Fig. 1. Cellular effects of E1 enzyme inhibitors vs. proteasome inhibitors. (A) A fraction of newly synthesized proteins misfold, followed by their polyubiquitination and proteasomal degradation. Thus, proteasome inhibitors cause the accumulation of the polyubiquitinated misfolded proteins, which induces ER stress and contributes to cell death. However, misfolded polyubiquitinated proteins can be cleared by an alternative degradation pathway, which requires ubiquitin tags on misfolded proteins and involves the formation of aggresomes. In contrast to proteasome inhibitors, E1 enzyme inhibitors would induce the accumulation of misfolded proteins, yet would not cause the formation of aggresomes due to the lack of ubiquitin tags on the misfolded proteins. (B) Dual inhibitors of ubiquitin and Nedd8 E1 enzymes inhibit ubiquitin conjugation. Ubiquitin is activated by E1 enzyme, transferred onto the catalytic cysteine of E2, and conjugated to the lysine of protein substrates in the presence of RING or Cullin-RING E3s (CRL E3s). Alternatively E2–Ub thioesters can transfer ubiquitin onto the catalytic cysteine of HECT or RBR E3s (not shown), which then conjugate the ubiquitin onto the lysine of protein substrates. CRL E3s require the covalent modification with Nedd8 to be activated. Therefore, dual inhibition of ubiquitin E1 and Nedd8 E1 would efficiently inhibit substrate ubiquitination.