While a relatively limited number of known oncogenes underlie a large percentage of cancers, a variety of new genes have emerged as low-frequency cancer drivers. Each of these new oncogenes represents a frontier for targeted therapy. However, the discovery of low-frequency targetable oncogenic drivers is challenging. This study focuses on the poorly understood Tyrosine kinase non-receptor-1 (TNK1), which has been reported to have both oncogenic and tumor suppressive functions. TNK1 has been identified to promote cancer cells survival and promote chemoresistance in multiple independent studies. On the other hand, whole-body constitutive deletion of TNK1 in mice caused an increase in spontaneous carcinomas and lymphomas. All in all, with no known regulatory mechanism and substrates of TNK1, the precise biological role of TNK1 is still unclear. To understand how TNK1 is regulated, we employed a proteomic approach to identify TNK1 interactors. We found out that TNK1 interacts with the phospho-binding protein 14-3-3 and this interaction is mediated by a cluster of MARK-mediated phosphorylations within the proline-rich domain. 14-3-3 binding retains TNK1 in the cytosol and maintains TNK1 in an inactive state. Release of TNK1 from 14-3-3 binding drives TNK1 to a heavy membrane fraction, where it becomes highly active. One unique feature of TNK1 is an ubiquitin association domain (UBA) on its C-terminus. Our data suggest that the UBA domain of TNK1 binds to poly-ubiquitin chains in nondiscriminatory manner. Remarkably, point mutations within the UBA that disrupt ubiquitin binding abolish TNK1 activation and oncogenic signaling, suggesting, to our knowledge, a unique UBA-centric mechanism of tyrosine kinase regulation. Finally, we used a structure-guided approach to identify a small molecule inhibiting TNK1 with high potency and selectivity. Such compound, TP-5801, inhibits TNK1 dependent STAT3 phosphorylation. TP-5801 also prolongs the survival of mice injected via tail vein with TNK1-driven Ba/F3 cells and reduces tumor burden in a subcutaneous xenograft model. In conclusion, our data reveal a mechanism of TNK1 regulation that controls its oncogenic tyrosine kinase activity and a potential strategy for TNK1 inhibition.

We studied the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We observed novel properties in crystals of two TELSAM-target protein fusions. TELSAM as a crystallization chaperone shows rapid crystallization when it's fused to target proteins and possibly with a greater propensity. Some TELSAM-target fusions crystallized more rapidly than the same target protein alone. TELSAM-target proteins can be crystallized at relatively low protein concentrations such as 0.1 mg/mL. TELSAM requires no TELSAM polymers touching one another in the crystal lattice in order to form well-diffracting crystals. This lack of crystal contacts has not been observed in previously reported TELSAM crystal structures. Flexible TELSAM-target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. This study tested TELSAM linker lengths varying by the number of glycines, such as 2xGly, 4xGly, 6xGly, 8xGly, and 10xGly. Only TELSAM fused to UBA with 2 and 4 glycine linkers were crystalized. TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts, and fusion to TELSAM polymers increases avidity to stabilize weak inter-target protein crystal contacts. In conclusion, we report features of TELSAM-target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and define the best practices for its use.

Decades of research have begun to identify oncogenic mut-drivers that are responsible for driving a large percentage of cancers. These high frequency mut-drivers have therapeutics in the clinic for patient treatment. However, there is another group of low frequency mut-drivers that fail to rise above the noise of the high frequently drivers. These low frequency drivers represent a group of genes with untapped potential for new targeted therapies. However, identifying these drivers can be difficult. This study focuses on identifying new functional phosphorylations using the phospho-docking protein 14-3-3. The family of 14-3-3 proteins have been linked to many oncogenic pathways due to the diversity in their client protein interactions. One critical problem in studying 14-3-3 interactors is uncovering the docking site on the phospho-binding partner. Our work indicates that intrinsic disorder and unbiased mass spectrometry identification rate of a given phosphorylation are important for improving the selection of a 14-3-3 docking site. Using a machine learning model, we developed a tool that combines current available 14-3-3 prediction data and our observations about disorder and phosphorylation observation to predict 14-3-3 binding sites. Our publicly available tool "14-3-3-site-finder" produces a rank order list of potential 14-3-3 docking sites that could help overcome the time-consuming process of identifying the correct site. In our efforts of identifying functional phosphorylations with 14-3-3, we have observed that 14-3-3 interacts with a non-receptor tyrosine kinase, TNK1. TNK1 is a poorly characterized kinase that has essentially nothing known about its substrates, function or regulation. TNK1 has been implicated in both tumor suppressor and oncogenic roles. Particularly, a Hodgkin Lymphoma cell line is dependent on a truncated form of TNK1 for growth. Our work uncovers the first mechanism of regulation for this kinase. We found that MARK kinase phosphorylates TNK1 within the proline rich domain allowing 14-3-3 to dock on this phosphorylation. 14-3-3 binding restrains TNK1 in the cytosol and holds TNK1 in an inactive state. Upon the release of 14-3-3, TNK1 moves to a membrane fraction where it is active. One unique feature of TNK1 is that it has a c-terminal ubiquitin association domain (UBA). In vitro ubiquitin pulldowns indicate that the TNK1 UBA has no preference for linkage type or length of ubiquitin. Further, biolayer interferometry indicates that the UBA binds ubiquitin tightly. Mutation of residues within the ubiquitin:TNK1 interface prevent ubiquitin binding and decrease TNK1 activity, preventing downstream oncogenic signaling, suggesting a UBA-centric mechanism of regulation for TNK1. Finally, we developed a small molecule inhibitor, TP-5801, that selectively targets TNK1. TP-5801 prevents downstream TNK1 phosphorylation of STAT3. Further, TP-5801 prolonged the life of mice injected with TNK1 driven Ba/F3 cells. Taken together, our data reveal the first mechanism of kinase regulation for TNK1 involving 14-3-3 binding and ubiquitin association as well as the development of a TNK1 specific therapeutic

Structural biology uncovers life's secrets by studying protein structures via techniques like X-ray crystallography. This knowledge drives advancements in protein engineering for the improvement of human lives. Yet, obtaining high-quality crystals in X-ray crystallography is challenging. To overcome this, we used Translocation ETS Leukemia protein Sterile Alpha Motif domain (TELSAM), a promising polymer-forming crystallization chaperone (PFCC), to enhance protein crystallization. Human thirty-eight-negative kinase-1 (TNK1), a key player in cancer progression, possess a ubiquitin association (UBA) domain that binds polyubiquitin and regulates TNK1 activity and stability. Although sequence analysis hints at an unconventional TNK1 UBA domain architecture, its molecular structure lacks experimental validation. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Ã…. 1TEL enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our findings support a TELSAM fusion crystallization mechanism, highlighting fewer crystal contacts compared to traditional crystals. Both modeling and experimental validation indicate that the UBA domain exhibits selectivity towards polyubiquitin chain length and linkages. Radical S-adenosylmethionine (SAM) enzymes catalyze various radical-mediated substrate transformations. Despite the growing interest of computational enzyme design in industrial small molecule synthesis, radical SAM enzymes remain relatively unexplored. We used PyRosetta to leverage hydrogen bonding design (hbDes) and hydrophobic interaction design (hpDes) to enable a radical cyclization reaction on our selected substrate. Although the purified enzymes demonstrated activation potential with a reducing agent, enzymatic assays failed to exhibit activity against the reactant. To obtain successful results, addressing additional questions and issues is required, which may involve the implementation of machine learning.