Hyperglycemia impairs proteasome function by methylglyoxal.
OBJECTIVE
The ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo.


METHODS
MGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2(Akita) mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice.


RESULTS
In vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2(Akita), STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-beta2 subunit was increased.


CONCLUSIONS
Hyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions.
Queisser
Markus A
MA
School of Medicine, Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany.
Yao
Dachun
D
Geisler
Sven
S
Hammes
Hans-Peter
HP
Lochnit
Günter
G
Schleicher
Erwin D
ED
Brownlee
Michael
M
Preissner
Klaus T
KT
D000418:Albumins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005947:Glucose / Q000633:toxicity; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D066298:In Vitro Techniques; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D007791:Lactoylglutathione Lyase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011765:Pyruvaldehyde / Q000378:metabolism*
D000418:Albumins; D017127:Glycation End Products, Advanced; D007328:Insulin; D025821:Polyubiquitin; D011765:Pyruvaldehyde; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex; D007791:Lactoylglutathione Lyase; D005947:Glucose