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Protein-protein interactions shed light on blindness By analyzing the interactome of a protein called lebercilin, researchers have moved a step closer to understanding how a rare form of incurable blindness develops. Marius Ueffing, Karsten Boldt, and colleagues at Helmholtz Zentrum Munich used stable-isotope labeling by amino acids in cell culture (SILAC) to determine the changes in protein-protein interactions when lebercilin, which is encoded by the LCA5 gene, is mutated. The data suggest that lebercilin may be involved in interflagellar transport (IFT), a process that is vital to vision. Mutations in eight genes are associated with the development of Leber congenital amaurosis (LCA), a rare, inherited eye disease that causes children to go blind early in life. In 2007, Ronald Roepman’s group at Radboud University Nijmegen Medical Centre (The Netherlands) collaborated with Ueffing’s laboratory and with teams at several institutions around the world. They identified and described the LCA5 gene, which has been implicated in specific families affected by the disease (Nat. Genet. 2007, DOI 10.1038/ ng2066). The researchers found that the corresponding protein localized to cilia. To discover interactors, they tagged the LCA5 protein with a new, smaller version of the tandem affinity purification (TAP) tag called SF-TAP (Proteomics 2007, DOI 10.1002/pmic.200700038). With this tag, researchers could speed up the enrichment process. They performed a pull-down experiment, analyzed the bound proteins with MS, and identified 24 interacting proteins, many of which have cytoskeletal or ciliary functions. With these data in mind, the researchers called the protein lebercilin, says Ueffing. In the current work, Boldt conducted quantitative SILAC experiments to discover additional interactors and to see whether specific binding events change
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ments to replenish lost disks. “The hypothesis is that lebercilin [plays a] part in vesicular transport through the cilium,” he states. “If you have a defect there, then your photoreceptor outer segment gets shorter and shorter, and you lose eyesight. This is actually what you see in patients; they don’t have outer segments.”
when lebercilin is mutated. Either wild-type lebercilin or a mutant form that was discovered in an LCA patient was expressed in a cell line, and both forms were fused with the SF-TAP tag. Proteins bound by the mutant were compared with those bound by the wild-type. With this procedure, several new interactors were isolated, including MARIUS UEFFING
Katie Cottingham reports from the Proteomic Forum 2009— Berlin
Changes. Investigators compared the proteins that bound to wild-type lebercilin and a mutant variant and found that IFT proteins (red squares) have reduced binding to the mutant, whereas one protein (green square) showed increased binding.
members of the IFT complex B, as well as putative IFT proteins. Surprisingly, binding of the IFT proteins to the mutant form was greatly reduced compared with wild-type. “When this [result] came out, we were really struck,” says Ueffing. “It’s not one binder that falls off; it’s a whole part of a molecular machine for interflagellar transport that falls off the mutated protein.” He adds that, to his knowledge, this is the first study in which the impact of a discrete mutation in a specific protein is shown on the level of an entire network. Although the researchers still must conduct functional assays to verify lebercilin’s role in IFT, they know that losing this activity could cause blindness. Normally, disks within the outer segments of the retinal photoreceptors burn out during the day and are phagocytosed overnight by pigment epithelial cells, says Ueffing. When light hits the eye in the morning, vesicles transport proteins through cilia from the inner segments to the outer seg-
Journal of Proteome Research • Vol. 8, No. 6, 2009
After looking more closely at the interactome, the scientists realized that a connection may exist between LCA and diseases of other organ systems. For example, some of the ciliary proteins that interact with lebercilin also interact with proteins involved in kidney and brain diseases. “If you have the wrong mutation, you will have severe problems with the development of organs that depend on cilia,” explains Ueffing. “That’s why these diseases connect; that’s what we can see from the protein interactions.” By combining genetics and proteomics, Ueffing and colleagues hope to gain a better understanding of how vision works and find a treatment for LCA. Because LCA is a rare disease with a small potential market, pharmaceutical companies have not heavily invested in this area. Therefore, it is up to individual researchers and patients to take on the cause, he explains. And the patients refuse to be ignored. In mid-April, Ueffing and ∼50 other scientists attended a conference on blindness that was organized by a patient group. The members of the group organize regular meetings to learn about the latest research on blindness. “It’s nice to do science just to do science, but we also have a duty to give back to society what we have received,” he says. “I take that [duty] very seriously.”
10.1021/pr9003757
2009 American Chemical Society
