Towards Uncovering the Mechanisms of Electrophile Sensing and Signaling

Native reactive electrophile species (RES) such as 4-hydroxynonenal (HNE) act as cellular signals through covalent modification of kinetically-privileged electrophile sensor proteins. Research efforts in the field focus mainly on identifying these sensor proteins and investigating the functional repercussions of their modification by a given RES. However, we lack structural information and reliable in vitro studies on kinetically-privileged electrophile sensor proteins substoichiometrically modified by RES to understand the molecular mechanisms underlying electrophile sensing and signaling. In particular, principles governing RES modification site selectivity or protein activity/function modulation resulting from a single modification event remain elusive. Here, we first introduce the state-of-the-art tools recently developed to identify, validate and functionally study electrophile sensor proteins. We also evaluate the structural data available for some of these proteins modified with native electrophiles or related small molecule drugs. We then report our ongoing work towards structure determination of the electrophile sensor protein Ube2v2 substoichiometrically labeled by HNE. In particular, we discuss our efforts to optimize reaction conditions allowing selective labeling of Ube2v2's kinetically-privileged and functionally relevant electrophile sensing site Cys69, and to develop a pulldown-based enrichment strategy to isolate the HNE-modified protein state. Finally, we describe how we generated Ube2v2(C69L) by site-directed mutagenesis and validated it as the first functional electrophile-mimetic of a RES-modified sensor protein through activity-based assays. Protein NMR spectroscopy investigations into the structure of this electrophile-mimetic allowed us to pinpoint a handful of Ube2v2 residues playing a key role in either electrophile sensing or resulting signaling events in a purified system. Following functional validation, we gained unique and valuable insights into the structure/function relationships underlying Ube2v2's HNE sensing ability and resulting signaling. Our work paves the way for structural investigations into other kinetically-privileged electrophile sensor proteins which are essential to expand our knowledge on the mechanisms underlying electrophile sensing and signaling.