Technologies for DNA sequencing and protein analysis have accelerated so rapidly that generation of infor-mation now outpaces our ability to completely understand it. As a result, many novel genes and proteins havebeen discovered, but their functions remain unknown. The intent of the current proposal is to illuminate thefunction of one group of these uncharacterized ("dark") proteins, the kinases, which are likely to have criticalactivities.Human cells have over 600 kinases, but the role played by a third of them is unknown. Kinases regulate allaspects of intracellular signaling, affecting everything from growth and differentiation to protein localization andDNA repair. One key activity of kinases is to activate or inhibit transcription factors (TFs), which regulate theactivity of genes in the nucleus, thereby controlling the fate and activity of the cell. Many kinases therefore aretargets of pharmaceutical drug development, particularly as cancer therapeutics, due to their role in regulatingTFs and other key molecules.Talus has developed the ChESS-DIA technology to follow the localization of the entire population of proteins(the "proteome") in the nucleus that are either unbound or bound to chromatin. In the current proposal, theChESS-DIA method will be adapted to measure the effect of specific "dark" kinases on the nuclear and genomiclocalization of the proteome. In Aim 1 we will chemically deplete known kinases by targeted protein degradationand use ChESS-DIA to observe the effects on all TFs. The method will be optimized to ensure the results areconsistent with known effects of these kinases on TFs. We will then use the optimized ChESS-DIA protocol toanalyze the effect of dark kinases on TF localization. This information is critically valuable for developing noveldrugs that target cellular activities of each affected TF. In Aim 2 will analyze the effects of dark kinases in moredetail, by determining the exact amino acid sites on TFs that are recognized and modified (i.e., phosphorylated)by dark kinases. As in Aim 1, the ChESS-DIA technology will be tested and optimized first with known kinases,and the subsequently used to analyze understudied dark kinases.Drugs against kinases are relatively straightforward to develop, because the protein structure is well defined andaccessible for interaction with small molecule drugs. In contrast, the structures of most TFs generally are difficulttargets for drug development. The information obtained through this project will not only show which dark kinasesare likely to be strong drug targets, but it will also be the first identification of the specific protein sequences onTFs that represent valuable drug targets. We anticipate partnering with academic and pharmaceutical partnersto develop novel therapeutics to treat a variety of diseases for which there are no effective therapies.
Public Health Relevance Statement: NARRATIVE
Many novel proteins are believed to be clinically important, but they remain uncharacterized (i.e., "dark"). The
dark kinases are one such family, which may regulate a wide range of cellular activities in normal and diseased
cells. Talus has developed the ChESS-DIA technology to demonstrate the effect of unknown proteins such as
the dark kinases on activation of transcription factors.
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