3 years ago

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1
Rajesh K. Singh, Yaniv Kazansky, Donald Wathieu, David Fushman
Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.6b04194

DOI: 10.1021/acs.analchem.6b04194

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.