Activation Dynamics of Ubiquitin Specific Protease 7.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

Nuclear magnetic resonance spectral data of the USP7 TRAF and UBL1-2 domains in complex with DNA polymerase ι peptides.

This data article is related to the publication 'DNA polymerase ι interacts with both the TRAF-like and UBL1-2 domains of USP7' [1]. Ubiquitin-specific protease 7 (USP7) is an essential deubiquitinating enzyme with characterized substrates in many cellular pathways. Established USP7 substrates interact with one of two major binding sites, located on the N-terminal TRAF-like (TRAF) domain and the first and second UBL domains (UBL1-2) within the C-terminal tail. In this article, we present complete nuclear magnetic resonance (NMR) spectroscopy data used to characterize direct interactions between USP7 and its novel substrate DNA polymerase iota (Pol ι), that binds both TRAF and UBL1-2 domains. The detailed description of the NMR data, and the methodology used for processing and analysis, will add to the reproducibility and transparency of the companion research article, as well as aid in the reuse of these data.

DNA Polymerase ι Interacts with Both the TRAF-like and UBL1-2 Domains of USP7.

Reversible protein ubiquitination is an essential signaling mechanism within eukaryotes. Deubiquitinating enzymes are critical to this process, as they mediate removal of ubiquitin from substrate proteins. Ubiquitin-specific protease 7 (USP7) is a prominent deubiquitinating enzyme, with an extensive network of interacting partners and established roles in cell cycle activation, immune responses and DNA replication. Characterized USP7 substrates primarily interact with one of two major binding sites outside the catalytic domain. These are located on the USP7 N-terminal TRAF-like (TRAF) domain and the first and second UBL domains (UBL1-2) within the C-terminal tail. Here, we report that DNA polymerase iota (Pol ι) is a novel USP7 substrate that interacts with both TRAF and UBL1-2. Through the use of biophysical approaches and mutational analysis, we characterize both interfaces and demonstrate that bipartite binding to both USP7 domains is required for efficient Pol ι deubiquitination. Together, these data establish a new bipartite mode of USP7 substrate binding.

USP7 Is a Master Regulator of Genome Stability.

Genetic alterations, including DNA mutations and chromosomal abnormalities, are primary drivers of tumor formation and cancer progression. These alterations can endow cells with a selective growth advantage, enabling cancers to evade cell death, proliferation limits, and immune checkpoints, to metastasize throughout the body. Genetic alterations occur due to failures of the genome stability pathways. In many cancers, the rate of alteration is further accelerated by the deregulation of these processes. The deubiquitinating enzyme ubiquitin specific protease 7 (USP7) has recently emerged as a key regulator of ubiquitination in the genome stability pathways. USP7 is also deregulated in many cancer types, where deviances in USP7 protein levels are correlated with cancer progression. In this work, we review the increasingly evident role of USP7 in maintaining genome stability, the links between USP7 deregulation and cancer progression, as well as the rationale of targeting USP7 in cancer therapy.