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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 12, 2002 as doi:10.1096/fj.01-0696fje. |
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-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function1
Departments of Pharmacology, Medicine, and Pathology, Vanderbilt University, Nashville, Tennessee, USA
2Correspondence: Department of Pharmacology, 522 RRB, Vanderbilt University, Nashville, TN 27232-6602, USA. E-mail: jack.roberts{at}mcmail.vanderbilt.edu
SPECIFIC AIMS
We recently demonstrated the formation of a series of highly reactive 
-ketoaldehydes, isoketals (IsoKs), and neuroketals formed by the isoprostane and neuroprostane pathways of free radical-induced lipid peroxidation. IsoKs are regio- and stereoisomers of levuglandins formed by the cyclooxygenase pathway. We addressed the hypothesis that adduction of peptides and proteins by IsoKs would significantly reduce their clearance by the 20S proteasome and inhibit proteasome activity.
PRINCIPAL FINDINGS
1. Adduction of a synthetic isoketal (E2-IsoK) to proteasomal substrates markedly retards their degradation
Adduction of the fluorogenic proteasomal substrate val-lys-met-amourilide methyl courain (VKM-AMC) with an equimolar concentration of E2-IsoK essentially eliminated degradation of this peptide by the 20S proteasome in RAW 264.7 cell lysates (Fig. 1
A). This effect was specific for adducted substrate, as reduction of E2-IsoK with sodium borohydride before addition to VKM-AMC eliminated the effect but reduction after adduction of VKM-AMC did not. Adduction of ovalbumin (OVA) with an equimolar concentration of E2-IsoK significantly reduced degradation of this protein by recombinant Methanosarcina thermophila 20S proteasome; adduction with 10 molar equivalents E2-IsoK eliminated its degradation (Fig. 1B
).
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2. Adduction of amyloid beta by E2-IsoK enhances its inhibitory effect on proteasome activity
To test the effect of IsoK-adducted protein on proteasomal chymotrypsin-like activity, we measured hydrolysis of the unadducted fluorogenic proteasomal substrate leu-leu-val-tyr-AMC (LLVY-AMC) by RAW lysates after incubation with the adducted or unadducted peptide amyloidß1–40 (Aß1–40). Unadducted Aß1–40 dose-dependently inhibited proteasome chymotrypsin-like activity (IC50 6.9 µM). This inhibition was significantly enhanced by Aß1–40 that had been adducted with 2 molar equivalents E2-IsoK (IsoK-Aß 1–40) (IC50 1.3 µM). Reduction of IsoK-Aß1–40 with sodium borohydride did not significantly change its effects on proteasome activity. To test whether enhanced proteasome inhibition was limited to Aß1–40,, we incubated RAW lysates with OVA or OVA adducted with 10 molar equivalents E2-IsoK. Adducted OVA significantly increased proteasome inhibition compared with unadducted OVA. Adducted Aß1– 40 and adducted OVA both inhibited proteasomal chymotrypsin-like activity in a competitive fashion.
3. Direct adduction of proteasome by E2-IsoK only weakly inhibits proteasome activity
To test whether direct adduction of the proteasome by E2-IsoK would inhibit proteasome activity, we added E2-IsoK directly to 10 nM of recombinant M. thermophila 20S proteasome. E2-IsoK inhibited LLVY-AMC hydrolysis dose-dependently but required high concentrations (IC50 68 µM). Based on these data, we hypothesized that adduction of E2-IsoK with other intracellular proteins might inhibit proteasome function to a greater extent. We added E2-IsoK to RAW cell lysates and assessed proteasome function. In contrast to our results with recombinant M. thermophila 20S proteasome, E2-IsoK dose-dependently inhibited LLVY-AMC hydrolysis in RAW cell lysates with an IC50 of 2.1 µM. The results of varying LLVY-AMC concentration on inhibition under these conditions were not consistent with a simple competitive inhibition model.
4. E2-IsoK added exogenously to P19 neuroglial cultures inhibits their proteasomal activity and induces cell death
To determine whether E2-IsoK impairs proteasome activity in intact cells, we added E2-IsoK exogenously to retinoic acid differentiated P19 neuroglial cells. After 24 h, treatment media was removed, the cells were lysed, and the proteasome activity of the lysate was measured. Viability of wells treated identically was measured using the MTT conversion assay. E2-IsoK potently inhibited the chymotrypsin-like activity of P19 cells in a dose-dependent manner (IC50 of 330 nM) (Fig. 2
). E2-IsoK induced cell death in a dose-dependent manner at slightly higher concentrations (LC50 of 670 nM).
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CONCLUSIONS AND SIGNIFICANCE
Oxidative stress has been shown to cause proteasome dysfunction. Impaired proteasome function, which leads to the accumulation of aggregated proteins, is a feature of neurodegenerative disorders, such as Alzheimer’s disease, in which oxidative stress and overproduction of isoprostanes and neuroprostanes has been demonstrated. A role for cyclooxygenase has been suggested in the pathogenesis of Alzheimer’s disease. Therefore, we explored the effect of a highly reactive E2-IsoK -ketoaldehyde, which can be formed by the isoprostane and cyclooxygenase pathways and is similar to -ketoaldehydes formed by the neuroprostane pathway (neuroketals), on proteasome function.
Adduction of proteasome substrates with E2-IsoK markedly impaired their ability to be degraded by the proteasome. Similar results have been shown with 4-hydroxynonenal (HNE), but much higher ratios of HNE to protein substrate are required. These results are in keeping with our previous demonstration that IsoKs are more than an order of magnitude more reactive than HNE and that IsoKs exhibit a unique proclivity to cross-link proteins. Adduction of OVA with only 1 molar equivalent of E2-IsoK, which produced only few visible intermolecular cross-links, reduced its degradation by as much as 50%. Although the underlying mechanism for this effect remains to be established, it is reasonable to speculate that the bulky IsoK adduct sterically hinders the processive passage of substrate through the peptide tunnel of the proteasome into the buried catalytic site. Consistent with this notion was the finding that adduction of OVA with higher concentrations of E2-IsoK (10 molar equivalents), which produced extensive cross-linking, essentially abolished its degradation.
Since binding of nondegradable E2-IsoK adducted proteins to the proteasome might competitively inhibit proteasome function, we explored whether IsoK-adducted peptides/proteins inhibit the ability of the proteasome to degrade unadducted substrates. Aß1–40 has been reported to bind in the peptide tunnel of the 20S proteasome and to inhibit proteasome activity. Consistent with previous reports, we found that Aß1–40 inhibited proteasome chymotrypsin-like activity with an IC50 of 6.9 µM. This degree of inhibition was increased significantly (
fivefold) by E2-IsoK adducted Aß1–40. Similar results were obtained with adducted OVA, suggesting that enhanced inhibition of proteasome function is a general effect of IsoK protein adduction.
We examined whether E2-IsoK directly inhibits proteasome function. Addition of E2-IsoK to purified recombinant M. thermophila proteasome dose-dependently inhibited LLVY-AMC hydrolysis, although high concentrations were required (IC50 68 µM). This suggests that the IsoK does not have ready access to the catalytic lysine residues of the buried beta subunits or that because of its high reactivity, it readily adducts to surface lysine residues, precluding its access to the catalytic site. However, when E2-IsoK was added directly to RAW cell lysates, inhibition of LLVY-AMC hydrolysis occurred at much lower concentrations (IC50 2.1 µM). In light of the above observations that adducted OVA and Aß1–40 potently inhibited proteasome function, a reasonable explanation for this finding was that the inhibition of proteasome function under these conditions was primarily mediated by IsoK adducted cellular proteins present in the RAW cell lysate preparation.
Our finding that exogenous addition of submicromolar concentrations of E2-IsoK to P19 neuroglial cells completely abolished chymotrypsin-like activity demonstrates the potentially important contribution of IsoKs to the inhibition of proteasome function in settings of oxidative stress. Consistent with previous observations that selective inhibitors of proteasome function such as lactacystin induce apoptosis in neuronal cells, we found that lactacystin induced cell death in P19 cells. We found that E2-IsoK induced cell death with an LD50 (670 nM) only twofold higher than its IC50 (330 nM) for inhibition of proteasome function. However, additional studies are required to determine whether proteasome inhibition is the principal mechanism by which IsoKs induce cell death. Regardless of the mechanism(s) involved, IsoK-induced cytotoxicity in P19 cells was observed at concentrations < 100-fold lower than we previously reported for HNE. This suggests that IsoKs, neuroketals, and levuglandins are among the most potent neurotoxic products of lipid oxidation identified.
Collectively, these findings suggest a possible mechanism that links the occurrence of lipid peroxidation, protein aggregation, and proteasome inhibition observed in neurodegenerative diseases such as Alzheimer’s disease (Fig. 3
). Oxidative stress with attendant lipid peroxidation generates IsoKs via the isoprostane pathway. Development of an inflammatory response could also lead to formation of levuglandins by the cyclooxygenase pathway. These highly reactive -ketoaldehydes rapidly adduct to intracellular proteins, including proteasomes. The adducted proteins are then targeted to the proteasome, but their degradation is impaired, resulting in their accumulation. The adducted proteins inhibit proteasome activity, leading to impaired degradation of other, nonadducted proteins. Key proteins not degraded could include proapoptic proteins, such as p53. If such proteins accumulate, it could lead to neurodegeneration by activation of programmed cell death pathways. Although IsoKs, neuroketals, and levuglandins are not the only products of lipid peroxidation likely to be involved in protein aggregation, proteasome inhibition, and cell death, their unique reactivity and potent effects on the proteasome suggest they may be important contributors to this phenomenon.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0696fje; to cite this article, use FASEB J. (March 12, 2002) 10.1096/fj.01-0696fje 
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