Structural Basis of Interaction of Herpesvirus Proteins with the Deubiquitinase USP7
Ubiquitin Specific Protease 7 (USP7) has emerged as a key regulator of many cellular pathways, including those critical for oncogenesis. USP7 is a deubiquitinase that regulates the stability or localization of specific substrate proteins by cleaving ubiquitin from them. USP7 consists of an N-terminal TRAF-like domain (NTD), a catalytic domain and five C-terminal ubiquitin-like (CTD) domains. USP7 was discovered as a target of a herpesvirus protein and later shown to be manipulated by multiple proteins from several different herpesviruses, earning its alternative name of Herpesvirus Associated USP (HAUSP). EBNA1 is the only Epstein-Barr virus protein expressed in infected cells and has important roles in EBV latent infection. We previously showed that the EBNA1 protein uses an EGPS motif to bind strongly to the NTD and blocks USP7 stabilization of p53. Using a combination of pull-down, binding and structural studies, we recently identified that USP7 interacts with vIRF1 of Kaposi’s sarcoma herpesvirus via the identical EGPS motif. These studies also demonstrated that vIRF1 destabilizes p53 by hijacking USP7 similar to what was previously observed with EBNA1. We mapped the interactions between USP7 and herpes simplex virus 1 ICP0 using a combination of GST pull-down and fluorescence polarization studies. We determined the crystal structure of the CTD domain with an ICP0 peptide and identified USP7 residues mediating the interaction with ICP0. We have identified mechanisms of USP7 substrate recognition by its NTD and CTD that are used by both viral and cellular proteins. Further details and analysis of the crystal structures will be presented.
11:30-12:30 BioSci Rm. 3110
11.29.2016 // Steven Smith, Department of Biomedical and Molecular Science, Queen's University
Molecular basis of gene deregulation by the oncogenic transcription factor E2A-PBX1
The E2A gene is also involved in a chromosomal translocation that results in the oncogenic transcription factor E2A-PBX1. The two activation domains of E2 (AD1 and AD2) display redundant, independent, and cooperative functions in a cell-dependent manner, at least in part through an interaction with the transcriptional co-activator CBP/p300. The E2A-PBX1:CBP/p300 interaction is critical for oncogenesis. However, a molecular understanding of this interaction and associated function has been lacking. Here, we describe our use of structural biology, biophysical and biochemical approaches, and complementary cell-based assays and mouse studies to characterize the interactions of E2A-PBX1 with CBP/p300, and our ability to disrupt this interaction by an engineered peptide. Our studies are defining the molecular basis for transcriptional activation and oncogenesis by E2A and E2A-PBX1, and serve as a structural foundation for inhibitor design.
11:30-12:30 BioSci Rm. 3110
The living dead: metabolic arrest and the control of biological time
The Storey lab studies the molecular mechanisms and regulatory principles that provide the common basis for animal metabolic arrest. Although not part of the human experience, torpor or dormancy is widespread across the animal kingdom and represents a key survival strategy in the face of daunting environmental challenges. Indeed, strong metabolic rate depression underlies multiple phenomena including estivation, diapause, freezing and anoxia tolerance, anhydrobiosis, and hibernation. Mammalian hibernation has perhaps the greatest relevance to humans as molecular adaptations imparting survival of hibernator organs at low temperatures have numerous medical applications including improvement of the hypothermic preservation of excised organs for transplant, ischemia resistance, and prevention of muscle atrophy. Current research in the Storey lab also focuses on signal transduction and gene expression in multiple systems, including the action of microRNAs as translational regulators, and novel epigenetic mechanisms (histone and DNA modifications). Supported by NSERC.
For more information visit http://kenstoreylab.com/
Biography: Prof. Kenneth B. Storey, Ph.D., F.R.S.C holds the Canada Research Chair in Molecular Physiology. He received a B.Sc. from the University of Calgary and Ph.D. from the University of British Columbia. Ken is an international authority in the field of biochemical adaptation. His lab integrates the tools of enzymology, metabolic biochemistry, protein chemistry, and molecular biology to identify evolved adaptations underpinning amazing animal phenomena including hibernation, estivation, and freeze and anoxia tolerance. Ken is a prolific author and speaker – he has authored over 750 publications and has given hundreds of talks around the world. Among his many tributes Ken was awarded the 2010 Flavelle Medal in Biological Sciences from the Royal Society of Canada, the 2011 Fry Medal from the Canadian Society of Zoologists, and the 2014 CryoBiology Society Medal.
***Note Special Location: BioSci Rm 1103***
Chill susceptible insects: a slow battle against entropy
The timing and severity of the winter season are key predictors of insect distribution and abundance, but we lack an integrative understanding of why chilling causes tissue damage and death in insects. Most insect species are chill susceptible, meaning they enter a cold-induced coma (chill coma) and suffer irreversible chilling injury well before any freezing of their body fluids occurs. I will give a overview of our recent efforts to connect natural variation in chilling tolerance to the capacity of insects to maintain osmotic homeostasis in the cold. Cold-adapted or cold-acclimated flies and crickets can maintain ion balance at low temperatures and survive prolonged cold exposure. By contrast, warm-adapted species or warm-acclimated individuals lose Na+ and water balance when chilled, which causes a progressive rise in extracellular [K+] that depolarizes cell membranes and causes cell death. The gut and Malpighian tubule epithelia of insects are central to the maintenance of whole animal ion and water balance, and appear to play a critical role in the ability to survive chilling. As an example, I will discuss how cold acclimation reduces the tendency for solute leak through the paracellular septate junctions of the Drosophila gut before and during chronic cold stress. This tightening of paracellular pathways appears to primarily be driven by plastic changes in the abundance of several major structural components of the junctions, and is likely to have cascading impacts on metabolism, growth, and development.
11:30-12:30 BioSci Rm. 3110
*** And Free Pizza Meet & Greet after 12:30-1:30 in BioSci 3rd floor lunch room (Rm 3406)
Macrophage activation and fusion on polymeric surfaces in the presence of damage-associated molecular patterns
Synthetic polymers have widespread use in current and emerging biomedical applications, as components of medical devices, tissue engineering scaffolds, drug delivery devices and sensors. However, the success of these applications is dependent on the cellular response to the implanted material. Essentially all materials elicit an inflammatory response following implantation that can have detrimental affects on the performance of the material, including material degradation leading to failure, chronic inflammation at the implant site and poor tissue integration at the material-tissue interface. Protein adsorption from the biomaterial’s local environment is considered a key factor in determining the cellular, and thus overall host response, to implanted materials. While effect of adsorbed proteins derived from blood has been investigated extensively, the role of endogenous danger signals in biomaterial-induced inflammatory responses is not well understood. These damage associated molecular patterns (DAMPs) are released upon tissue injury (e.g. during implant placement) and are known to induce inflammatory responses in macrophages, a key cell type of biomaterial host responses.
The work described here will focus on an in vitro model of macrophage-biomaterial interactions that investigates the adsorption of DAMPs, and subsequent macrophage activation, on polymer surfaces. Using cell lysate as a complex source of DAMPs, we have demonstrated that DAMP-adsorbed surfaces have increased Toll-like receptor (TLR)-2-mediated NF-kB activity and cytokine expression, compared to serum-adsorbed surfaces. Furthermore, we have observed rapid macrophage fusion to form multinucleated giant cells, independent of the addition of fusogenic factors interleukin (IL)-4, IL-13 and interferon (IFN)-g, suggesting the lysate-mediated fusion occurs through a novel fusion mechanism. The research aims to improve our understanding of in vivo material-cell interactions, which will guide the development of novel strategies to control biomaterial host responses current clinical materials and improve the lifespan of current medical implants.