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 1-3 hours following 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 provides cells with increased capacity to degrade oxidatively damaged proteins and to survive the initial effects of stress exposure. Subsequent adaptive (hormetic) processes (3-24 hours following 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.

Lon now emerges as a major regulator of multiple mitochondrial functions in human beings. Lon catalyzes the degradation of oxidatively modified matrix proteins, chaperones the assembly of inner membrane complexes, and participates in the regulation of mitochondrial gene expression and genome integrity. An early result of Lon downregulation in WI-38 VA-13 human lung fibroblasts is massive caspase 3 activation and extensive (although not universal) apoptotic death. At a later stage, the surviving cells fail to divide, display highly abnormal mitochondrial function and morphology, and rely almost exclusively on anaerobic metabolism. In a selected subpopulation of cells, the mitochondrial mass decreases probably as a result of mitochondrial inability to divide. At this final point the Lon-deficient cells are not engaged anymore in apoptosis, and are lost by necrosis or "mitoptosis." Our results indicate that mitochondrial Lon is required for normal survival and proliferation; a clear impetus for Lon's evolutionary conservation.

A decline in mitochondrial biogenesis and mitochondrial protein quality control in skeletal muscle is a common finding in aging, but exercise training has been suggested as a possible cure. In this report, we tested the hypothesis that moderate-intensity exercise training could prevent the age-associated deterioration in mitochondrial biogenesis in the gastrocnemius muscle of Wistar rats. Exercise training, consisting of treadmill running at 60% of the initial V̇o2max, reversed or attenuated significant age-associated (detrimental) declines in mitochondrial mass (succinate dehydrogenase, citrate synthase, cytochrome- c oxidase-4, mtDNA), SIRT1 activity, AMPK, pAMPK, and peroxisome proliferator-activated receptor gamma coactivator 1-α, UCP3, and the Lon protease. Exercise training also decreased the gap between young and old animals in other measured parameters, including nuclear respiratory factor 1, mitochondrial transcription factor A, fission-1, mitofusin-1, and polynucleotide phosphorylase levels. We conclude that exercise training can help minimize detrimental skeletal muscle aging deficits by improving mitochondrial protein quality control and biogenesis.

The targeted removal of damaged proteins by proteolysis is crucial for cell survival. We have shown previously that the Lon protease selectively degrades oxidized mitochondrial proteins, thus preventing their aggregation and cross-linking. We now show that the Lon protease is a stress-responsive protein that is induced by multiple stressors, including heat shock, serum starvation, and oxidative stress. Lon induction, by pre-treatment with low-level stress, protects against oxidative protein damage, diminished mitochondrial function, and loss of cell proliferation, induced by toxic levels of hydrogen peroxide. Blocking Lon induction, with Lon siRNA, also blocks this induced protection. We propose that Lon is a generalized stress-protective enzyme whose decline may contribute to the increased levels of protein damage and mitochondrial dysfunction observed in ageing and age-related diseases.

The elimination of oxidatively modified proteins is a crucial process in maintaining cellular homeostasis, especially during stress. Mitochondria are protein-dense, high traffic compartments, whose polypeptides are constantly exposed to superoxide, hydrogen peroxide, and other reactive species, generated by ‘electron leakage’ from the respiratory chain. The level of oxidative stress to mitochondrial proteins is not constant, but instead varies greatly with numerous metabolic and environmental factors. Oxidized mitochondrial proteins must be removed rapidly (by proteolytic degradation) or they will aggregate, cross-link, and cause toxicity. The Lon Protease is a key enzyme in the degradation of oxidized proteins within the mitochondrial matrix. Under conditions of acute stress Lon is highly inducible, possibly with the oxidant acting as the signal inducer, thereby providing increased protection. It seems that under chronic stress conditions, however, Lon levels actually decline. Lon levels also decline with age and with senescence, and senescent cells even lose the ability to induce Lon during acute stress. We propose that the regulation of Lon is biphasic, in that it is up-regulated during transient stress and down-regulated during chronic stress and aging, and we suggest that the loss of Lon responsiveness may be a significant factor in aging, and in age-related diseases.

Mitochondria are a major intracellular source of free radicals and related oxidants. It is generally agreed that the mitochondrial production of such reactive oxygen and nitrogen species increases with age. Antioxidant systems in the mitochondria play an important role in limiting the amount of oxidative damage to tolerable levels. The Lon protease degrades oxidatively modified proteins in the mitochondrial matrix, a function similar to that of the 20S proteasome in the cytoplasm. Recently it was shown that inactive aconitase, a preferential substrate for the Lon protease, might be involved in the maintenance of the mitochondrial genome. Lon protease expression and activity declines with age, which may contribute to the accumulation of the oxidatively modified protein aggregates typically observed in aging and diseased cells. In addition, Lon has multiple functions, such as DNA binding and chaperone activity, for the assembly of respiratory complexes in the Electron Transport Chain. Taken together, Lon and aconitase may be key players in the maintenance of mitochondrial homeostasis under conditions of stress, and (partial) compromise of their function may contribute to both aging and degenerative diseases.

Oxidative damage to mitochondrial proteins is thought to contribute to the aging process, but the Lon protease normally degrades such proteins. In early-passage WI-38 human lung fibroblasts, Lon expression is rapidly induced during H(2)O(2) stress, which prevents the accumulation of oxidized proteins and protects cell viability. In contrast, middle passage cells exhibit only sluggish induction of Lon expression in oxidative stress, and oxidized proteins initially accumulate. Late-passage, or senescent, cells have low basal levels of Lon and high levels of accumulated oxidized proteins; in response to oxidative stress, they fail to induce Lon expression and exhibit continually increasing accumulation of oxidized proteins. Senescent cells separated into two populations, one exhibiting normal mitochondrial mass and a second displaying significant loss of mitochondria; both populations had diminished mitochondrial transmembrane potential. These senescent changes are similar to the effects of Lon silencing in young cells. We suggest that loss of Lon stress inducibility is part of a pattern of diminishing stress adaptability that predisposes cells to senescence.

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