l.pone.0064042.g003 subunit a1) chromatographically purified from cerebrum and cerebellum of young and aged rats and measured the order BIRB-796 substrate degradation by means of immunoblotting as described previously . Cerebral 26S proteasomes of aged rats exhibited an approximately 2.7-fold increased poly-Ub-substrate degradation activity when compared with the corresponding 26S proteasomes of young rats. Whereas the 26S proteasomes from aged rats had degraded 50% of the substrate after 80 min, the enzyme of young animals degraded only 18% within the same period of time. 26S proteasomes from cerebellum exhibited, independent of age, a considerably higher poly-Ubsubstrate degradation activity than cerebral 26S proteasomes. Nevertheless, 26S proteasomes from cerebellum of aged rats also possessed an approximately 2-fold 9570468 increased poly-Ub-substrate degradation activity compared with 26S proteasomes from young rats. To substantiate this finding we additionally used polyubiquitinated GST-tagged UbcH5 as a substrate. This GSTtagged ubiquitin-conjugating enzyme can be isolated as an auto-poly-ubiquitinated protein when preincubated with purified E1 and ubiquitin. As shown in Fig S4 this protein exists in various poly-ubiquitinated forms. There was no age-dependent difference in the rate of degradation of this substrate by 26S proteasomes from cerebrum and cerebellum. These data show that 26S proteasomes from aged rats despite their decreased activity towards short fluorogenic peptide substrates exhibited the same or even enhanced poly-Ub-substrate degradation activity when compared with 26S proteasomes of young rats. Previously it was shown that binding of a poly-ubiquitinated substrate to the 26S proteasomes results in the activation of the proteasomal peptide hydrolysing activity. When we tested the effect of Ub5Muc4 substrate binding to 26S proteasomes isolated from cerebrum of young and aged animals a similar result was obtained. 10760364 Strikingly, the peptide hydrolysing activity of 26S proteasomes of aged animals was stronger activated by binding of Ub5Muc4 substrate than 26S proteasomes of young animals. This activating effect was even higher when poly-UbGST-UbcH5 was used as a substrate, however with this substrate no difference was observed between 26S proteasomes from young and aged rats. Discussion The process of aging most likely combines an intrinsic cellular senescence program with environmental effects potentially imposing harmful attacks on the organism. Aging also includes changes of the finely tuned cellular proteostasis that is based on the balance between protein biosynthesis and degradation. Any alteration of this balance may also affect the cellular protein degradation machinery. Thus, impairment of the protein degradation rate can result in an accumulation of proteins that may be non-functional any more or mis-folded. If not eliminated such defective proteins Unimpaired 26S Proteasome Activity in Aging Brain are prone to forming aggregates, which eventually will induce cell death, a mechanism proposed to be responsible for the development of neurodegenerative diseases. In the present study we have analysed and compared the molecular composition and protein degradation activity of 20S/ 26S proteasomes in three different parts of the brain from young and aged rats. Our analyses show that proteasomes in young and Unimpaired 26S Proteasome Activity in Aging Brain dependent alterations of proteasomes take place in the brain. These may also go al