James Bowie of the University of California, Los Angeles, has not previously worked on neurodegeneration but ran smack into AD research this year. It happened when Bowie’s group tested a deceptively simple algorithm they had developed to model how certain membrane proteins fold. This research grew out of a frustrated acknowledgement that, despite 40 years of trying, scientists still have not cracked the old problem of de novo structure prediction.
Far fewer membrane proteins than cytosolic proteins have yielded insight into their atomic structure, largely because membrane protein biochemistry is so complex. Bench science aside, however, mere computational prediction of how a protein will fold ought to be easier for membrane proteins than for proteins in watery solutions, Bowie said. That’s because in water, a protein can assume a huge number of conformations, and sorting through them to find the right one remains daunting. With a membrane protein, its sequence implies where its transmembrane regions are, and once scientists have packed these, the possible structures for arranging the rest of the polypeptide chain become greatly restricted, Bowie said.
In this spirit, his group developed a three-step algorithm for folding homo-oligomeric transmembrane helices. Starting with helices in random orientation, the program runs 200 iterations of a procedure that finds conformations of minimal energy determined by the protein’s many van der Waals forces. Next, the algorithm filters out asymmetric conformations, then it clusters similar structures together. The largest cluster contains the structure most likely to be the correct one. “If someone had told me this approach a while ago I would have dismissed it as too simple. But as it turns out, this works really well,” Bowie said.
Bowie presented examples of validated or predicted structures, for example for glycophorin, for an influenza virus proton channel, and for the H. pylorum cytotoxin VacA. Like many of the proteins in this study, VacA appears to form a pore, and the commonality between these proteins turned out to be a packing motif Bowie called a glycine zipper. “We think this is an important mode of creating channel structures,” he said. One in four membrane proteins contains at least one glycine zipper motif—too many to examine. A more stringent database search, for proteins that contain a single transmembrane helix and at least four glycine zippers in a row, dredged up Aβ, Bowie said, as well as major prion protein precursor, which is also thought to form channels.
The idea that misfolded pathogenic proteins form pores in neuronal membranes has been around in the Alzheimer’s, Parkinson’s, Huntington’s, and prion fields for a decade without garnering wide support (see Kawahara et al., 1997, Lin et al., 1997, Kagan et al., 2002, Lashuel et al, 2003, Lashuel et al., 2002, Hirakura et al., 2000, and Kourie and Henry, 2002). Bowie said that his lab first reproduced some of the earliest data reported on the topic (Arispe and Rojas, 1993). Next, scientists began testing their own hypothesis that the glycine zipper motifs drive formation of the purported channels. They mutated different glycine positions on Aβ, and initial results suggest that wild-type Aβ forms channels, but the mutants do not, Bowie said. The mutants also appear less toxic to cultured neurons, but that work is even more preliminary. Bowie cautioned that he has no evidence as yet on whether his work is relevant to Alzheimer disease, and invited the field to come up with ways of finding out.
Ironically, Bowie’s research opens up a fresh vein of support for the channel hypothesis just as prominent work in the AD field appears to weaken it. This website has reported extensively on Charlie Glabe’s and Rakez Kayed’s studies of an antibody that is specific to small oligomers of different amyloidogenic peptides regardless of their amino acid sequence (see ARF related news story). This surprising study had suggested that the antibody recognizes a shared structural motif on the peculiar oligomeric intermediates of these proteins. At the NBA conference, Glabe, who is at the University of California, Irvine, recapped published data and noted that the list of amyloidogenic proteins known to react with the antibody has since grown to 24.
This September, the scientists further reported that these intermediates all damage synthetic lipid bilayers by a common mechanism that increases the membrane conductance, but that the oligomers do not form pores or specific ion channels in the process (see ARF related news story). At the main Society for Neuroscience conference, Erene Mina, a graduate student in Glabe’s lab (who contributes occasional news summaries to Alzforum), presented a poster describing how treating cultured cells with Aβ oligomers greatly increases calcium influx and disrupts the integrity of the membrane. Soluble oligomers of other amyloidogenic proteins do this, as well, but their respective monomers or fibrils do not. The anti-oligomer antibody reverses the change in conductance. However, much as the scientists had expected to find oligomer pores, they could not. “We see no evidence for discrete conductivity, we see no evidence for open and closed states, and we see no ion specificity. We looked a broad range of inhibitors described as Aβ channel inhibitors, to no effect,” Glabe said at the NBA symposium. “I wanted to see a channel, but this is what we are left with. It’s an urgent issue for us to sort out, and we’ll do it soon.” Other recent work also supports the notion that amyloidogenic oligomers damage the lipid bilayer but not by forming channels (see Green et al., 2004.)
In related news, Glabe also reported these data: immunocytochemistry experiments performed to see whether these oligomers exist in human brain found no immunoreactivity in controls, but a punctate pattern in AD brain. The anti-oligomer antibody detected only a small fraction of total amyloid and did not colocalize with astrocytes or microglia. It occurred largely in extracellular regions that contained plaques but did not overlap with plaques or with diffuse Aβ deposits. Rather, it stained the outer rim of diffuse deposits. The oligomer antibody did not colocalize with neurons, though some intraneuronal staining was apparent. Having solved technical problems with Western blots using this antibody, the lab has now detected the oligomers in soluble extracts of people with MCI and AD, but not controls, Glabe added.
Glabe’s laboratory has begun collaborations with groups studying other diseases. One, with Jeffrey Robbins at Cincinnati Children’s Hospital, shows that human tissue from conditions not previously thought to be amyloid aggregation diseases actually show massive staining with the anti-oligomer antibody. Glabe mentioned forms of idiopathic dilated cardiomyopathy as examples, where oligomeric intermediates might exert their toxicity early and formal fibril aggregation never fully progresses (see Sanbe et al., 2004). The antibody may identify further diseases involving amyloid, Glabe said.
In short, Glabe proffered this working hypothesis: The primary mechanism of all degenerative amyloid diseases lies in the oligomer-induced leakiness of membranes, possibly because oligomers disrupt the way membrane lipids are packed (see also Glabe, 2004).—Gabrielle Strobel.
To be continued Monday, 20 December 2004.