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"Ozone Holes" in Human Brain? New Twists on Protein Folding
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19 March 2004. Ozone protects us from solar radiation, sterilizes, and even cleans our clothes. It’s also a summer hazard in areas where its levels in surface air rise beyond safe levels. And here is a new environmental concern to ponder, if admittedly a far-fetched one at this point: If Jeffrey Kelly and colleagues are correct, ozone may precipitate formation of the plaques found in the brains of Alzheimer's patients.
Why do amyloid peptides misfold in some people but not in others? This question is at the heart of explaining late-onset AD from a protein misfolding perspective. In this week’s PNAS online, Kelly, at the Scripps Research Institute, La Jolla, and colleagues put forth the hypothesis that ozone can convert relatively inert lipids—cholesterol, in particular—to highly reactive aldehydes. These aldehydes can then covalently modify amino acid side chains, turning hydrophilic soluble peptides into hydrophobic insoluble ones. To find out if this theory could have any physiological significance, Kelly tested if products of lipid ozonolysis can react with the Aβ peptide to influence aggregation, and if such products can be formed in the human brain.
Lead author Qinghai Zhang tackled the first question by incubating cholesterol ozonolysis products with Aβ in vitro. Zhang found that two of these cholesterol-derived aldehydes accelerated Aβ aggregation, as judged by both thioflavin T fluorescence and atomic force microscopy. To confirm that this was due to covalent modification of Aβ, Zhang separated the aggregates and analyzed the proteins by both HPLC and mass spectrometry. The results showed that Aβ was indeed modified, at lysine 16, lysine 28, and at the N-terminus.
But what about in vivo? Zhang and colleagues tested samples taken postmortem from brains with and without AD pathology. They found traces of the two cholesterol derivatives in almost all samples. However, they found no statistical difference between levels in the two groups. The researchers did not report if any modified Aβ could be found in AD or even in normal brain tissue. Tiny, indeed, barely detectable amounts of modified Aβ may be able to “seed” aggregation, the authors argue, making it difficult to test definitively whether they influence pathology in human disease. “Once nucleated, the propagation of amyloidogenesis is fast, making the initiating event traceless,” the scientists write.
The authors state that this work suggests a "new paradigm where a ubiquitous metabolite (e.g., cholesterol) is transformed into an abnormal metabolite with unusual reactivity…pathology is initiated only when the new functional group(s) on the reactive metabolite forms a covalent bond with a protein or related macromolecule." If it holds up in future experiments, the theory could explain the link among high cholesterol, inflammation, and the risk of developing AD. Cholesterol ozonolysis, for example, has been shown to occur during atherosclerosis (see Wentworth et al., 2003), while the same authors showed in 2002 that antibodies can actually produce ozone (see Wentworth et al., 2002). Thus, immune mechanisms in AD may cause production of ozone, resulting in more Aβ aggregation and more inflammation, and leading to a vicious cycle of pathology, the authors speculate. Mark Smith and others have shown previously that the lipid peroxidation product 4-hydroxynonenal is elevated in AD brains (see Sayre et al., 1997), and Zhang and colleagues were able to show that this aldehyde also accelerates aggregation of Aβ in vitro.
In a separate paper on the basic science of how lipids affect protein folding, Heedeok Hong and Lukas Tamm at the University of Virginia, Charlottesville, report in the 27 February PNAS online that they have succeeded in measuring the thermodynamic stability of a membrane β-barrel protein. Methods for measuring the stability of membrane proteins have remained elusive, writes James Bowie from University of California, Los Angeles, in an accompanying commentary, primarily due to the difficulty of getting the proteins to fold reversibly.
Hong and Tamm overcame those difficulties by using urea to drive bacterial outer membrane protein A (ompA) from small unilamellar vesicles. Urea does not disrupt the membrane but helps solubilize ompA in the aqueous phase. Using the procedure, Hong shows that folding/refolding is essentially a two-state process, and that the free energy of the protein in the membrane is highly dependent on the lipid environment. Membrane curvature and hydrophobic mismatch, where the head and tail of the lipid are different widths, can create an unstable environment for the protein.
"The work opens the door to a more quantitative description of the energetics of protein-protein and protein-lipid interactions in the bilayer," writes Bowie. It may also be useful for optimizing conditions for resolving three-dimensional structures by crystallography or NMR, suggest Hong and Tamm.—Tom Fagan.
References:
Zhang Q, Powers ET, Nieva J, Huff ME, Dendle MA, Bieschke J, Glabe CG, Eschenmoser A, Wentworth P, Lerner RA, Kelly JW. Metabolite-initiated protein misfolding may trigger Alzheimer's disease. PNAS Early Edition. 2004 March 18. Abstract
Hong H, Tamm LK. Elastic coupling of integral membrane protein stability to lipid bilayer forces. PNAS Early Edition. 2004 February 24. Abstract
Bowie JU. Membrane proteins: A new method enters the fold. PNAS 2004 March 23;101:3995-3996. Abstract
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Comments on News and Primary Papers |
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Primary Papers: Metabolite-initiated protein misfolding may trigger Alzheimer's disease.
Comment by: Kazuhiro Honda, Quan Liu, Paula Moreira, George Perry, ARF Advisor (Disclosure), Mark A. Smith (Disclosure)
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Submitted 30 April 2004
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Posted 2 May 2004
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Embalming Amyloid-β: The Role for Aldehyde Stress in Alzheimer's Disease
Zhang and colleagues present novel findings demonstrating that cholesterol derivative-aldehydes resulting from ozonolysis can accelerate the aggregation of Aβ into fibers. This finding importantly extends earlier work showing that aldehydes from lipid peroxidation (Sayre et al., 1997; Takeda et al., 2000; Gómez-Ramos et al., 2003; Pocernich and Butterfield, 2003; Woltjer et al., 2003) and glycation (Smith et al., 1994) play a pivotal role in the genesis of AD pathology.
Oxidative damage to lipids results in the production of several reactive aldehydes that can attack and modify DNA (Marnett, 2002; Luczaj and Skrzydlewska, 2003), proteins (Wataya et al., 2002; Zhang et al., 2003), lipids, and sugars (Woltjer et al., 2003). Aldehyde modifications may contribute to changes of biomolecule properties in two distinct ways: 1) render biomolecules more hydrophobic and 2) potentiate protein crosslinks. These alterations predispose to the occurrence of protein misfolding processes that accumulate...
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Embalming Amyloid-β: The Role for Aldehyde Stress in Alzheimer's Disease
Zhang and colleagues present novel findings demonstrating that cholesterol derivative-aldehydes resulting from ozonolysis can accelerate the aggregation of Aβ into fibers. This finding importantly extends earlier work showing that aldehydes from lipid peroxidation (Sayre et al., 1997; Takeda et al., 2000; Gómez-Ramos et al., 2003; Pocernich and Butterfield, 2003; Woltjer et al., 2003) and glycation (Smith et al., 1994) play a pivotal role in the genesis of AD pathology.
Oxidative damage to lipids results in the production of several reactive aldehydes that can attack and modify DNA (Marnett, 2002; Luczaj and Skrzydlewska, 2003), proteins (Wataya et al., 2002; Zhang et al., 2003), lipids, and sugars (Woltjer et al., 2003). Aldehyde modifications may contribute to changes of biomolecule properties in two distinct ways: 1) render biomolecules more hydrophobic and 2) potentiate protein crosslinks. These alterations predispose to the occurrence of protein misfolding processes that accumulate during aging and age-related processes such as AD.
Among common reactive aldehydes, 4-hydroxy-2-nonenal (HNE) is probably the most widely studied (Sayre et al., 1997; Takeda et al., 2000; Uchida, 2000; Wataya et al., 2002; Dianzani, 2003; Liu et al., 2003). It is considered one of the most neurotoxic aldehydes produced in vivo (Montine et al., 1996) and is a highly reactive electrophile that can form adducts onto several nucleophilic groups on biomacromolecules (Liu et al., 2003). In oxidative stress conditions, HNE accumulates in membranes at concentrations from 10 µM to 1 mM (Dianzini, 2003).
Recent studies demonstrated that HNE promotes the increase of intracellular Aβ protein production, probably via the induction of BACE expression and activity (Tamagno et al., 2002). However, it seems that HNE also affects tau protein and, therefore, may play an important role in neurofibrillary degeneration. The new data presented by Zhang et al. alert us to the existence of other potentially deleterious aldehydes that can be produced in vivo (hypercholesterolemia and inflammation) and have an environmental source. These cholesterol derivate-aldehydes resulting from ozonolysis are worthy of further investigation.
References:
Dianzani MU. 4-hydroxynonenal from pathology to physiology. Mol Aspects Med 2003;24:263-272. Abstract
Gomez-Ramos A, Diaz-Nido J, Smith MA, Perry G, Avila J. Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells. J Neurosci Res. 2003 Mar 15;71(6):863-70. Abstract
Liu Q, Raina AK, Smith MA, Sayre LM, Perry G. Hydroxynonenal, toxic carbonyls, and Alzheimer disease. Mol Aspects Med. 2003 Aug-Oct;24(4-5):305-13. Abstract
Luczaj W, Skrzydlewska E. DNA damage caused by lipid peroxidation products. Cell Mol Biol Lett. 2003;8(2):391-413. Abstract
Marnett LJ. Oxy radicals, lipid peroxidation and DNA damage. Toxicology. 2002 Dec 27;181-182:219-22. Review. Abstract
Montine TJ, Amarnath V, Martin ME, Strittmatter WJ, Graham DG. E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures. Am J Pathol. 1996 Jan;148(1):89-93. Abstract
Pocernich CB, Butterfield DA. Acrolein inhibits NADH-linked mitochondrial enzyme activity: implications for Alzheimer's disease. Neurotox Res. 2003;5(7):515-20. Abstract
Sayre LM, Zelasko DA, Harris PL, Perry G, Salomon RG, Smith MA. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J Neurochem. 1997 May;68(5):2092-7. Abstract
Smith MA, Taneda S, Richey PL, Miyata S, Yan SD, Stern D, Sayre LM, Monnier VM, Perry G. Advanced Maillard reaction end products are associated with Alzheimer disease pathology.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5710-4. Erratum in: Proc Natl Acad Sci U S A 1995 Feb 28;92(5):2016. Abstract
Takeda A, Smith MA, Avila J, Nunomura A, Siedlak SL, Zhu X, Perry G, Sayre LM. In Alzheimer's disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification. J Neurochem. 2000 Sep;75(3):1234-41. Abstract
Tamagno E, Bardini P, Obbili A, Vitali A, Borghi R, Zaccheo D, Pronzato MA, Danni O, Smith MA, Perry G, Tabaton M. Oxidative stress increases expression and activity of BACE in NT2 neurons. Neurobiol Dis. 2002 Aug;10(3):279-88. Abstract
Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res. 2003 Jul;42(4):318-43. Review. Abstract
Wataya T, Nunomura A, Smith MA, Siedlak SL, Harris PL, Shimohama S, Szweda LI, Kaminski MA, Avila J, Price DL, Cleveland DW, Sayre LM, Perry G. High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal. J Biol Chem. 2002 Feb 15;277(7):4644-8. Epub 2001 Dec 03. Abstract
Woltjer RL, Maezawa I, Ou JJ, Montine KS, Montine TJ. Advanced glycation endproduct precursor alters intracellular amyloid-beta/A beta PP carboxy-terminal fragment aggregation and cytotoxicity. J Alzheimers Dis. 2003 Dec;5(6):467-76. Abstract
Zhang Q, Powers ET, Nieva J, Huff ME, Dendle MA, Bieschke J, Glabe CG, Eschenmoser A, Wentworth P Jr, Lerner RA, Kelly JW. Metabolite-initiated protein misfolding may trigger Alzheimer's disease. Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4752-7. Epub 2004 Mar 19. Abstract
Zhang WH, Liu J, Xu G, Yuan Q, Sayre LM. Model studies on protein side chain modification by 4-oxo-2-nonenal. Chem Res Toxicol. 2003 Apr;16(4):512-23. Abstract
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