carbobenzoxy-leucyl-leucyl-norvalinal and ubenimex

The cyclic expression of the period (PER) and timeless (TIM) proteins is critical for the molecular circadian feedback loop in Drosophila. The entrainment by light of the circadian clock is mediated by a reduction in TIM levels. To elucidate the mechanism of this process, the sensitivity of TIM regulation by light was tested in an in vitro assay with inhibitors of candidate proteolytic pathways. The data suggested that TIM is degraded through a ubiquitin-proteasome mechanism. In addition, in cultures from third-instar larvae, TIM degradation was blocked specifically by inhibitors of proteasome activity. Degradation appeared to be preceded by tyrosine phosphorylation. Finally, TIM was ubiquitinated in response to light in cultured cells.

Topics: Acetylcysteine; Animals; Biological Clocks; Cells, Cultured; Circadian Rhythm; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Darkness; Drosophila; Drosophila Proteins; Feedback; Insect Proteins; Leucine; Leupeptins; Light; Multienzyme Complexes; Neurons; Phosphorylation; Phosphotyrosine; Protease Inhibitors; Proteasome Endopeptidase Complex; Ubiquitins

A method was investigated for monitoring the activity of protease(s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation, which is known to be mediated by proteasome. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself.

Topics: Adenine; Aminopeptidases; Animals; Cell Membrane Permeability; Coumarins; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Fluorescent Dyes; Hydrolysis; Kinetics; Leucine; Leupeptins; Meiosis; Mesylates; Multienzyme Complexes; Oligopeptides; Oocytes; Proteasome Endopeptidase Complex; Spectrometry, Fluorescence; Starfish