Protein ubiquitination requires the concerted action of the E1, E2, and E3 ubiquitin-conjugating enzymes. Ubiquitin is first activated through ATP-dependent formation of a thiol ester with ubiquitin-activating enzyme E1. The activated ubiquitin is then transferred to a thiol group of ubiquitin-carrier enzyme E2. The final step is the transfer of ubiquitin from E2 to an ε-amino group of the target protein lysine residue, which is mediated by ubiquitin-ligase enzyme E3. E2-25K (Hip2) is a member of the E2 protein family that catalyzes multiubiquitin chain synthesis via Lys48 of ubiquitin. E2-25K is reportedly involved in Alzheimer's disease, Huntington's disease and antigen processing through its interaction with amyloid-β, huntingtin, and MHC-heavy chain proteins. Lys14 of E2-25K can be modified by SUMOylation, with this modification resulting in inhibited E2 activity.