Protein Oxidative Modifications and Replicative Senescence of WI-38 Human Embryonic Fibroblasts

摘要

The age-related accumulation of oxidized proteins is de-pendent on the balance between the generation of oxidatively modifiedproteins and their elimination by protein degradation and repair sys-tems. Previous studies have demonstrated that replicative senescencerepresents a valid model of in vitro aging and that senescent cells do ac-cumulate oxidized proteins while both proteasome, which is the major in-tracellular proteolytic system implicated in the removal of abnormal andoxidized proteins, and the oxidized protein-repair enzymes, methioninesulfoxide reductases, are being impaired. Declining proteasome activitywith age has been attributed to decreased proteasome subunits expres-sion and/or inactivation upon alteration of proteasome subunits, as wellas accumulation of endogeneous inhibitors, such as highly oxidized andcross-linked proteins. To gain further insight into the mechanisms thatmight be implicated in the decreased activity of the proteasome withreplicative senescence, the occurrence of proteins modified by glycoxi-dation and conjugation by lipid peroxidation products has been investi-gated in senescent cells. Indeed, such modification as the formation ofprotein adducts with the lipid peroxidation product 4-hydroxy-2-nonenalcan generate cross-linked proteins that become resistant to degradationby the proteasome and can act as inhibitors of the proteasome. Usingspecific antibodies that recognize glycoxidation and lipid peroxidationadducts on proteins, both modifications were demonstrated and foundto increase in senescent cells when compared with young fibroblasts.Moreover, the patterns of modified proteins obtained after separationby SDS gel electrophoresis were indicative of preferential protein targetsfor both modifications.