-targeting candidate genes in NOD/NckH and/or NOD/NckL backgrounds using CRISPR/Cas9 to create germline knockout alleles or replacement alleles (exact re-creations of the ENU-induced alleles) or both, to verify or exclude causation. Once verified, the encoded proteins will be studied in their mutant and WT forms to identify other proteins with which they associate, what effects the mutation might have on such associations, and also effects on cell signaling. Interactions will be directly tested in cells known to have altered function as a result of the mutation (based on studies from Project 1). Mass spectrometry pulldown will be performed using primary cells isolated from mice in which we have engineered epitope-tagged versions of the WT and/or mutant proteins in the germline. Verification of direct and indirect interactions will depend upon reciprocal pulldown of the target protein and putative interactors in HEK 293 cells. We will identify direct interactions using purified proteins and GST pulldown studies in vitro, as well as size-exclusion chromatography. Structural studies will be performed as warranted to determine precisely how each implicated protein interacts with its binding partners, and how the ENU-induced mutation alters this interaction. The effects of mutations on post-translational modification of cellular proteins will be examined when signaling abnormalities are suspected. These studies will, in many cases, reveal the molecular basis of acceleration or suppression of T1D. Frequent communication of our findings with Project 1, and vice versa, will mutually inform studies in both Projects, resulting in the understanding and development of the very best candidates. Our conclusions may point to alterations of signaling pathways affecting immune or β-cell function in a relatively subtle way. Yet in each case, there will be a highly significant effect on T1D development. Particularly in the case of strong and novel suppressor mutations, we hope to identify therapeutic opportunities for prevention or mitigation of T1D in human patients. Project 2 will undertake construction of inducible conditional alleles to test such therapeutic opportunities, instituting gene knockout or conversion immediately after the onset of T1D to determine whether remission may be established. This would point to the potential treatment of human patients during the honeymoon phase of T1D. We will make data on all modifier mutations publicly ...