Characterization of CAA0225, a novel inhibitor specific for cathepsin L, as a probe for autophagic proteolysis.
We screened a series of new epoxysuccinyl peptides for the development of a lysosomal cathepsin L-specific inhibitor. Among the compounds tested, (2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide} (compound CAA0225) was the most potent inhibitor of cathepsin L. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). To assess the contribution of cathepsin L to lysosomal proteolysis, we evaluated autophagy, which is the process of lysosomal self-degradation of cell constituents. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions CAA0225 significantly inhibited degradation of long-lived proteins; however, the magnitude of inhibition was comparable to that in the presence of CA-074-OMe, which is a cathepsin B-specific inhibitor. Thus the contributions of cathepsin L and cathepsin B to autophagic protein degradation of cytoplasmic proteins are nearly equal. During autophagy, microtubule-associated protein IA/IB light chain 3-II (LC3-II) and gamma-aminobutyric acid (A) receptor-associated protein (GABARAP)-II, which are specific markers of autophagosomal membranes that engulf cytoplasmic components, also undergo degradation upon fusion of autophagosomes with lysosomes. CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore we conclude that cathepsin L does not play a general role in the degradation of proteins in the lumen of autophagosomes, but rather is involved specifically in the degradation of autophagosomal membrane markers.
Takahashi
Katsuyuki
K
Clinical Laboratory Department, Nihon University School of Medicine, Tokyo, Japan.
Ueno
Takashi
T
Tanida
Isei
I
Minematsu-Ikeguchi
Naoko
N
Murata
Mitsuo
M
Kominami
Eiki
E
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy; D001593:Benzyl Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002401:Cathepsin B / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004151:Dipeptides; D004852:Epoxy Compounds; D005027:Ethylene Oxide / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives; D051381:Rats; D017208:Rats, Wistar; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
C540134:(2S,3S)-oxirane-2,3-dicarboxylic acid 2-(((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide) 3-((2-(4-hydroxy-phenyl)-ethyl)-amide); D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D001593:Benzyl Compounds; D004151:Dipeptides; D004852:Epoxy Compounds; C116741:GABARAP protein, human; D008869:Microtubule-Associated Proteins; D010649:Phenylalanine; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse; C534280:Ctsl protein, rat; D005027:Ethylene Oxide