Decoding E6AP/UBE3A function: Structural revelations and small molecule modulators

• Applicants: Professor Dr. Martin Scheffner; Professor Dr. Florian Stengel
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406631249

• Final Report Year: 2023

Final Report Abstract

The activity of many cell regulatory proteins is controlled by covalent modification with ubiquitin. The specificity of the ubiquitin-conjugation system is mediated by the action of E3 ubiquitin-protein ligases. Notably, E3s are frequently deregulated in human diseases including cancer, viral infections, and cardiovascular, immunological as well as neurological disorders. E6AP, which is a member of the family of HECT E3s and encoded by the UBE3A gene, represents a prime example for this notion: Dysregulation or stimulation of E6AP's E3 activity by the E6 oncoprotein of human papillomaviruses (HPVs) decisively contributes to HPV-induced cervical carcinogenesis; inactivation of E6AP by genetic alterations of the UBE3A gene is the cause of the Angelman syndrome, a neurodevelopmental disorder; UBEA3 gene amplification resulting in E6AP overexpression is the genetic hallmark of the Dup15q syndrome. Thus, a thorough analysis of how the E6 oncoprotein or missense mutations in the UBE3A gene affect the E3 activity of E6AP is of critical importance for our understanding of the pathophysiological functions of E6AP and for the eventual design of new strategies in the treatment of E6AP-associated disorders. In this collaborative project, we combined the different, yet complementing expertise of our groups (biochemistry and cell biology of E6AP; structural biology with emphasis on analyzing structures of E6 oncoproteins and protein-inhibitor complexes; mass spectrometry with focus on deciphering conformational changes and protein-protein interfaces in macromolecular complexes) to decipher the structure-function relationships of E6AP and its interaction partners. Thereby, we obtained structural insights into the interaction of E6AP with two known interacting proteins - PSMD4 and HERC2 - that appear to be of critical importance for the pathophysiologic role of E6AP. Furthermore, we developed and refined several methods that eventually will contribute to dissect the complex network of E6AP interactions in the different disease settings. In parallel, in a dedicated high-throughput screen, we identified several compounds including isoalloxazine derivatives that not only stimulate the E3 ligase activity of wild-type E6AP, but also rescue the E3 ligase activity of distinct E6AP variants derived from individuals with Angelman Syndrome. Moreover, by mass spectrometry-based approaches we obtained evidence that the E6 oncoprotein as well as the activating compounds induce similar structural rearrangements in both wild-type E6AP and AS-derived E6AP variants, presumably resulting in the stabilization of an enzymatically active conformation. Thus, our data lay the foundation for the design of small molecules that affect E6AP function and eventually pave the way for new strategies in the treatment of E6AP-associated disorders.

Publications

• Structural dynamics of the E6AP/UBE3A-E6-p53 enzyme-substrate complex. Nature Communications, 9(1).
  Sailer, Carolin; Offensperger, Fabian; Julier, Alexandra; Kammer, Kai-Michael; Walker-Gray, Ryan; Gold, Matthew G.; Scheffner, Martin & Stengel, Florian
  
• Identification of Small-Molecule Activators of the Ubiquitin Ligase E6AP/UBE3A and Angelman Syndrome-Derived E6AP/UBE3A Variants. Cell Chemical Biology, 27(12), 1510-1520.e6.
  Offensperger, Fabian; Müller, Franziska; Jansen, Jasmin; Hammler, Daniel; Götz, Kathrin H.; Marx, Andreas; Sirois, Carissa L.; Chamberlain, Stormy J.; Stengel, Florian & Scheffner, Martin
  
• Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms. Nature Communications, 12(1).
  Gogl, Gergo; Tugaeva, Kristina V.; Eberling, Pascal; Kostmann, Camille; Trave, Gilles & Sluchanko, Nikolai N.
  
• Author Correction: Quantitative fragmentomics allow affinity mapping of interactomes. Nature Communications, 13(1).
  Gogl, Gergo; Zambo, Boglarka; Kostmann, Camille; Cousido-Siah, Alexandra; Morlet, Bastien; Durbesson, Fabien; Negroni, Luc; Eberling, Pascal; Jané, Pau; Nominé, Yves; Zeke, Andras; Østergaard, Søren; Monsellier, Élodie; Vincentelli, Renaud & Travé, Gilles
  
• Native holdup (nHU) to measure binding affinities from cell extracts. Science Advances, 8(51).
  Zambo, Boglarka; Morlet, Bastien; Negroni, Luc; Trave, Gilles & Gogl, Gergo