Insights into the regulation of CHIP E3 ligase-mediated ubiquitination of neuronal protein BNIP-H.

BCL2/adenovirus E1B 19-kDa protein-interacting protein 2 homolog (BNIP-H or Caytaxin), a pivotal adaptor protein that facilitates cerebellar cortex growth and synaptic transmission, is posttranslationally modified to regulate neuronal function. This study reports the ubiquitination of BNIP-H by Carboxyl terminus of Hsc70-Interacting Protein (CHIP), a U-box containing E3 ligase that is also regulated autoubiquitination. Specifically, it was observed that CHIP autoubiquitinated itself primarily at Lys23 and Lys31 . Mutation of these residues shows the autoubiquitination of successive lysines of CHIP. In total, nine lysines on CHIP were identified as the autoubiquitination sites, the collective mutation of which almost completely terminated its autoubiquitination. Additionally, CHIP-mediated ubiquitination of BNIP-H is completely inhibited when BNIP-H bears arginine mutations at four key lysine residues. Next, using hydrogen deuterium exchange mass spectrometry, a model of a plausible mechanism was proposed. The model suggests transient N-terminal interactions between the CHIP and BNIP-H which allows for the swinging of U-box domain of CHIP to ubiquitinate BNIP-H. Following complex dissociation, BNIP-H population is regulated the ubiquitin-proteasome pathway. Collectively, these results aid in our understanding of CHIP-mediated BNIP-H ubiquitination and provide further insight into the roles of these proteins in neuritogenesis and neurotransmission.