br Acknowledgements This research was supported by Slovenian
Acknowledgements This research was supported by Slovenian Research Agency (ARRS). We thank prof. R. Marinšek Logar for helpful advice, Dr. T. Kranjc for help with data management, N. Vrhovnik and G. Lavrič for technical help with preliminary growth experiments and dr. J. Burkeljca for help with graphics.
Introduction Living cells constantly synthetize proteins, and dispose proteins that are misfolded, aggregated and no longer needed. Maintenance of proper protein homeostasis is essential for cell growth and survival. In eukaryotes, protein degradation is carried out by the ubiquitin–proteasome system (UPS). In this multi-step pathway, a polyubiquitin chain is conjugated to a protein substrate and serves as a signal for the substrate recognition and degradation by the 26S proteasome . Protein ubiquitination can be reversed by action of deubiquitinating enzymes (DUBs)  that remove ubiquitin moieties from their substrates (Fig. 1). Human ubiquitin-specific protease 7 (USP7) also known as Herpes virus associated protease (HAUSP) is a cysteine peptidase that belongs to the largest USP family of DUBs (Fig. 2) . Located primarily in the nucleus, USP7 regulates the stability of multiple proteins involved in diverse cellular processes including DNA damage response, transcription, epigenetic control of gene expression, immune response, and viral infection (Table 1). USP7 has been extensively studied for its ability to regulate the cellular level of tumor suppressor p53 affected in the majority of solid tumors [, , , ]. USP7 knockout was shown to be lethal in mice [8,9]. However, several children have been recently identified carrying USP7 mutations and deletions. The 46 individuals identified so far suffer from neurodevelopmental disorders such as autism spectrum disorder, intellectual disability, and speech/motor impairments  (www.usp7.org).
USP7 and cancer Given involvement of USP7 in multiple cellular pathways, it is not surprising that its expression is often dysregulated in human malignancies. USP7 overexpression contributes to tumor progression through changes in DNA damage response, apoptosis and pri-724 sale control. In particular, USP7 is upregulated in chronic lymphocytic leukemia  and its overexpression in human prostate cancer correlates with the tumor aggressiveness . USP7 expression level was shown to gradually increase with the tumor progression from grade I to grade IV in glioma patients . The enzyme is also overexpressed in breast carcinomas , as well as in lung squamous cell carcinoma and large cell carcinoma . Its dysregulation in non-small cell lung cancers leads to disruption of the HDM2–p53 axis and is associated with induced cell epithelial mesenchymal transition (EMT), (metastasis and overall poor prognosis . Similarly, USP7 overexpression in patients with epithelial ovarian cancer was found to induce cell invasion and correlate with poor survival [16,17]. Because of the USP7 aberrant expression in many human cancers and its role in important cell signaling pathways, this enzyme has emerged as a promising target for cancer therapy.
Cellular function of USP7
Regulation of USP7 in the cell USP7 is an important component of UPS and its activity in the cell is tightly regulated to avoid uncontrolled stabilization of its multiple substrates. There are several levels of USP7 regulation including intramolecular mechanisms, post-transcriptional modifications, and protein–protein interactions. Intramolecular mechanisms include domain reorganization required for the enzyme activation and active site rearrangement [, , , , , , ]. Post-transcriptional modifications can further tune the activity of USP7. In particular, the enzyme was shown to be phosphorylated at Ser18, Ser963, and ubiquitinated at Lys869 . Phosphorylation at Ser18 by the protein kinase CK2 alters affinity of USP7 towards its substrates HDM2 and p53 in a way that the phosphorylated enzyme preferably binds to HDM2, while its dephosphorylation results in the higher affinity to p53 . USP7 ubiquitination at Lys869 by E3 ligase TRIM27 promotes the TNF-α-induced apoptosis through deubiquitination of RIPK1  and the role of phosphorylation at Ser963 remains to be determined. In addition, USP7 is aberrantly phosphorylated at Tyr243 by chimeric protein p210 BCR-ABL in chronic myeloid leukemia (CML) cells. This post-translational modification enhances deubiquitinase activity of the enzyme towards the tumor suppressor PTEN whose dysregulation is linked to CML pathogenesis . Efficient USP7 regulation is mediated by several proteins that interact with the enzyme and mediate its stability and/or activity. Thus, Trip12 was recently identified as an E3 ligase for USP7 ubiquitination . In the absence of DNA damage, the DAXX protein binds to USP7 and HDM2, facilitating the HDM2 deubiquitination . DAXX–USP7 complex also regulates stability of the E3 ligase CHFR and Aurora-A kinase involved in mitosis . In the Wnt/β-catenin signaling pathway, the USP7 mediated stabilization of β-catenin depends on the E3 ligase RNF220 that forms a ternary complex with both USP7 and β-catenin and promotes deubiquitination of the latter . Another protein, MBD4, recruits USP7 to heterochromatin foci where it co-localizes with UHRF1 and DNMT1 promoting their interaction . Finally, at least one protein, TRAF4, negatively regulates USP7 activity. It binds to the same region of USP7 as p53 blocking p53 accesses to the enzyme. This inhibition mechanism was shown to play an important role in breast cancers where TRAF4 overexpression prevents USP7-mediated p53 stabilization and diminishes cytotoxic stress response .