There is a new ingredient in the complex mix that yields masses of malformed proteins. From a C. elegans mutant screen, scientists have plucked out a gene they call MOAG-4, for modifier of aggregation 4. When MOAG-4 is inactivated, aggregation drops, suggesting the protein promotes conglomeration. Researchers led by Ellen Nollen, of the University of Groningen in The Netherlands, report their finding in this week’s Cell. Although preliminary—MOAG-4’s function is still a mystery—the finding suggests MOAG-4 might be worth investigating as a drug target or new tool to analyze amyloid formation.
Nollen searched for modifiers of aggregation before, but aimed for mutations that would increase aggregation, she told ARF (ARF related news story on van Ham et al., 2008; Nollen et al., 2004). The modifiers she found were primarily essential genes, meaning knocking them down was not a valid therapeutic strategy. She reasoned that mutations that diminish aggregation would point her toward aggregation-promoting, possibly druggable targets. Joint first authors Tjakko van Ham, Mats Holmberg, Annemieke van der Goot, and Eva Teuling took on the challenge.
The scientists worked with nematodes carrying a YFP-tagged 40-mer polyglutamine peptide, or Q40, which is prone to aggregation. They chemically mutagenized the animals and sought those that would make fewer aggregates than did wild-type. One strain, identified to have a missense mutation in the gene christened MOAG-4, evinced a 75 percent drop in protein inclusions.
Biochemical experiments pointed to a job for MOAG-4 in shepherding aggregation-prone proteins on the pathway to full-fledged inclusions. Using native gel electrophoresis to detect of YFP-tagged polyglutamines (Q40, Q24), van Ham and colleagues observed bands that ran faster than fully formed aggregates, but slower than monomeric polyglutamine. The researchers took these mid-speed bands for intermediates in the aggregation process.
Nollen and colleagues also examined the polyglutamines and aggregation intermediates in denaturing, SDS gel electrophoresis. Worms expressing mutated MOAG-4 had more detergent-soluble intermediates, but half as much of the detergent-insoluble aggregates. The data suggest that without MOAG-4, polyglutamines were stuck midway through the aggregation pathway, unable to fully aggregate.
In Q40 animals with wild-type MOAG-4, the researchers observed a band that ran faster than Q40 monomers normally do. But when MOAG-4 was absent, the faster Q40 nearly disappeared. The researchers hypothesize that the faster-moving species is made up of one or more polyglutamines with an altered, compacted conformation, and suggest this compaction could be a step toward aggregation. Therefore, the authors suggest that MOAG-4’s role is to help generate these compacted forms that are on the way to aggregation. Some researchers believe that a switch from a globular, disordered structure to an ordered one, such as a β-sheet, is the first step in the formation of protein aggregates, such as those formed by the amyloid-β peptide.
In fact, the researchers wondered whether MOAG-4’s actions were specific to polyglutamine proteins such as huntingtin, or more general. Accordingly, they deleted MOAG-4 in worms expressing other aggregation-prone proteins in their muscles. Animals carrying the amyloid-β(1-42) of Alzheimer’s, the α-synuclein linked to Parkinson’s, or the mutant superoxide dismutase 1 (SOD1) associated with amyotrophic lateral sclerosis, accumulate inclusions and have motility problems. In the Aβ and α-synuclein worms, loss of MOAG-4 resulted in fewer aggregates and better motility. SOD1 animals, however, were unaffected by MOAG-4’s absence. The researchers concluded that if aggregating proteins are around, MOAG-4 worsens the situation. Nollen speculated that the MOAG-4 pathway might only act on amyloid structures. If SOD1 does not form amyloid—that is currently debated, said Nollen—MOAG-4 might be irrelevant to its conglomeration.
The scientists still do not know what MOAG-4 actually does. In the paper, they suggest it might be involved in stress responses or protein quality control, or perhaps simply promote aggregation. It does not appear to be involved in the chaperone or proteasome pathways that frequently deal with misfolded proteins, Nollen said.
MOAG-4 is also a highly conserved gene, with human orthologs SERF1A and SERF2. SERF1A has already been linked to neurodegenerative disease; previous research pointed to it as a potential modifying gene in spinal muscular atrophy (Scharf et al., 1998).
Nollen and colleagues found that the SERF proteins do fulfill an aggregation-related function. When the researchers overexpressed the SERF genes in human neuroblastoma cells containing mutant human huntingtin, they observed increased protein aggregation and cell death. Likewise, when they knocked down SERF levels in human embryonic kidney cells expressing the same huntingtin fragment, the cells suffered fewer aggregates and less toxicity.
Despite the preliminary nature of the findings, there are interesting implications, said Dagmar Ringe of Brandeis University in Waltham, Massachusetts. Ringe was not an author on the current paper. For one, if diminishing MOAG-4 activity prevents toxicity in human tissue as it does in worm muscles, a drug that interferes with MOAG-4 could be useful for several neurodegenerative conditions.
“At this point, to say it is a potential drug target is to underline ‘potential’…but that does not mean we can’t think about it,” Ringe said. In addition, she noted, knowing about MOAG-4 provides an opportunity for scientists studying the basic aggregation process: “This particular gene product is a very interesting potential tool to study the pathways by which aggregation occurs.”—Amber Dance
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