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MOLECULAR BIOLOGY Enzyme Mends Fibers that Give Stretch to Tissues Uterus, Other Organs Depend on Maintenance of Elastin Flexibility has its advantages. Trees that sway in the wind don't snap in a storm, suspension protects cars, and spandex keeps some people looking good. But from a physiological perspective, flexibility is essential. It allows lungs to fill with air and ensures that with each heart beat blood vessels don't rupture. It is indispensable for mammalian survival because during pregnancy, the uterus must grow severalfold without bursting and then must shrink back to normal size after parturition. All these biomechanical feats are made possible by elastin, a protein polymer that works like a rubber band, repeatedly stretching and contracting without suffering any damage. Stretching out elastin. The enzyme LOXL1 is necessary to keep elastin in shape. Findings by Tiansen Li (below right), Xiaoqing Liu, and colleagues predict that the enzyme is recruited to sites of elastin by the extracellular-matrix protein fibulin-5. LOXL1 then primes tropoelastin monomers (TE) for incorporation into the larger polymer. Liu found that LOXL1 is necessary to prevent age-related loss of elasticity in tissues such as arteries and lungs. (Image modified from original by Tiansen Li; Photo by Graham Ramsay) Elastin polymers are formed by the cross-linking of tropoelastin monomers. While there are as many as five enzymes that can catalyze this process--lysyl oxidase and four lysyl oxidase-like proteins--it remains sketchy which one does the job and when. In the February Nature Genetics, HMS associate professor of ophthalmology Tiansen Li, of the Berman-Gund Laboratory at the Massachusetts Eye and Ear Infirmary, and colleagues report that one of these enzymes, lysyl oxidase-like 1 (LOXL1) is essential for remodeling elastin fibers and keeping tissues, literally, in their place. LOX and Labels For years Li has been studying macular degeneration, a major cause of blindness in the elderly. This disorder leads to vision loss when blood vessels in the eye penetrate the Bruch's membrane, an elastin film that normally separates the vasculature from the retina. Li postulated that an age-related weakening of this layer may contribute to the degenerative process and wondered if LOXL1 might have a role to play in maintaining the elastin. To test this idea, Xiaoqing Liu, a postdoctoral fellow in Li's lab, made knockout mice that are missing the Loxl1 gene. Unlike knockouts of lysyl oxidase (LOX), which do not survive past birth, the Loxl1 knockouts at first seemed normal. "In fact, even their retinas appeared undamaged," said Li, who was initially disappointed with the results of the experiment. But then he saw something curious. Female mutant mice, being fertile, conceived and delivered healthy-looking pups. But within two days of giving birth, almost all the mothers were found to have a uterine prolapse. "...Our work clearly shows that elastin remodeling, or mending, goes on throughout life." --Tiansen Li "We thought that LOXL1 may be playing a special role in female animals," said Li. Subsequent experiments would show that the enzyme was essential for the maintenance of elastin in male mice, too. First, however, to understand how LOXL1 prevents prolapse, Liu compared the uterine tissue of normal and mutant mice. Prior to pregnancy, there was little or no difference, but Liu found that postpartum, the elastin polymers in the Loxl1 knockouts were fragmented even though fibrils of collagen, which give connective tissue strength rather than elasticity, were normal. "This suggested to us that while LOX may be essential for collagen cross-linking, LOXL1 is essential for maintenance and remodeling of elastin," said Li. To confirm this, Liu used fluorescently labeled antibodies, which specifically recognize either LOX or LOXL1, to localize the enzymes immunohistochemically. Sure enough, they found that LOXL1, but not LOX, colocalizes with elastin in virtually all tissues examined. Location, Location, Location This finding helps settle the outstanding question of which oxidase acts on which protein substrate. LOX and the four LOX-like proteins all catalyze the same chemical reaction, the oxidative deamination of lysine amino acids. "In a test tube, all five enzymes will deaminate lysyl groups on collagen, elastin, or for that matter, on probably any protein," explained Li. In vivo, however, such indiscriminate deamination could be problematic. Instead, these oxidases are thought to acquire a degree of substrate specificity by virtue of their proximity to a particular fiber. In the case of LOXL1, Li's data suggested that it was being targeted to sites of elastin deposition and may therefore be exclusively a tropoelastin deaminase. To understand how this topographical specificity might work, Liu used the well-known yeast two-hybrid assay to search for proteins that have high affinity for LOXL1 and which might recruit the oxidase to elastin fibers. This dragnet pulled in fibulin-5, one of a family of extracellular matrix proteins with diverse functions. But would this fibulin act as the matchmaker between elastin and LOXL1? Indications are that it does. Again using immunohistochemistry, Liu was able to show that fibulin-5, LOXL1, and elastin all colocalize. But more importantly, he found that in fibulin-5 knockouts, LOXL1 fails to find elastin whereas in the Loxl1-negative mice, fibulin-5 and elastin still end up together. These results indicate that it is indeed fibulin-5 that acts as the go-between. The data have allowed Li and colleagues to propose a model in which fibulin-5 anchors LOXL1 to extracellular-matrix sites where elastin fibers are being mended (see figure). The model explains why LOXL1 is crucial for maintenance of perhaps all elastin fibers. In addition to uterine prolapse, for example, Li found a host of elastin-related problems in the Loxl1 knockouts, female and male. Rectal prolapse, emphysema, and sagging skin all got progressively worse as the animals aged. These findings may change how people think about diseases that can be traced to elastin fiber loss, according to Li. "Much attention has been given to elastolytic factors like metalloproteinases and smoking," he said. "Little is known about the role of renewal, but our work clearly shows that elastin remodeling, or mending, goes on throughout life." How this balance of loss and renewal fits in with weakening of the Bruch's layer is unclear at present, but this is an area that Li and his colleagues are currently pursuing in the hope of providing new insight into age-related macular degeneration. --Tom Fagan