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. 2020 Jun 8;117(25):14220–14230. doi: 10.1073/pnas.2003277117

Fig. 1.

Fig. 1.

Pharmacological agents that raise cGMP stimulate 26S proteasome activities without changing proteasome amount. (A) Treatment of human neuroblastoma cells (SH-SY5Y) with the PDE5 inhibitor tadalafil (100 nM), the soluble guanylyl cyclase stimulator BAY41-2272 (100 nM), or a combination of the two increased proteasomal chymotrypsin-like activity in cell lysates. The linear rate of Suc-LLVY-AMC hydrolysis was used here and below as the measure of activity. In this and subsequent figures, error bars represent the means ± SEM. n = 4. (B) Levels of the 20S proteasome subunit β5 or the 19S subunit Rpn6 were not changed by treatments with pharmacological agents that raise cGMP. SH-SY5Y cells were treated with tadalafil (100 nM), BAY41-2272 (100 nM), or both, and the levels of proteasome subunits were analyzed by western blot. Representative western blots for one of three experiments is shown. (C) The amount of assembled 26S proteasomes was not changed by exposure of SH-SY5Y cells to the PDE5 inhibitor tadalafil. Cell lysates were analyzed by native PAGE and western blot with an antibody against the 19S subunit Rpn1. The same samples were also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot with an antibody against GAPDH to evaluate protein loading. Representative western blots for one of four experiments is shown. (D) After treatment of SH-SY5Y cells with these same agents (100 nM) for 30 min to raise cGMP, 26S proteasomes were affinity purified by the UBL method and exhibited greater chymotrypsin-like, caspase-like, and trypsin-like activities than those from control cells. n = 3 proteasome purifications. One-way ANOVA with Dunnett multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (E) 26S proteasomes purified from SH-SY5Y cells treated to raise cGMP as in B hydrolyzed ATP more quickly than proteasomes from control (Ctrl) cells. n = 3 proteasome purifications. One-way ANOVA with Dunnett multiple comparison test. *P ≤ 0.05, **P ≤ 0.01. (F) 26S proteasomes purified from SH-SY5Y cells treated with tadalafil (Tad) or BAY41-2272 (Bay) as in B degraded the fluorescent protein mEOS3.2 conjugated to a single K48-linked polyubiquitin chain more rapidly than proteasomes from control cells. n = 3 proteasome purifications. One-way ANOVA with Dunnett multiple comparison test. *P ≤ 0.05.