The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf 2 activity through the interaction with Nrf 2 Neh2 domain. However, the mechanism(s) of Nrf 2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule.

Novel HIV protease inhibitors containing a hydroxyethylamine dipeptide isostere as a transition state-mimic king structure were synthesized by combining substructures of known HIV protease inhibitors. Among them, TYA5 and TYB5 were proven to be not only potent enzyme inhibitors (K(i) = 0.12 nM and 0.10 nM, respectively) but also strong anti-HIV agents (IC(50) = 9.5 nM and 66 nM, respectively), even against viral strains with multidrug resistance. Furthermore, insertion of an (E)-alkene dipeptide isostere at the P(1)-P(2) position of TYB5 led to development of a purely nonpeptidic protease inhibitor, TYB1 (K(i) = 0.38 nM, IC(50) = 160 nM).

A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine-128 residue in SV40 T-antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78 þ 0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm 2 ). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally. ß