Mitochondrial Lon protease is a human stress protein

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 pretreatment 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 aging and age-related diseases.     

OGG1 is degraded by calpain following oxidative stress and cisplatin exposure

Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.     

DNA ligase III is degraded by calpain during cell death induced by DNA-damaging agents

A yeast two-hybrid screen identified the regulatory subunit of the calcium-dependent protease calpain as a putative DNA ligase III-binding protein. Calpain binds to the N-terminal region of DNA ligase III, which contains an acidic proline, aspartate, serine, and threonine (PEST) domain frequently present in proteins cleaved by calpain. Recombinant DNA ligase III was a substrate for calpain degradation in vitro. This calpain-mediated proteolysis was calcium-dependent and was blocked by the specific calpain inhibitor calpeptin. Western blot analysis revealed that DNA ligase III was degraded in human fibrosarcoma HT1080 cells following exposure to gamma-radiation. The degradation of DNA ligase III was prevented by pretreatment with calpeptin, which protected irradiated cells from death. Calpeptin treatment also blocked 9-amino camptothecin-induced DNA ligase III proteolysis and simultaneously protected the cells from death. HT1080 clones expressing a modified DNA ligase III that lacked a recognizable PEST domain were significantly more resistant to killing by gamma-radiation or 9- amino camptothecin than were cells that overexpressed the wild-type form of DNA ligase III. These data show that calpain-mediated proteolysis of DNA ligase III plays an essential role in DNA damage-induced cell death in human cells.     

Quality control of a molybdoenzyme by the Lon protease

Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site.     

Mitochondrial calpain 10 activity and expression in the kidney of multiple species

Calpains, Ca²⁺-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in Ca²⁺-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75 kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75 kDa (calpain 10a), 56 kDa (calpain 10c or 10d), and 50 kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice.     

Mitochondrial Tim9 protects Tim10 from degradation by the protease Yme1

Translocase of IM (inner membrane; Tim)9 and Tim10 are essential homologue proteins of the mitochondrial intermembrane space (IMS) and form a stable hexameric Tim9–Tim10 complex there. Redox-switch of the four conserved cysteine residues plays a key role during the biogenesis of these proteins and, in turn, the Tim proteins play a vital chaperone-like role during import of mitochondrial membrane proteins. However, the functional mechanism of the small Tim chaperones is far from solved and it is unclear whether the individual proteins play specific roles or the complex functions as a single unit. In the present study, we examined the requirement and role for the individual disulfide bonds of Tim9 on cell viability, complex formation and stability using yeast genetic, biochemical and biophysical methods. Loss of the Tim9 inner disulfide bond led to a temperature-sensitive phenotype and degradation of both Tim9 and Tim10. The growth phenotype could be suppressed by deletion of the mitochondrial i-AAA (ATPases associated with diverse cellular activities) protease Yme1, and this correlates strongly with stabilization of the Tim10 protein regardless of Tim9 levels. Formation of both disulfide bonds is not essential for Tim9 function, but it can facilitate the formation and improve the stability of the hexameric Tim9–Tim10 complex. Furthermore, our results suggest that the primary function of Tim9 is to protect Tim10 from degradation by Yme1 via assembly into the Tim9–Tim10 complex. We propose that Tim10, rather than the hexameric Tim9–Tim10 complex, is the functional form of these proteins.     

Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism

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.     

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.     

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.     

Imaging calpain protease activity by multiphoton FRET in living mice

Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.     

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000muM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.     

TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain

m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes.     

Adenosine triphosphate-dependent degradation of a fluorescent lambda N substrate mimic by Lon protease

Escherichia coli Lon exhibits a varying degree of energy requirement toward hydrolysis of different substrates. Efficient degradation of protein substrates requires the binding and hydrolysis of ATP such that the intrinsic ATPase of Lon is enhanced during protein degradation. Degradation of synthetic tetrapeptides, by contrast, is achieved solely by ATP binding with concomitant inhibition of the ATPase activity. In this study, a synthetic peptide (FRETN 89-98), containing residues 89-98 of lambda N protein and a fluorescence donor (anthranilamide) and quencher (3-nitrotyrosine), has been examined for ATP-dependent degradation by E. coli and human Lon proteases. The cleavage profile of FRETN 89-98 by E. coli Lon resembles that of lambda N degradation. Both the peptide and protein substrates are specifically cleaved between Cys93 and Ser94 with concomitant stimulation of Lon's ATPase activity. Furthermore, the degradation of FRETN 89-98 is supported by ATP and AMPPNP but not ATPgammaS nor AMPPCP. FRETN 89-98 hydrolysis is eight times more efficient in the presence of 0.5 mM ATP compared to 0.5 mM AMPPNP at 86 microM peptide. The ATP-dependent hydrolysis of FRETN 89-98 displays sigmodial kinetics. The k(cat), [S](0.5), and the Hill coefficient of FRETN 89-98 degradation are 3.2 +/- 0.3 s(-1), 106 +/- 21 microM, and 1.6 respectively.     

Platelet factor XIII is activated by calpain

The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII.     

Induction of peroxisomal Lon protease in rat liver after di-(2-ethylhexyl)phthalate treatment

Previously, we identified a peroxisome-specific isoform of Lon protease using subcellular proteomics. In the present study, we investigated changes in the level of the Lon protease in peroxisomes during recovery from peroxisomal proliferation induced by di-(2-ethylhexyl)phthalate (DEHP) to elucidate the function of peroxisomal Lon protease (PSLP). Following a 2-week treatment with DEHP, the level of PSLP was monitored for 15 days. The amount of protease was greatly increased after the 2-week treatment, followed by a further increase 3 days after cessation of the treatment. Afterward, it decreased and reached the control level on day 15. On the other hand, level peroxisomal β-oxidation enzymes induced to express by DEHP started to decrease soon after discontinuation of treatment. The results suggest that PSLP functions to degrade β-oxidation enzymes induced by DEHP during recovery from perxisomal proliferation.