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
- Wentworth P, Nieva J, Takeuchi C, Galve R, Wentworth AD, Dilley RB, DeLaria GA, Saven A, Babior BM, Janda KD, Eschenmoser A, Lerner RA. Evidence for ozone formation in human atherosclerotic arteries. Science. 2003 Nov 7;302(5647):1053-6. PubMed.
- Wentworth P, McDunn JE, Wentworth AD, Takeuchi C, Nieva J, Jones T, Bautista C, Ruedi JM, Gutierrez A, Janda KD, Babior BM, Eschenmoser A, Lerner RA. Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation. Science. 2002 Dec 13;298(5601):2195-9. PubMed.
- 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. PubMed.
No Available Further Reading
- Hong H, Tamm LK. Elastic coupling of integral membrane protein stability to lipid bilayer forces. Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4065-70. PubMed.
- Bowie JU. Membrane proteins: a new method enters the fold. Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):3995-6. PubMed.
- 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. Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4752-7. PubMed.