Nitric oxide-mediated inhibition of the PA28 proteasome activator

Abstract

Introduction: The 26S proteasome degrades most cell cycle proteins and is centrally involved in regulating cell proliferation. We previously showed that nitric oxide (NO) inhibits the 26S proteasome in vascular smooth muscle cells (VSMC). The enzymatic activity of the 26S proteasome is further regulated by the proteasome activator, PA28. However, the stimulatory effect of the PA28 activator has not been studied in VSMC. The purpose of this study was two-fold: 1) to evaluate the effect of the PA28 activator in VSMC; and 2) to determine the effect of NO on the activity and expression of the PA28 activator in VSMC. Methods: A proteasome activity assay was used to determine the functional activity of the 26S proteasome with and without the PA28 activator. Whole cell lysate was collected from rat aortic VSMC. Reaction buffer, ATP, +/− recombinant PA28, and +/− increasing concentrations of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) were added to the lysate. To this reaction, one of three fluorogenic 26S proteasome-specific peptide substrates (Suc-LLVY-AMC[chymotrypsin-like], Bz-VGR-AMC[trypsin-like], or Z-LLE-AMC[caspase-like]) was added. Proteasome activity was determined using a fluoroscent microplate reader with excitation and emission wavelengths of 380s460, respectively. Additionally, protein expression of the PA28 subunits (alpha, beta, and gamma) were examined by Western blot analysis in VSMC treated with and without SNAP. Data is expressed as the mean +/− standard error of the mean. Statistical significance was determined using analysis of variance and assumed for P-values < 0.05. Results: In VSMC, the PA28 activator (0.1-100 nM) increased the chymotrypsin- (10,925+/−403 PA28 1nM vs 6,797+/−82 control), trypsin- (16,005+/−203 PA28 1nM vs 10,146+/−139 control), and caspase-like (28,107+/−285 PA28 1nM vs 12,041+/−552 control) proteolytic activities of the 26S proteasome in a time and concentration-dependent manner. This increase in activity was greatest for caspase-like activity (133%) and least for chymotrypsin- and trypsin-like activity (60.7s57.7%, respectively). The NO-donor SNAP inhibited all three enzymatic activities of the 26S proteasome by 33.1 - 40.8% in a time (0-120 minutes) and concentration-dependent (5-500 uM) manner (P<0.05). However, SNAP-mediated inhibition of the PA28 activator was much more dramatic: SNAP (500 uM) completely inhibited PA28-induced proteasome activity for all three substrates (P<0.05, see Figure). This inhibition was time (0-120 minutes) and concentration-dependent (5-500 uM). Western blot analysis of VSMC treated with SNAP (125-500 uM) x 24 hours showed no difference in the protein expression of the alpha, beta, or gamma subunits of the PA28 activator versus control VSMC. Conclusions: This study shows that the PA28 activator is a potent stimulator of all three proteolytic activities of the 26S proteasome in VSMC but increases caspase-like activity to the greatest degree. NO effectively inhibits the activity of the PA28 activator but has no effect on the protein expression of the different PA28 subunits. This data provides further insight into the role of the 26S proteasome in VSMC and one mechanism by which NO may inhibit VSMC proliferation.