The formation and accumulation of D-aspartate residue (D-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55-65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human alpha A-crystallin and its corresponding diastereoisomers in which L-alpha-Asp was replaced with L-beta-, D-alpha- or D-beta-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at D-alpha-Asp(58), independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal D-alpha-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious D-Asp-containing protein in the long life span of mammals. (C) 2011 Published by Elsevier B.V.