cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases

Significance Most studies of the regulation of proteolysis by the ubiquitin proteasome system have focused on the control of ubiquitination. However, it is now clear that the activity of the 26S proteasome and rates of protein degradation in cells are also tightly regulated through proteasome phosphorylation. Here we demonstrate that agents that raise cGMP and activate cGMP-dependent protein kinase (e.g., widely used phosphodiesterase 5 inhibitors) stimulate proteasome activities and intracellular proteolysis without affecting autophagy. Furthermore, we showed that raising cGMP reduced the levels of the disease-causing mutant tau in a zebrafish model by increasing its degradation, and also decreased the associated morphological abnormalities. Thus, activating the proteasome via cGMP is a promising strategy to prevent the progression of neurodegenerative diseases.

was disposed and the pellet (nuclear fraction) was washed once more in STM buffer (1,000 x g for 10 minutes) and then resuspended in NET buffer (25 mM HEPES-KOH pH 7.5, 5 mM MgCl2, 100 mM NaCl, 10% glycerol, 1 mM ATP, 0.5 mM PMSF, 25 nM Calyculin A, 1 mM NaF, and 1 mM β-glycerophosphate). The crude nuclear fraction was incubated for 30 minutes on ice, sonicated, and then centrifuged at 9,000 x g for 30 minutes at 4 o C. The supernatant was collected as the nuclear fraction. The cytosolic/mitochondrial fraction was centrifuged at 10,000 x g for 10 minutes to remove the mitochondria and sonicated identically as the nuclear fraction. 5M NaCl was added to the cytosolic fraction to a final concentration of 100 mM, to make it equimolar with the nuclear fractions.
Relative protein concentration of cytosolic and nuclear fractions was measured by Bradford assay (ThermoFisher Scientific) and buffer was added to each sample to bring the fractions to the same relative protein concentration. 26S proteasomes were then purified from each fraction using the UBL-method (5).
Because lower amounts of 26S proteasomes were purified from the nuclear fractions of SH-SY5Y cells, 26S proteasomes from the cytosolic fractions were diluted to the concentration obtained from the nuclear fractions. An equal amount of proteasomes from each fraction was used in all assays.

Activation of PKG and ubiquitination in cytosolic fractions:
The cytosolic fraction from HEK293 cells was prepared in STM buffer as described above, plus IBMX (45 µM). The reaction was prepared on ice in STM buffer plus 0.5 mM EGTA and the following agents were added as indicated: cGMP (Promega), Bortezomib (UBPbio)(1µM), PR-619 (SelleckChemical)(10µM), 1,10phenanthroline (Sigma)(250µM). After the addition of all agents, the samples were incubated at RT for 5 minutes, and then incubated at 37 o C for 30 minutes.
The reaction was stopped by the addition of Laemmli buffer and boiling.
Measuring deubiquitination of proteins in cytosolic fractions: HEK293 cells were treated for 30 minutes with 1 µM Bortezomib, and then cytosolic fraction was prepared in STM buffer plus IBMX. The reaction was prepared on ice in STM buffer plus 0.5 mM EGTA plus Bortezomib (UBPbio)(1µM), Tak243 (1µM), and cGMP (1µM) where indicated. The samples were vortexed, incubated at RT for 5 minutes, and then incubated at 37 o C for the indicated times. The reaction was stopped by the addition of Laemmli buffer and boiling.
Measuring activity of deubiquitinases in cell lysates: SH-SY5Y cells were treated for 30 minutes with 100 nM of tadalafil, cinaciguat, or both and then lysed as described for measurements of proteasome activity. Active site modification of DUBs by HA-Ub-VS and hydrolysis of Ub-amc were performed as previously described (6).

Degradation of long-or short-lived cell proteins:
The degradation rate of long-or short-lived cell proteins was measured after labeling with [ 3 H]phenylalanine for different lengths of time, and the conversion of radiolabeled cell proteins to TCA-soluble radiolabeled amino acids in the media was assayed as described (7).

Affinity purification and activity measurements of 26S proteasomes: 26S
proteasomes were purified from cells in culture and zebrafish larvae using the ubiquitin-like domain (Ubl) method described previously (5). Cells were lysed in 25 mM HEPES-KOH pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM ATP, 1 mM DTT, and 0.5 mM PMSF plus the phosphatase inhibitors, 25nM Calyculin A, 10mM NaF, and 20mM β-glycerophosphate. Proteasomal peptidase activities were measured as described previously (8), and ATPase activity was assayed using the EnzCheck Phosphate Assay Kit (ThermoFisher Scientific). The ATPase assay was performed at 32 o C with 2 nM 26S proteasomes in 100 µL of the reaction buffer: 50 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM ATP, 1 mM DTT, 2% glycerol, and 0.1 mg/mL BSA. The rate of ATP hydrolysis was measured from the linear portion of the reaction. The fluorescent protein EOS was used to assay degradation of ubiquitinated proteins by 26S proteasomes. EOS was expressed, purified, ubiquitinated, and fractionated to achieve substrates of some uniform style as described previously (9). Polyubiquitinated EOS conjugated to ubiquitin chains containing 5-10 ubiquitins (50 nM) was incubated with 26S proteasomes

Compound treatment in zebrafish transgenic lines and Dendra-tau
phenotype determination: Embryos were collected in EM and reared at 28.5 °C. Larvae from the outcross of Rho::EGFP-TauWT, Rho::EGFP-TauP301L and Rho::EGFP-HD71Q with wild type fish were treated from 1 day post fertilization (d.p.f.) with EM containing 0.03% phenylthiourea (PTU) to prevent pigmentation. At 3 d.p.f, larvae were screened for EGFP expression in the rod photoreceptors and then removed from PTU and reared in EM. Larvae were treated either with 1 µM or 10 µM sildenafil, 3 µM or 30 µM rolipram or 0.1 % DMSO as a control from d.p.f. Drugs and medium were replenished daily. The percentage of larvae with different morphological phenotypes was quantified at 3 d.p.f. Phenotypes were classified either as normal; mild (when slight torsion of dorsal spine was observed or when head axis was not aligned to the dorsal spine but the larvae could swim straight); or severe (when fish showed a complete torsion of whole body in 'U'-shape) as previously described (14).

Supplemental Figure 1: PKG is predominantly localized to the cytosol in SH-SY5Y cells and co-purifies with 26S proteasomes in HEK293 cells with overexpression of PKG or treatment to raise levels of cGMP.
A.) Proteasomal chymotrypsin-like activity is increased by transient overexpression of PKG or the catalytic subunit of PKA. 24 hours after transfection with the indicated vectors, proteasomal chymotrypsin-like activity was measured in the cell lysates with suc-LLVY-AMC. n=3. Averages +/-SEM are shown. One-way ANOVA with Dunnett multiple comparison test. **p≤.0.01.
B.) PKG is predominantly localized to the cytosol. SH-SY5Y cells were treated with DMSO or tadalafil for 30 minutes and then separated by differential centrifugation into cytosolic and nuclear fractions. Equal amounts of protein from each fraction were analyzed by SDS-PAGE and western blot for PKG, the 20S proteasome subunit β5, GAPDH (cytosol), and LaminB1 (nucleus). Representative western blots from one of two experiments are shown.
C.) 26S proteasomes purified from HEK293 cells overexpressing PKG exhibit increased levels of co-purifying PKG and phosphorylated proteins. However, PKG overexpression did not increase the levels of phosphorylated Rpn6-S14, the proteasome subunit phosphorylated by PKA, or Rpt3-Thr25, the proteasome subunit phosphorylated by DYRK2. HEK293 cells stably over-expressing the proteasome subunit Rpn11 with both his-and biotin-tags (Rpn11-HTBH) were transfected with PKG, PKA catalytic subunit, or an empty vector. 24 hours after transfection, 26S proteasomes were purified via the biotin tag, and analyzed by SDS-PAGE and western blot with the indicated antibodies. Representative western blots of one of two proteasome purifications are shown.
D.) Increased levels of PKG co-purify with 26S proteasomes from HEK293 cells treated with cinaciguat, an activator of soluble guanylyl cyclase. HEK293 (Rpn11-HTBH) cells were exposed to DMSO or cinaciguat for 1 hour and then proteasomes were purified as in Supplemental Figure 1C. Representative western blots are shown for one of two proteasome purifications. A.) Puromycin-containing polypeptides were degraded more rapidly in cells treated with tadalafil. To generate incomplete proteins, SH-SY5Y cells were exposed for 1 hour to puromycin (5 μg/mL), which is incorporated into newly synthesized proteins and causes premature termination of their translation. The degradation of these puromycin-containing polypeptides was then followed in the presence of cycloheximide (150 µg/mL) with or without the addition of tadalafil. The puromycin-containing polypeptides were detected with an antibody against puromycin and the signal throughout the entire lane was quantified. Representative western blots of one of three experiments are shown. n=3. Averages +/-SEM.

Supplemental Figure 2
B.) The degradation of p53 was faster when cGMP is raised with tadalafil or BAY41-2272. SH-SY5Y cells were exposed to cycloheximide to block protein synthesis and pharmacological agents to raise cGMP. The amounts of p53 in the cell lysates were analyzed by western blot. Representative western blots are shown from one of three experiments. Averages +/-SEM. A.) The degradation of long-lived proteins is increased in SH-SY5Y cells by exposure to the indicated PDE5 inhibitors or soluble guanylyl cyclase stimulators. Cells were exposed to 3H-Phenylalanine for 16 hours, chased with excess non-labeled phenylalanine for 2 hours, and then the conversion from radiolabeled proteins to labeled-amino acids in the media was measured. These agents were added to the media after the chase period. n=3. Averages +/-SEM are shown.

Supplemental Figure 4
One-way ANOVA with Dunnett multiple comparison test. **p≤ 0.01. ***p≤0.001. A.) The breakdown of short-lived proteins is increased by exposure of BJ5A cells to cinaciguat (100nM), but not tadalafil (100 nM). This lack of stimulation by tadalafil is likely due to a low rate of cGMP synthesis. Combining cinaciguat and tadalafil to raise cGMP even further resulted in a greater increase in the degradation of the short-lived proteins. The degradation of short-lived proteins was performed as in  A.) Adding cGMP to cytosolic extracts of HEK293 cells increases the phosphorylation of VASP (Ser239), a well-characterized PKG substrate. HEK293 cells were lysed in a hypotonic buffer, and cytosolic extract was prepared by pelleting the nucleus (15 minutes at 800 x g) and then the mitochondria (10 minutes at 10,000 x g). After the addition of cGMP at the indicated concentrations, the extracts were incubated for 30 minutes at 37oC.  B.) The amount of assembled 26S proteasomes was not changed by 5 day treatment with 1 µM sildenafil or 3 µM rolipram. Lysates of zebrafish larvae were analyzed by native-PAGE and western blot with an antibody against the 19S subunit Rpn1. The same samples were also analyzed by SDS-PAGE and western blot for Rpn1 to evaluate loading. Representative western blots are shown.