Differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine.
To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition of profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25 microM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (Suc-LLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl -7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100 nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50 1.20 microM), but the inhibition of proteasome was weak (IC50S for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110 microM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA- and Suc-LLVY-MCA-degrading activities in proteasome were 1,100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20 nM and 10 microM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentration of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100 microM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology.
Tsubuki
S
S
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science (Rinsko-ken). tsubuki@rinshoken.or.jp
Saito
Y
Y
Tomioka
M
M
Ito
H
H
Kawashima
S
S
D000818:Animals; D001329:Autolysis; D002154:Calpain / Q000037:antagonists & inhibitors*; D002417:Cattle; D003374:Coumarins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D007976:Leupeptins / Q000378:metabolism*; D008858:Microscopy, Phase-Contrast; D009097:Multienzyme Complexes / Q000378:metabolism*; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure; D009842:Oligopeptides / Q000378:metabolism*; D016716:PC12 Cells; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D051381:Rats
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; C084513:benzyloxycarbonylleucyl-leucyl-leucyl-4-methyl-coumaryl-7-amide; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde