RNA binding proteins (RBPs) regulate many important cellular processes through their interactions with RNA molecules and are critical for posttranscriptional mechanisms keeping gene regulation in a fine equilibrium. The dysregulation of many RBPs are an established hallmark of tumorigenesis and biologically relevant targets for drug development. Human nucleolin (NCL) is a multifunctional RBP that interacts with different types of RNA molecules, in part through its four RNA binding domains (RBDs). One of the RNA species of interest are microRNAs (miRNAs) as NCL interacts directly with miRNAs and is involved in their aberrant processing linked with many cancers, including breast cancer. However, the molecular details of the NCL-miRNA interaction remain obscure. In this study, we used an in silico approach to characterize the molecular details of how NCL targets miRNAs. Here, we present structural models of NCL-RBDs and miRNAs, as well as predict scenarios of NCL-miRNA interactions generated using docking algorithms. Our study suggests for the first time, a predominant role of NCL RBDs 3 and 4 (RBD3-4) in miRNA binding. We provide detailed analyses of specific motifs/residues at the NCL-substrate interface in both these RBDs and miRNAs. Finally, we propose that the evolutionary emergence of more than two RBDs in NCL in higher organisms coincides with its additional role/s in miRNA processing. Our study shows that RBD3-4 display sequence/structural determinants to specifically recognize miRNA precursor molecules. The insights from this study can ultimately support the design of novel drugs aimed at regulating NCL-dependent biological pathways with a causal role in cancer.
The NEK kinase family of proteins comprises of eleven serine/threonine kinases that participate in the disjunction of the centrosome, mitotic spindle assembly, and primary cilium formation. NEK10 is the most divergent member of the NEK family. It has a catalytic domain that is centrally positioned and flanked by two coiled-coil domains, while all the other NEKs have their catalytic domain near the N-terminus. In its place, NEK10 has four armadillo repeats of unidentified functions in its N-terminus. NEK10 plays a key role in carcinogenesis and has been associated to melanoma, breast cancer, and numerous ciliopathies. As part of our long-term goal to create interactomes of all the NEK members, we have previously reported data for known and predicted NEK10 interacting proteins, containing novel protein-protein interactions such as HSPB1 and MEKK, which have not been previously reported in the literature. In this study, we focused on understanding the molecular mechanism underlying NEK10’s interaction with MEKK (MEKK, MEKK1, MAPKKK1, MAP3K1) and its functional consequences. MEKK is a serine/threonine kinase and ubiquitin ligase that plays a key role in a network of enzymes integrating cellular receptor responses to various mitogenic and metabolic stimuli. Recent studies have shown that MEKK mutations are seen in a substantial number of different cancers, being most prominent in luminal breast cancer. MEKK has a protein kinase domain, and a PHD finger that serves as an E3 ubiquitin ligase, and scaffold protein regions that mediate protein-protein interactions. We utilized homology and ab-initio modeling tools to model the full-length of NEK10 and MEKK protein and provide robust models of NEK10, MEKK, along with their biophysical characterization. Docking prediction software and analysis/visualization tools were used to predict the interaction scenarios for the two proteins and characterize the interaction interface. Our docking analysis shows that I693 of NEK10 (necessary for kinase function) situated within its kinase domain, interacts with well-known phosphosite S275 of MEKK. Our results, thus, suggest a scenario in which, upon UV irradiation, NEK10 phosphorylates and activates MEKK, which in turn phosphorylates MAP2K1/2-ERK1/2 promoting cell survival. Overall, this study shows novel and intriguing information about NEK10’s structure-function relationships, particularly in the context of its interaction with MEKK. Additionally, it establishes the framework for elucidating the detailed molecular mechanisms of NEK10 interactions with other proteins to further investigate its potential as a therapeutic target.