Circadian modulation of proteasome activity and accumulation of oxidized protein in human embryonic kidney HEK 293 cells and primary dermal fibroblasts.

The circadian system orchestrates the timing of physiological processes of an organism living in daily environmental changes. Disruption of circadian rhythmicity has been shown to result in increased oxidative stress and accelerated aging. The circadian regulation of antioxidant defenses suggests that other redox homeostasis elements such as oxidized protein degradation by the proteasome, could also be modulated by the circadian clock. Hence, we have investigated whether proteasome activities and oxidized protein levels would exhibit circadian rhythmicity in synchronized cultured mammalian cells and addressed the mechanisms underlying this process. Using synchronized human embryonic kidney HEK 293 cells and primary dermal fibroblasts, we have shown that the levels of carbonylated protein and proteasome activity vary rhythmically following a 24h period. Such a modulation of proteasome activity is explained, at least in part, by the circadian expression of both Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the proteasome activator PA28αß. HEK 293 cells showed an increased susceptibility to oxidative stress coincident with the circadian-dependent lower activity of the proteasome. Finally, in contrast to young fibroblasts, no circadian modulation of the proteasome activity and carbonylated protein levels was evidenced in senescent fibroblasts. This paper reports a novel role of the circadian system for regulating proteasome function. In addition, the observation that proteasome activity is modulated by the circadian clock opens new avenues for both the cancer and the aging fields, as exemplified by the rhythmic resistance of immortalized cells to oxidative stress and loss of rhythmicity of proteasome activity in senescent fibroblasts.

Noncovalent fluorescent probes of human immuno- and constitutive proteasomes.

We report here the synthesis and biological evaluation of fluorescent probes functioning as inhibitors that noncovalently block human immuno- and constitutive proteasomes. These cell-penetrating linear analogues of the natural cyclopeptide TMC-95A were efficient on cells at the nanomolar level and assessed by confocal microscopy and flow cytometry. They may constitute an alternative to previously reported fluorescent probes that all bind covalently to proteasomes.

Circadian modulation of proteasome activities and removal of carbonylated proteins.

The circadian clock generates rhythms with a periodicity of 24hours of various biochemical and physiological processes. Recent data suggest a mutual influence between the circadian clock and the cell cycle, and provide a functional connection between the circadian clock, cancer and ageing. In addition, the established link between the circadian clock and anti-oxidative defence suggests that elements of the redox homeostasis, including oxidized protein degradation pathways such as the proteasome, could be modulated by the circadian clock. Using HEK cells synchronized by a serum shock as an initial cellular model for studying the circadian influence on protein maintenance, we have shown that the level of carbonylated protein varies rhythmically following a 24hours period as well as the level of ROS. The proteasome also exhibits circadian rhythmicity in either its expression levels or activities. The rhythms match the circadian oscillations observed for protein oxidative damage. Moreover, adaptation to a Nrf2-dependent oxidative stress has been associated with an increase in the cellular capacity to degrade oxidized proteins that is attributable to an increased expression of the 20S proteasome and its activator Pa28αß. Therefore, using our synchronized cellular models to investigate more precisely the modulation of proteasome function mediated by the circadian clock, we have shown that both Nrf2 and Pa28αß exhibit a circadian expression. Interestingly, the circadian variation in ROS precedes the Nrf2 protein level and the transcript level of proteasome catalytic subunits and activators. If as we envisage, circadian rhythmicity is involved in damaged protein degradation, the age-associated alteration of the circadian system may therefore contribute to the accumulation of oxidized proteins and the decline of intracellular protein maintenance. Hence, strategies that could restore this vital function may be effective in slowing ageing and the onset of diseases for which a defect in the protein homeostasis has been proposed to play a key role.

Dimerized linear mimics of a natural cyclopeptide (TMC-95A) are potent noncovalent inhibitors of the eukaryotic 20S proteasome.

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors used in cancer therapy. Starting from a noncovalent linear mimic of TMC-95A, a series of dimerized inhibitors using polyaminohexanoic acid spacers has been designed and optimized to target simultaneously two of the six active sites of the eukaryotic 20S proteasome. The homodimerized compounds actively inhibited chymotrypsin-like (Ki = 6-11 nM) and trypsin-like activities, whereas postacid activity was poorly modified. The noncovalent binding mode was ascertained by X-ray crystallography of the inhibitors complexed with the yeast 20S proteasome. The inhibition of proteasomal activities in human cells was evaluated. The use of the multivalency inhibitor concept has produced highly efficient and selective noncovalent compounds (no inhibition of calpain and cathepsin) that have potential therapeutic advantages compared to covalent binders such as bortezomib and carfilzomib.

Carbonylhydrazide-based molecular tongs inhibit wild-type and mutated HIV-1 protease dimerization.

We have designed and synthesized new molecular tongs based on a rigid naphthalene scaffold and evaluated their antidimer activity on HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide (azatide) fragments into their peptidomimetic arms to reduce hydrophobicity and increase metabolic stability. These fragments are designed to disrupt the protein-protein interactions by reproducing the hydrogen bond pattern found in the antiparallel ß-sheet formed between the N- and C-ends of the two monomers in the native PR. Kinetic analyses and fluorescent probe binding studies showed that several molecular tongs can inhibit PR dimerization. The best nonpeptidic molecular tongs to date were obtained with an inhibition constant K(id) of 50 nM for PR and 80 nM for the multimutated protease ANAM-11. The PR inhibition was selective, the aspartic proteases renin and pepsin were not inhibited.