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NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.
Dimerization of Taspase1 activates an intrinsic serine protease function that leads to the catalytic Thr234 residue, which allows to catalyze the consensus sequence Q−3X−2D−1⋅G1X2D3D4, present in Trithorax family members and TFIIA. Noteworthy, Taspase1 performs only a single hydrolytic step on substrate proteins, which makes it impossible to screen for inhibitors in a classical screening approach. Here, we report the development of an HTRF reporter assay that allowed the identification of an inhibitor, Closantel sodium, that inhibits Taspase1 in a noncovalent fashion (IC50 = 1.6 μM). The novel inhibitor interferes with the dimerization step and/or the intrinsic serine protease function of the proenzyme. Of interest, Taspase1 is required to activate the oncogenic functions of the leukemogenic AF4-MLL fusion protein and was shown in several studies to be overexpressed in many solid tumors. Therefore, the inhibitor may be useful for further validation of Taspase1 as a target for cancer therapy.
The FUBP1-FUSE complex is an essential component of a transcription molecular machinery that is necessary for tight regulation of expression of many key genes including c-Myc and p21. FUBP1 utilizes its four articulated KH modules, which function cooperatively, for FUSE nucleotide binding. To understand molecular mechanisms fundamental to the intermolecular interaction, we present a set of crystal structures, as well ssDNA-binding characterization of FUBP1 KH domains. All KH1-4 motifs were highly topologically conserved, and were able to interact with FUSE individually and independently. Nevertheless, differences in nucleotide binding properties among the four KH domains were evident, including higher nucleotide-binding potency for KH3 as well as diverse nucleotide sequence preferences. Variations in amino acid compositions at one side of the binding cleft responsible for nucleobase resulted in diverse shapes and electrostatic charge interaction, which might feasibly be a contributing factor for different nucleotide-binding propensities among KH1-4. Nonetheless, conservation of structure and nucleotide-binding property in all four KH motifs is essential for the cooperativity of multi KH modules present in FUBP1 towards nanomolar affinity for FUSE interaction. Comprehensive structural comparison and ssDNA binding characteristics of all four KH domains presented here provide molecular insights at a fundamental level that might be beneficial for elucidating the mechanisms of the FUBP1-FUSE interaction.