Mitochondrial aconitase is sensitive to oxidative inactivation and can aggregate and accumulate in many age-related disorders. Here we report that Lon protease, an ATP-stimulated mitochondrial matrix protein, selectively recognizes and degrades the oxidized, hydrophobic form of aconitase after mild oxidative modification, but that severe oxidation results in aconitase aggregation, which makes it a poor substrate for Lon. Similarly, a morpholino oligodeoxynucleotide directed against the lon gene markedly decreases the amount of Lon protein, Lon activity and aconitase degradation in WI-38 VA-13 human lung fibroblasts and causes accumulation of oxidatively modified aconitase. The ATP-stimulated Lon protease may be an essential defence against the stress of life in an oxygen environment. By recognizing minor oxidative changes to protein structure and rapidly degrading the mildly modified protein, Lon protease may prevent extensive oxidation, aggregation and accumulation of aconitase, which could otherwise compromise mitochondrial function and cellular viability. Aconitase is probably only one of many mitochondrial matrix proteins that are preferentially degraded by Lon protease after oxidative modification.

The mitochondrial genome encodes just a small number of subunits of the respiratory chain. All the other mitochondrial proteins are encoded in the nucleus and produced in the cytosol. Various enzymes participate in the activation and intramitochondrial transport of imported proteins. To finally take their place in the various mitochondrial compartments, the targeting signals of imported proteins have to be cleaved by mitochondrial processing peptidases. Mitochondria must also be able to eliminate peptides that are internally synthesized in excess, as well as those that are improperly assembled, and those with abnormal conformation caused by mutation or oxidative damage. Damaged mitochondrial proteins can be removed in two ways: either through lysosomal autophagy, that can account for at most 25-30% of the biochemically estimated rates of average mitochondrial catabolism; or through an intramitochondrial proteinolytic pathway. Mitochondrial proteases have been extensively studied in yeast, but evidence in recent years has demonstrated the existence of similar systems in mammalian cells, and has pointed to the possible importance of mitochondrial proteolytic enzymes in human diseases and ageing. A number of mitochondrial diseases have been identified whose mechanisms involve proteolytic dysfunction. Similar mechanisms probably play a role in diminished resistance to oxidative stress, and in the aging process. In this paper we review current knowledge of mammalian mitochondrial proteolysis, under normal conditions and in several disease states, and we propose an etiological classification of human diseases characterized by a decline or loss of function of mitochondrial proteolytic enzymes.

The Mitochondrial Lon protease, also called LonP1 is a product of the nuclear gene LONP1. Lon is a major regulator of mitochondrial metabolism and response to free radical damage, as well as an essential factor for the maintenance and repair of mitochondrial DNA. Lon is an ATP-stimulated protease that cycles between being bound (at the inner surface of the inner mitochondrial membrane) to the mitochondrial genome, and being released into the mitochondrial matrix where it can degrade matrix proteins. At least three different roles or functions have been ascribed to Lon: 1) Proteolytic digestion of oxidized proteins and the turnover of specific essential mitochondrial enzymes such as aconitase, TFAM, and StAR; 2) Mitochondrial (mt)DNA-binding protein, involved in mtDNA replication and mitogenesis; and 3) Protein chaperone, interacting with the Hsp60-mtHsp70 complex. LONP1 orthologs have been studied in bacteria, yeast, flies, worms, and mammals, evincing the widespread importance of the gene, as well as its remarkable evolutionary conservation. In recent years, we have witnessed a significant increase in knowledge regarding Lon's involvement in physiological functions, as well as in an expanding array of human disorders, including cancer, neurodegeneration, heart disease, and stroke. In addition, Lon appears to have a significant role in the aging process. A number of mitochondrial diseases have now been identified whose mechanisms involve various degrees of Lon dysfunction. In this paper we review current knowledge of Lon's function, under normal conditions, and we propose a new classification of human diseases characterized by a either over-expression or decline or loss of function of Lon. Lon has also been implicated in human aging, and we review the data currently available as well as speculating about possible interactions of aging and disease. Finally, we also discuss Lon as potential therapeutic target in human disease.

Adaptive homeostasis is defined as the transient expansion or contraction of the homeostatic range following exposure to subtoxic, non-damaging, signaling molecules or events, or the removal or cessation of such molecules or events (Mol. Aspects Med. (2016) 49, 1-7). Adaptive homeostasis allows us to transiently adapt (and then de-adapt) to fluctuating levels of internal and external stressors. The ability to cope with transient changes in internal and external environmental stress, however, diminishes with age. Declining adaptive homeostasis may make older people more susceptible to many diseases. Chronic oxidative stress and defective protein homeostasis (proteostasis) are two major factors associated with the etiology of age-related disorders. In the present paper, we review the contribution of impaired responses to oxidative stress and defective adaptive homeostasis in the development of age-associated diseases.

Increasing oxidative stress, a major characteristic of aging, has been implicated in a variety of age-related pathologies. In aging, oxidant production from several sources is increased, whereas antioxidant enzymes, the primary lines of defense, are decreased. Repair systems, including the proteasomal degradation of damaged proteins, also decline. Importantly, the adaptive response to oxidative stress declines with aging. Nrf2/EpRE signaling regulates the basal and inducible expression of many antioxidant enzymes and the proteasome. Nrf2/EpRE activity is regulated at several levels, including transcription, posttranslation, and interactions with other proteins. This review summarizes current studies on age-related impairment of Nrf2/EpRE function and discusses the changes in Nrf2 regulatory mechanisms with aging.

We present arguments for an evolution in our understanding of how antioxidants in fruits and vegetables exert their health-protective effects. There is much epidemiological evidence for disease prevention by dietary antioxidants and chemical evidence that such compounds react in one-electron reactions with free radicals in vitro. Nonetheless, kinetic constraints indicate that in vivo scavenging of radicals is ineffective in antioxidant defense. Instead, enzymatic removal of nonradical electrophiles, such as hydroperoxides, in two-electron redox reactions is the major antioxidant mechanism. Furthermore, we propose that a major mechanism of action for nutritional antioxidants is the paradoxical oxidative activation of the Nrf2 (NF-E2-related factor 2) signaling pathway, which maintains protective oxidoreductases and their nucleophilic substrates. This maintenance of "nucleophilic tone," by a mechanism that can be called "para-hormesis," provides a means for regulating physiological nontoxic concentrations of the nonradical oxidant electrophiles that boost antioxidant enzymes, and damage removal and repair systems (for proteins, lipids, and DNA), at the optimal levels consistent with good health.

We compared Lon protease expression in murine skeletal muscle of young and old, wild-type and Sod2(-/+) heterozygous mice, and studied Lon involvement in the accumulation of damaged (oxidized) proteins. Lon protease protein levels were lower in old and oxidatively challenged animals, and this Lon deficiency was associated with increased levels of carbonylated proteins. We identified one of these proteins as aconitase, and another as an aconitase fragmentation product, which we can also generate in vitro by treating purified aconitase with H(2)O(2). These results imply that aging and oxidative stress down-regulate Lon protease expression which, in turn, may be responsible for the accumulation of damaged proteins, such as aconitase, within mitochondria.

Transforming growth factor-β1 (TGF-β) is involved in multiple cellular processes through Src activation. In the canonical pathway, Src activation is initiated by pTyr530 dephosphorylation followed by a conformational change allowing Tyr419 auto-phosphorylation. A non-canonical pathway in which oxidation of cysteine allows bypassing of pTyr530 dephosphorylation has been reported. Here, we examined how TGF-β activates Src in H358 cells, a small cell lung carcinoma cell line. TGF-β increased Src Tyr419 phosphorylation, but surprisingly, Tyr530 phosphorylation was increased rather than decreased. Vanadate, a protein tyrosine phosphatase inhibitor, stimulated Src activation itself, but rather than inhibiting Src activation by TGF-β, activation by vanadate was additive with TGF-β showing that pTyr530 dephosphorylation was not required. Thus, the involvement of the non-canonical oxidative activation was suspected. TGF-β increased extracellular H2O2 transiently while GSH-ester and catalase abrogated Src activation by TGF-β. Apocynin, a NADPH oxidase inhibitor, inhibited TGF-β-stimulated H2O2 production. Furthermore, mutation of cysteines to alanine, 248C/A, 277C/A, or 501C/A abrogated, while 490C/A significantly reduced, TGF-β-mediated Src activation. Taken together, the results indicate that TGF-β-mediated Src activation operates largely through a redox dependent mechanism, resulting from enhanced H2O2 production through an NADPH oxidase and that cysteines 248, 277, 490, and 501 are critical for this activation.

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.

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.