USP7 (Ubiquitin-Specific Protease 7)
Proteins are vital to the structure and functioning of cells, and so the regulated turnover of proteins is an absolute essential of cellular metabolism. This necessity for the major part has been found to be taken care of by the ubiquitin-proteasome system (UPS). The system was discovered as a result of the work carried out by Avram Hershko, Aaron Ciechanover, and Irwin Rose, for which they shared the 2004 Nobel Prize in Chemistry. The UPS in its most simplified form consists of a tagging factor in the form of the small protein ubiquitin, enzymes that mediate the tagging of unwanted or damaged proteins, and the proteasome, a large molecular shredder that cleaves tagged proteins in to smaller peptides for use in other anabolic processes. More than 80% of all cellular proteins undergo degradation by the UPS, highlighting its importance in regulating various eukaryotic cellular processes such as cell cycle progression, stress response, apoptosis, viral infection, transcriptional activation, and DNA repair.
Ubiquitination can be simply defined as the process leading to the covalent linkage of ubiquitin to itself or other proteins either as a single molecule or as poly-ubiquitin chains. Ubiquitin, a highly conserved 76 amino acid protein gets attached to the ε-amino group of lysine residues of the target proteins through its C-terminal glycine (G76) via an isopeptide bond. Ubiquitination is a multistep, ATP-dependent process and is carried out by three distinct enzymes – E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligases). Unlike E1 and E2, the E3 ligases represent a highly diverse class of enzymes. Substrate specificity is conferred by the E3 ligases which interact with target proteins and mediate the covalent linkage of ubiquitin.
Ubiquitination is a reversible process and is undone by specific deubiquitinating enzymes or deubiquitinases (DUBs). There are around 90 different DUBs encoded by the human genome that are grouped in five different families according to the architecture of their catalytic domains – ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado Joseph disease (MJDs), monocyte chemotactic protein-induced protein (MCPIP), and JAMM/MPN domain-associated metalloproteases (JAMMs), of which the first four are cysteine proteases, whereas the JAMMs are metalloproteases. USPs represent the most abundant group of DUBs, consisting of about 50 members. Sequence conservation among USPs have been observed to be limited to the catalytic domain which is characterized by an active site catalytic triad consisting of Cys, His, and Asp/Asn residues. The sequences of the non-catalytic domains vary greatly between enzymes and have been hypothesized to confer substrate specificity and to regulate activity of the catalytic domain.
USP7 was originally identified as an interacting partner of the ICP0/Vmw110 protein of the herpes simplex virus (HSV) (Everett et al. 1997). ICP0 is a virus-encoded E3 ubiquitin ligase, involved in the ubiquitination and degradation of itself and certain host cell proteins with antiviral properties. USP7 was shown to interact with ICP0 and regulate its auto-ubiquitination and degradation (Canning et al. 2004). In subsequent years, USP7 was shown to interact with other virus-encoded proteins such as the EBNA1 protein of Epstein-Barr virus (EBV) and the LANA protein of Kaposi’s sarcoma-associated herpes virus (KSHV), thereby establishing it as a common target of herpesviruses and hence the alias – herpesvirus-associated USP (HAUSP). Apart from its involvement in viral infections, USP7 is also famous for regulating the turnover of a critical tumor suppressor protein – p53 (Li et al. 2002). Later on USP7 was also shown to deubiquitinate and stabilize MDM2/HDM2 which is a negative regulator of p53, indicating a complex regulatory mechanism (Li et al. 2004; Cummins et al. 2004; Meulmeester et al. 2005). Subsequent research revealed the identity of many more USP7 substrates having pivotal roles in various physiological and disease processes, thus marking the protein an attractive target for pharmacological intervention.
The protein is coded by the USP7 gene located on the short arm of chromosome 16 (16p13.2; Ensembl). The gene gives rise to 21 transcripts/ splice variants, 12 of which are protein coding (Ensembl).
Structure of USP7
N-terminal poly-glutamine (poly Q) stretch that has been implicated in neurodegenerative disorders.
Tumor necrosis factor receptor-associated factor (TRAF) domain, which is mainly involved in protein-protein interactions utilizing its own “DWGF” motif and the “(P/A/E)XXS” motif on the substrates. The domain is also involved in the nuclear localization of the protein.
Catalytic (CAT) domain, which is conserved throughout USPs.
Carboxy-terminal domain (CTD), which contains about 5 ubiquitin-like folds and facilitates interactions with other proteins like ICP0, ubiquitin like PHD and RING finger domain-containing protein 1 (UHRF1), DNMT1, etc. A second site for interaction with p53 and MDM2 has also been mapped to this domain.
Regulation of USP7
Post-translational regulation of USP7
Destabilization and proteasome-mediated degradation. Reduced half-life.
Boutell et al. 2005
Export of USP7 from nucleus to cytoplasm.
Daubeuf et al. 2009
Caesin kinase 2 (CK2)
Stabilization and improved half-life.
Khoronenkova et al. 2012
Reverse of CK2-mediated phosphorylation. Reduced half-life.
Khoronenkova et al. 2012
The poly-neddylation and dimerization of rat USP7 (rHAUSP) has also been reported, but their mediators and their effects on protein activity remains largely unknown.
Apart from these, USP7 gene expression has been reported to be enhanced by FOXO6 and contributes in inhibiting proliferation of lung carcinoma (Hu et al. 2015). The miRNA, miR-205 has been reported to negatively regulate USP7 mRNA levels by targeting its 3′-UTR in hepatocellular carcinoma (Zhu et al. 2015).
Role of USP7 in Physiological Processes
Host-virus interactions – As discussed earlier, USP7 was originally identified as an interacting partner of the HSV early protein ICP0. USP7 deubiquitinates and stabilizes ICP0 which goes on to facilitate various events necessary for productive infection such as disruption of host cell microtubule networks, activation of transcription of essential viral and host cell genes, degradation of key host cell pathways producing antiviral responses, and degradation of PML nuclear bodies. Other viral proteins, such as EBNA1 of EBV, have also been found to interact with USP7, and these interactions have been found to either stimulate lytic infection and reactivation of quiescent viral genomes or maintain latency depending upon the infective phase.
p53-MDM2/HDM2 axis – USP7 is involved in a dynamic relationship with the two proteins, p53 and MDM2/HDM2. Overexpression of USP7 leads to p53 stabilization; however, disruption of the USP7 gene produces a similar effect. This is because USP7 also stabilizes MDM2/HDM2 which is the antagonist of p53. Both proteins interact with the TRAF domain of USP7, with similar affinity. The p53-USP7 interaction has been reported to be enhanced in response to genotoxic stress. Thus USP7 has to consistently juggle between saving MDM2 and promoting cell proliferation or saving p53 at the time of genotoxic stress and promoting growth arrest or apoptosis. Apart from its role in the p53-MDM2/HDM2 axis, USP7 has also been shown to increase levels of mono-ubiquitinated p53 in cells exhibiting DNA damage. Mono-ubiquitinated p53 can translocate to the mitochondria and trigger the mitochondrial apoptotic cascade.
DNA replication – Posttranslational modification of chromatin proteins by ubiquitin and ubiquitin-like modifiers is essential for the regulation of DNA replication. It has been reported that the chromatin around replisomes are rich in SUMO but lack ubiquitin, whereas in the case of mature or heterochromatin, it is the reverse. USP7 has been identified as a SUMO deubiquitinase (SDUB) enriched in replisomes and is essential for DNA replication. By interacting with sumoylated proteins, probably through a SUMO interaction motif (SIM), USP7 reverses their ubiquitination and allow these proteins to accumulate at sites of DNA replication and promote replication fork formation (Lecona et al. 2016).
DNA repair – USP7 is involved in the regulation of the base excision repair (BER) mechanism for repairing oxidative DNA lesions. USP7 achieves this by regulating MDM2 levels. MDM2 ubiquitinates the histone H2B, resulting in opening up of chromatin and assembly of BER machinery. USP7 has also been reported to regulate transcription-coupled nucleotide excision repair (TC-NER) through its interaction with and stabilization of the UV-stimulated scaffold protein A (UVSSA) which is an essential component of the TC-NER machinery (Higa et al. 2016). The UVSSA protein also functions in stabilizing the TC-NER master organizing protein ERCC6 by recruiting USP7 to the TC-NER complex.
Epigenetics – USP7 plays a major role in the epigenetic regulation of gene expression. USP7 deubiquitinates components of the polycomb repressive complex (PRC1) such as MEL18 and BMI1, thereby reducing their turnover and increasing their accumulation on chromatin. This in turn leads to repression of expression of the tumor suppressor gene p16INK4a, some homeostatic genes, and also hormone-regulated genes. USP7 also associates with GMP synthetase, and this stimulates the former to deubiquitinate mono-ubiquitinated H2B thus leading to gene silencing (Knaap et al. 2005). A multi-protein complex comprising of USP7, DNMT1, UHRF1, HDAC, Tip60, and PCNA has been reported to regulate the global methylation status of genes. USP7 has also been shown to play a part in the repression of p53-responsive genes. In unstressed cells, USP7 deubiquitinates and protects the methyltransferase SUV39H1 from MDM2-mediated degradation. This in turn leads to the tri-methylation of histone H3 (H3K9me3) at p53-responsive promoters and the repression of their respective genes (Mungamuri et al. 2016). More recently it has been reported that hypoxia-induced K3-polyubiquitinated USP7 promotes CBP-mediated H3K56 acetylation on HIF-1α target gene promoters to induce EMT/metastasis.
Immune functions – USP7 has been implicated in thymocyte apoptosis, linking it with the caspases and apoptosis (Vugmeyster et al. 2002). The principal regulator of inflammatory and immune signaling NF-κB is deubiquitinated by USP7 (Collaran et al. 2013). This essentially increases the nuclear localization and transcriptional activity of the former protein. FOXP3, a transcription factor and also a characteristic marker protein of regulatory T cells (Treg), is also stabilized via USP7 mediated deubiquitination.
Role of USP7 in Disease
Cancer – As discussed in the earlier section, USP7 is involved in a number of physiological processes, particularly those that are frequently altered during tumorigenesis. The tumor suppressor p53 is arguably the most famous protein associated with cancer, and so it is not surprising that USP7 being a key regulator of p53 has gained significant fame during the last decade in the field of cancer research. Apart from p53, extensive research has led to the discovery of several USP7 substrates having either oncogenic or tumor suppressive potential such as MDM2 (Li et al. 2004), DAXX (Song et al. 2008), PTEN (Song et al. 2008), Rb (Bhattacharya et al. 2014b), Gli (Zhou et al. 2015), PHF8 (Wang et al. 2016), N-myc (Tavana et al. 2016), and HIF-1α (Wu et al. 2016). Although a direct role of USP7 as either an oncogene or tumor suppressor has yet to be established, in today’s scenario, USP7 is generally treated a negative prognostic factor for a number of cancers.
Other diseases – The presence of a polyglutamine stretch in USP7 links it to neurodegenerative disorders like spinocerebellar ataxia, Huntington’s disease, spinobulbar muscular atrophy, and Machado-Joseph disease. It has been suggested that haploinsufficiency or duplications of USP7 disrupt neuronal homeostasis, giving rise to neurological and behavioral aberrations such as seizures, aggressiveness, hypotonia, intellectual deficit, and hypogonadism with clinical features similar to Schaaf-Yang syndrome. USP7 has been linked to other diseases like UV-sensitive syndrome, bone diseases, cardiovascular disorders, etc., but the exact mechanistic details are lacking.
In conclusion, USP7 has emerged as a major deubiquitinase with roles in a number of biological processes ranging from cell division, genome stability, epigenetic regulation, immunity to viral infection, carcinogenesis, and other pathologies. USP7 shows a profound variation in expression in various cancers. This molecule is overexpressed in colon, bladder, lung, prostate, and liver cancer while it is found to be downregulated in head and neck and breast cancer. According to a recent study which shows a grade-wise upregulation of USP7 in glioma in a Chinese population. Both upregulation and downregulation of USP7 result in increased radiation sensitivity of mouse xenograft tumor due to p53 stabilization under both conditions, and this suggests novel strategies in cancer therapy of cells carrying wild-type p53 through modulating the HAUSP activity. Targeting USP7 by application of specific small molecule inhibitor have yielded significant results by triggering p53-dependent pathway while showing very less specificity towards other DUBs. Use of synthetic peptide which targets and inhibits HAUSP-MDM2 interaction leading to stabilization of p53 may also serve as an alternative.
The most important criteria in this scenario is a better understanding of HAUSP functionality under different contexts by identifying novel interacting partners as well as deciphering the key modes of regulation of HAUSP in different cancer types.
Financially supported by grants from CSIR, India (EMPOWER-OLP-002, MEDCHEM-BSC0108 & CSIR-MAYO: MLP-0017), and DST Nano Mission program (SR/NM/NS-1058/2015) to Dr. Mrinal K Ghosh.
Conflict of Interest The authors declare no conflict of interest.
- Bhattacharya S, Ghosh MK. Cell death and deubiquitinases: perspectives in cancer. BioMed Res Int. 2014a. Article ID 435197.Google Scholar
- Canning M, Boutell C, Parkinson J, Everett RD. A RING finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitin-specific protease USP7. J Biol Chem. 2004;279: 38160–8.Google Scholar
- Cummins JM, Rago C, Kohli M, Kinzler KW, Lengauer C, Vogelstein B. Tumour suppression: disruption of HAUSP gene stabilizes p53. Nature. 2004;428:1 p following 486.Google Scholar
- Daubeuf S, Singh D, Tan Y, Liu H, Federoff HJ, et al. HSV ICP0 recruits USP7 to modulate TLR-mediated innate response. Blood. 2009;113:3264–75.Google Scholar
- Everett RD, Meredith M, Orr A, Cross A, Kathoria M, Parkinson J. A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J. 1997;16:1519–30.Google Scholar
- Hu M, Li P, Li M, Li W, Yao T, Wu J-W, Gu W, Cohen RE, Shi Y. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell. 2002;111:1041–54.Google Scholar