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Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure, and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate because of impaired lysosomal proteases we studied the effects of advanced glycation end products on the endosomal-lysosomal system. Five different types of AGEs were generated by bovine serum albumin incubation with glyoxal, methylglyoxal, glucose, fructose, and ribose. The first experiments revealed the uptake of AGEs by the macrophage cell line RAW 264.7. Further investigations demonstrated an increase in cathepsin D and L activity and an increase in mature cathepsins D and L. Increased activities were accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the roles of cathepsins D and L we used knockout cells to test the roles of both cathepsins on the toxicity of advanced glycation end products. In summary we conclude that both cathepsins are required for a reduction in advanced glycation end product-induced cytotoxicity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report an entirely new role for the HSP70 chaperone in dissociating 26S proteasome complexes (into free 20S proteasomes and bound 19S regulators), preserving 19S regulators, and reconstituting 26S proteasomes in the first 1-3h after mild oxidative stress. These responses, coupled with direct 20S proteasome activation by poly(ADP ribose) polymerase in the nucleus and by PA28αβ in the cytoplasm, instantly provide cells with increased capacity to degrade oxidatively damaged proteins and to survive the initial effects of stress exposure. Subsequent adaptive (hormetic) processes (3-24h after stress exposure), mediated by several signal transduction pathways and involving increased transcription/translation of 20S proteasomes, immunoproteasomes, and PA28αβ, abrogate the need for 26S proteasome dissociation. During this adaptive period, HSP70 releases its bound 19S regulators, 26S proteasomes are reconstituted, and ATP-stimulated proteolysis is restored. The 26S proteasome-dependent, and ATP-stimulated, turnover of ubiquitinylated proteins is essential for normal cell metabolism, and its restoration is required for successful stress adaptation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Lipofuscin, a highly oxidized aggregate, consists of covalently cross-linked proteins, lipids, and sugar residues and is one of the major life-span-limiting factors in postmitotic aging cells. An artificial model of this material, showing characteristics and effects comparable to those of the natural form, has turned out to be very useful for in vitro studies. Artificial lipofuscin was used to investigate its effects on the viability of human fibroblasts, its rate of uptake, and its ability to inhibit the proteasomal system. The inhibition of the proteasomal system is one of the major aspects of the cytotoxic effects of lipofuscin. We present here that this proteasomal inhibition is due to proteasomal binding to the lipofuscin surface motifs, degradable by protease K. Furthermore, removal of the surface peptide structures by protease K strongly reduces the cytotoxic effects of lipofuscin and binding of cellular proteins and proteasomes to intracellular protein aggregates., (2010 Elsevier Inc. All rights reserved.)