Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load.

In order to assess the activity of cellular proteasome, we developed a method to permeabilize keratinocyte monolayers and measure proteasome activities intracellularly, using fluorogenic peptide substrates. The observed K(m) did not differ significantly in situ and in soluble extracts, and the K(i) of proteasome inhibitor MG132 was slightly higher in situ (34nM instead of 4nM). Inhibition studies in permeabilized cells showed that MG132 followed competitive inhibition patterns, and clasto-lactacystin beta-lactone non-competitive patterns, as expected. The observed velocities in situ (500pmoles/min/mg protein) were comparable to the best values of proteasome activity in crude cellular extracts. These features altogether allowed to identify the in situ activity as that of proteasome. To characterize proteasome complexes present in human keratinocytes, we analyzed cellular lysates by ultracentrifugation and gel filtration: most proteasome activity was associated with PA700-bound, presumably 26S, particles. PA28 activator was detected only when cells were treated by gamma interferon. Proteasome activities were determined using the in situ method in keratinocytes at different stages of replicative senescence. Only a slight decrease of proteasome activity per cell was seen at intermediate passages, followed by a slight increase in senescent cells. In the same time, the amount of total proteins increased notably with cellular ageing. Thus, proteasome activity decreased relatively to total proteins, but not relatively to cell numbers. Flow cytometry confirmed that the size of aged keratinocytes increased with the ageing marker beta-galactosidase.

Changes in rat liver mitochondria with aging. Lon protease-like reactivity and N(epsilon)-carboxymethyllysine accumulation in the matrix.

Aging is accompanied by a gradual deterioration of cell functions. Mitochondrial dysfunction and accumulation of protein damage have been proposed to contribute to this process. The present study was carried out to examine the effects of aging in mitochondrial matrix isolated from rat liver. The activity of Lon protease, an enzyme implicated in the degradation of abnormal matrix proteins, was measured and the accumulation of oxidation and glycoxidation (Nepsilon-carboxymethyllysine, CML) products was monitored using immunochemical assays. The function of isolated mitochondria was assessed by measuring respiratory chain activity. Mitochondria from aged (27 months) rats exhibited the same rate of oxygen consumption as those from adult (10 months) rats without any change in coupling efficiency. At the same time, the ATP-stimulated Lon protease activity, measured as fluorescent peptides released, markedly decreased from 10-month-old rats (1.15 +/- 0.15 FU x micro g protein-1 x h-1) to 27-month-old-rats (0.59 +/- 0.08 FU x micro g protein-1 x h-1). In parallel with this decrease in activity, oxidized proteins accumulated in the matrix upon aging while the CML-modified protein content assessed by ELISA significantly increased by 52% from 10 months (11.71 +/- 0.61 pmol CML x micro g protein-1) to 27 months (17.81 +/- 1.83 pmol CML x micro g protein-1). These results indicate that the accumulation of deleterious oxidized and carboxymethylated proteins in the matrix concomitant with loss of the Lon protease activity may affect the ability of aging mitochondria to respond to additional stress.