ABSTRACT
Repeated mild heat shock (RMHS) has been shown to have several beneficial hormetic effects on human skin fibroblast undergoing aging in vitro. Because an age-related decline in proteasome activity is 1 of the reasons for the accumulation of abnormal proteins during aging, we have investigated the effects of RMHS on the 20S proteasome, which is the major proteolytic system involved in the removal of abnormal and oxidatively damaged proteins. Serially passaged human skin fibroblasts exposed to RMHS at 41 degrees C for 60 minutes twice a week had increased 3 proteasomal activities by 40% to 95% in early- and midpassage cultures. RMHS-treated cells also contained a 2-fold higher amount of the proteasome activator 11S, and the extent of the bound activator was double in early- and midpassage cells only. Furthermore, there was no difference in the content of the 19S proteasome regulator in the stressed and the unstressed cells. Therefore, RMHS-induced proteasome stimulation in early- and midpassage fibroblasts appears to be due to an induction and enhanced binding of 11S proteasome activators. In contrast to this, the proteasomal system in late-passage senescent cells appears to be less responsive to the stimulatory effects of mild heat shock.
Subject(s)
Adenosine Triphosphatases/metabolism , Cellular Senescence/physiology , Endopeptidases/metabolism , Fibroblasts/physiology , Heat-Shock Response/physiology , Proteasome Endopeptidase Complex/metabolism , Adenosine Triphosphatases/analysis , Blotting, Western , Cells, Cultured , Chromatography, Gel , Endopeptidases/analysis , Enzyme-Linked Immunosorbent Assay , Fibroblasts/chemistry , Fibroblasts/metabolism , Humans , Oligopeptides/metabolism , Proteasome Endopeptidase Complex/analysis , Protein Binding , Substrate SpecificityABSTRACT
A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing alpha-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that alpha-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric alpha-synuclein. Proteasome binding to insoluble alpha-synuclein filaments and soluble alpha-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Abeta peptide aggregates. Endogenous ligands of aggregated alpha-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.
Subject(s)
Multienzyme Complexes/antagonists & inhibitors , Nerve Tissue Proteins/chemistry , Chymotrypsin/chemistry , Cysteine Endopeptidases/chemistry , Dose-Response Relationship, Drug , Erythrocytes/metabolism , HSP70 Heat-Shock Proteins/chemistry , Humans , Immunohistochemistry , Lewy Bodies/metabolism , Ligands , Microscopy, Confocal , Microscopy, Electron , Multienzyme Complexes/chemistry , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Proteasome Endopeptidase Complex , Protein Binding , Recombinant Proteins/chemistry , Synucleins , Time Factors , alpha-SynucleinABSTRACT
In a series of experimental studies we have shown that repetitive mild heat stress has anti-aging hormetic effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. We have reported the hormetic effects of repeated challenge at the levels of maintenance of stress protein profile; reduction in the accumulation of oxidatively and glycoxidatively damaged proteins; stimulation of the proteasomal activities for the degradation of abnormal proteins; improved cellular resistance to ethanol, hydrogenperoxide, and ultraviolet-B rays; and enhanced levels of various antioxidant enzymes. We are now undertaking a detailed analysis of the signal transduction pathways to determine alterations in the phosphorylation and dephosphorylation states of extracellular signal-related kinase, c-Jun terminal kinase and p38 MAP-kinases as a measure of cellular responsiveness to mild and severe heat stress. Furthermore, we are also undertaking comparative studies using non-aging immortal cell lines, such as SV40-transformed human fibroblasts, spontaneous osteosarcoma cells, and telomerase-immortalized human bone marrow cells for establishing differences in normal and cancerous cells with respect to their responsiveness to mild and severe stresses.
ABSTRACT
Repeated mild heat-shock (RMHS) treatment has anti-aging hormetic effects on human fibroblasts undergoing aging in vitro. Since heat and various other stresses induce the transcription and translation of heat-shock proteins (Hsp), it was investigated if RMHS treatment affected the basal levels of four major stress proteins Hsp27, 70, 90 and Hsc70. The basal levels of Hsp27, Hsc70, and Hsp70 increased significantly in late passage senescent cells, which is indicative of an adaptive response to cumulative intracellular stress during aging. RMHS increased the levels of these Hsp even in early passage young cells and were maintained high throughout their replicative lifespan. In comparison, the amount of Hsp90 decreased both with aging and RMHS treatment in vitro. However, whereas the difference in the levels of Hsp70 and Hsp90 was statistically significant, the levels of Hsp27 and Hsc70 were statistically similar in normal and RMHS-treated serially passaged cells. These alterations were accompanied by an improved functional and survival ability of the cells in terms of increased proteasomal activities, increased ability to decompose H(2)O(2), reduced accumulation of lipofuscin and enhanced resistance to ethanol, H(2)O(2) and UV-A radiation.
