Hyperglycemia Impairs Proteasome Function by Methylglyoxal
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Figures
- FIG. 1.
Proteasome activity in endothelial cells under chronic hyperglycemia. Proteasome activity and 20S proteasome expression levels were analyzed in immortalized human endothelial Eahy.926 cells and in primary bovine retinal endothelial cells (BRECs) following incubation with low- and high-glucose medium for 14 days. Proteasomal trypsin-like (A) and caspase-like (B) activities following incubation with high-glucose medium were not significantly changed in comparison with those following low-glucose medium, whereas the proteasomal chymotrypsin-like activity was significantly reduced (C). The level of the catalytic 20S proteasome remained constant, while the 19S regulatory complex expression was decreased, as shown by Western blot (D). Data represent means ± SD from at least five separate experiments. *P ≤ 0.05. RFU, relative fluorescence unit.
- FIG. 2.
Resistance of glycated albumin against proteasomal degradation in vitro. AGE-BSA (10 μg) was incubated with isolated 20S proteasome to examine the influence on proteasomal activity. The degree in carboxy-methyl-lysine modification of BSA (as demonstrated by Western blotting) did not significantly affect the chymotrypsin-like activity of the 20S proteasome. Ponceau S staining is shown as a loading control. Data represent means ± SD from at least three separate experiments. RFU, relative fluorescence unit.
- FIG. 3.
Inhibition of proteasomal chymotrypsin-like activity in vitro by reactive carbonyl compounds. Reactive carbonyl compounds were incubated with the 20S proteasome and the enzymatic activities determined. Trypsin-like (A) and caspase-like (B) activities were not significantly changed, whereas the chymotrypsin-like activity was significantly reduced (C). Data represent means ± SD from at least three separate experiments. *P ≤ 0.01. GA, glyceraldehyde; GO, glyoxal; RFU, relative fluorescence unit.
- FIG. 4.
Inhibition of proteasomal chymotrypsin-like activity in endothelial cells and accumulation of ubiquitinated proteins. Immortalized human endothelial Eahy.926 cells and primary bovine retinal endothelial cells were each incubated for 12 h with 500 μmol/l reactive carbonyl compounds. Trypsin-like (A) and caspase-like (B) activities were not changed, whereas the chymotrypsin-like activity was significantly reduced after incubation with each of the three reactive carbonyl compounds (C). Incubation with reactive carbonyl compounds exhibited accumulation of ubiquitinated proteins in a dose-dependent manner (D). Data represent means ± SD from at least three separate experiments. *P ≤ 0.05. GA, glyceraldehyde; GO, glyoxal; RFU, relative fluorescence unit.
- FIG. 5.
Decreased proteasomal activity in organs from diabetic Ins2Akita mice. Proteasomal activity and proteasome expression was determined in kidney, heart, and aorta from diabetic Ins2Akita mice (n = 6) and control wild-type mice (n = 6). The three proteolytic activities of the proteasome were measured with fluorogenic substrates. Trypsin-like (A) and caspase-like (B) activities were significantly reduced in kidneys from diabetic mice. The chymotrypsin-like activity (C) was significantly reduced in kidney and aorta, whereas in the heart no significant changes were seen. The 20S proteasome protein levels were not significantly changed in any organs, as shown by Western blotting (D and E). Data represent means ± SD from at least three separate experiments. *P ≤ 0.05.
- FIG. 6.
Increased MGO- and ubiquitin-modified proteins with impaired proteasomal activity in GLO1 knockdown and diabetic mice. Kidney lysate protein (40 μg) from wild-type (WT), GLO1-knockdown (GLO1-KD), and STZ diabetic and GLO1-knockdown diabetic (GLO1-KD-STZ) mice were analyzed by Western blotting. GLO1-knockdown mice exhibited a significant increase in MGO- and ubiquitin-modified proteins in kidney homogenates compared with levels in wild-type mice (A and B). GLO1-knockdown and STZ-injected mice exhibited significantly reduced proteasomal chymotrypsin-like activity in kidney homogenates compared with that in wild-type mice (C). The protein band densities were scanned and quantified using the Odyssey imaging system. Data are expressed as means ± SD. *P < 0.05 vs. wild type; n = 4. RFU, relative fluorescence unit. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 7.
Decreased 19S-S5a protein levels and MGO-modified 20S-β2 proteasome subunit in GLO1-knockdown (GL01-KD) and STZ diabetic mice. Kidney lysate protein (40 μg) from indicated mice were analyzed on NUPAGE 4–12% Bis-Tris gels, transferred to PVDF membranes, and blotted by 19S-S5a antibody (A). The lysates from indicated mice kidney tissues were immunoprecipitated with 20S-β2 antibody and analyzed by Western blotting, and 10% whole lysates were used for β-actin detection as input. MGO modification of the 20S proteasome β2-subunit was detected in kidneys from GLO1-knockdown and STZ diabetic mice (B). The protein band densities were scanned and quantified using the Odyssey imaging system. Data are expressed as means ± SD. *P < 0.05 vs. wild type; n = 4. RFU, relative fluorescence unit. (A high-quality digital representation of this figure is available in the online issue.)
Tables
- TABLE 1
20S proteasome modification after incubation with MGO in vitro
Subunit Peptide Sequence Modification β5 117–123 LLARQCR 7:methylimidazolone β5 124–130 IYELRNK 5:methylimidazolone β2 30–41 DDHDKMFKMSEK 8:carboxyethyllysine β2 69–85 MRNGYELSPTAAANFTR 17:methylimidazolone β4 217–227 CMRVLYYRDAR 8:arg-pyrimidin β4 220–231 VLYYRDARSYNR 5:methylimidazolone β4 225–240 DARSYNRFQIATVTEK 7:methylimidazolone
