Nancy Jaiswal

UCCON HEALTH

ABSTRACT

Cellular levels of several key proteins involved in DNA damage response are regulated by Ubiquitin Proteasome System (UPS). Components of this pathway, including the proteasome, ubiquitinating enzymes and deubiquitinating enzymes, are highly specialized and tightly regulated. Amongst these, the deubiquitinating enzyme USP7 (Ubiquitin specific protease 7) has been extensively studied due to its ability to control the levels of tumor suppressor p53, a central regulator of the cell fate during DNA damage response. Microdeletions and mutations in the USP7 gene results in neurodevelopmental disorders characterized by intellectual disability, autism spectrum disorder, epilepsy and hypogonadism. Dysregulation of USP7 expression has been reported in a number of human malignancies, including human prostate cancer, ovarian cancer and non-small cell lung cancer. Furthermore, early studies in human colon cancer xenograft models showed that downregulation of USP7 suppresses cell proliferation and delays tumor growth due to p53 stabilization in the absence of cellular stress. Therefore, this enzyme has emerged as a promising target for cancer therapy.

USP7 (~130 kDa) is composed of seven domains including N- terminal TRAF-like domain followed by the catalytic domain (CD) and five ubiquitin-like domains. Remarkably, even small C-terminal truncations diminish the activity of the enzyme, suggesting that the C-terminus regulates USP7 activity via a yet unknown mechanism. According to the current model, USP7 adopts an L-shaped structure with N- and C-termini separated by 80-100 Å. This model, however, fails to explain regulation of CD by C-terminus of USP7. Speculatively, C-terminus and CD are brought together in close proximity to create an active conformation, although conclusive structural evidences are still missing, while the available biochemical and structural data are contradictory. Despite successful crystallography studies of various fragments of USP7, structural studies of the full length (FL) protein have been unsuccessful and represents a significant gap in our knowledge, which prevents a full understanding of the molecular mechanisms of USP7 activation and its substrate specificity. Here we report our progress in structural and functional characterization FL-USP7 enzyme using integrational approach combining NMR spectroscopy with Small Angle X-ray Scattering and computational molecular docking. A newly developed docking algorithm in HADDOCK allowed us to incorporate SAXS-derived spatial restrains into the modeling of the FL enzyme. Mapping of the inter-domain interaction sites using NMR chemical shift perturbation experiments validated the HADDOCK models. In our integrated FL- USP7 model, the catalytic domain and the regulatory C-terminal region of USP7 are located in close proximity, supporting previously hypothesized molecular mechanism of USP7 auto-activation.

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