Subcellular localization, protein-protein interactions and therapeutic targeting of human deubiquitinasesstudies on usp21 and usp1

Resumen

Deubiquitinases (DUBs), the enzymes that catalyze the removal of ubiquitin moieties from target proteins, participate in a wide range of cellular processes that are frequently altered in cancer, and hence are viewed as potential targets for the development of novel targeted therapies. The development of novel DUB-targeted drugs, though, is seriously hampered by the limited information still available about the basic biology of these enzymes. One of the unexplored issues is the regulation of their nucleocytoplasmic transport. Transport of proteins between the nucleus and the cytoplasm is carried out by importins or exportins, that respectively recognize and bind nuclear localization signals (NLSs) or nuclear export signals (NESs) in the cargo proteins. The best-characterized NESs generally consist of a stretch of hydrophobic amino acids with a poorly conserved consensus pattern and are recognized by the export receptor CRM1. By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human DUBs. These data were valuable to add novel information to the ¿loose¿ NES consensus, as well as to compare for the first time the performance of three programs widely used for NES prediction.The novel NESs identified in the present study reveal a subset of 22 DUBs bearing motifs that might mediate their binding to CRM1. Subsequent experiments on a subset of these DUBs led us to focus on the mechanisms that control the localization of two DUBs, USP21 and USP1. Consistent with its function in different subcellular compartments, our experiments reveal that USP21 is a dynamic DUB that shuttles between the nucleus, cytoplasm and centrosome, and can also localize to the microtubules. We validate the centrosomal kinase MARK4 as a USP21-interacting partner and identify the MARK4-binding region of USP21. Importantly, our results indicate that MARK4 interaction may regulate USP21 nucleocytoplasmic shuttling dynamics. In the case of the nuclear DUB USP1, its interaction with the cytoplasmic cofactor UAF1 has been shown to be essential for its function. Here we show that two active NLSs in USP1 mediate the nuclear import of the USP1/UAF1 complex, and by using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Importantly, we discuss the practical implications that these findings might have for the design and search for drugs that could target USP1. Recent evidence supports the view that USP1 could represent a valid target for the treatment of non-small cell lung cancer. However, current data on USP1 expression in this type of cancer are somewhat controversial. By combining the information on public databases with our own experimental results, we show that USP1 is overexpressed in lung cancer. In addition, we provide novel experimental evidence supporting the view that USP1 inhibition may contribute to revert cisplatin resistance in some preclinical models of NSCLC.