Enzymatic Alter-Egos Unmasked

Some proteins lead double or even quadruple lives. In 1994, researchers discovered the gene responsible for Wiskott Aldrich syndrome, an X-linked genetic disorder in which affected patients generally succumb to infections or cancer. Because of the observed immune dysfunction, the protein, called WASP, was believed to regulate lymphocytes or platelets.1 But, challenging the traditionally held one-protein-one-function notion, subsequent studies found that WASP had several jobs, among them organizing cellular actin skeletons and communicating between the immune system's T and B cells.2 This multifunctionality, considered an oddity a decade ago,3 might explain the disorder's wide array of manifestations. It turns out, however, that WASP's multitasking is not an anomaly at all. Multifunctional or "moonlighting" proteins have turned out to be surprisingly common, and examples of proteins with as many as five proposed functions have emerged.4 "People had had a notion for many, many years that some proteins had more than one function," says Gregory Petsko, professor of biochemistry and chemistry at Brandeis University in Waltham, Mass. "But most people thought it was an unusual situation, with perhaps one or two such molecules in an organism. Only recently, with the advent of modern genomics, has it become clear that in higher organisms, this may be the rule rather than the exception." Moonlighting could affect medical research for several reasons, says Constance J. Jeffery, an assistant professor of biological sciences at the University of Illinois at Chicago. A disease with varying and seemingly unrelated symptoms may result from defects in a single protein. Multifunctional proteins also may complicate the task of developing cures for illnesses. A given treatment might fail because it doesn't address all the functions of a missing protein, or a drug might have side effects because it interferes with protein activities distinct from the target function. Bioinformaticians also need to adapt to the prevalence of such proteins. Researchers using programs designed to predict protein function from structure must consider the possibility that multiple functions are possible.4,5 "Our annotation of the genome is incomplete until we know the functions of all the gene products," Petsko says. "We may be fooling ourselves" by continuing to assume each protein has one function. Multifunctional proteins shed light on the way evolution has economized on its inventions, reducing the amount of genetic material it needs, says Shelley D. Copley, an associate professor at the University of Colorado, Boulder. It also highlights a little-appreciated aspect of cellular complexity: the way cells employ ingenious mechanisms to switch the functions of a single molecule. WHAT'S KNOWN In two recent, separate reviews,6,7 Jeffery and Copley identified 38 examples of moonlighting proteins, and they acknowledged that their lists are only partial. These moonlighters are distinct from the common enzymes that can act on dozens of substrates using the same active site, cases of so-called "catalytic promiscuity."7 Such enzymes have been well known to researchers for a longer time because their multiple functions are relatively easy to identify, says Petsko. Moonlighting proteins, by contrast, conduct enzymatic and nonenzymatic activities, sometimes taking part in widely divergent pathways. Among the earlier known moonlighting proteins are several crystallins, the structural proteins in the eye's lens with various functions throughout the body. The crystallins include lactate dehydrogenase, a fermentation catalyst; glutathione S-transferase, a toxin neutralizer; and various heat-shock proteins. Other early examples of moonlighting proteins include several enzymes that work in elaborate feedback loops to regulate their own transcription.4 Newer examples of moonlighting may be even more striking for their ingenuity and simplicity. For instance, PMS2 is part of a proofreading system that in normal mammalian nuclei removes mismatched nucleotides when they are incorporated into new DNA strands. Yet B cells may co-opt the protein to vastly increase, rather than decrease, mutation rates in immunoglobulin genes, according to researchers at the University of California, San Francisco.8 A process called hypermutation raises the B-cell mutation rate by more than 100,000-fold over normal cells,9 allowing them to produce antibodies against the widest possible range of foreign substances. Here, PMS2 is again involved, but the system removes the nucleotide from the parental strand rather than the new strand. Thus, a nucleotide complementary to the mismatch is inserted and the mutation becomes entrenched. The system may distinguish parental from newly synthesized strands by the presence of methyl groups on the old strand. Although PMS2 would seem to conduct these opposing activities using basically the same mechanism, other moonlighting proteins have functions whose relationship is unclear, if they exist at all. Phosphoglucose isomerase is a common enzyme that catalyzes a step in glycolysis, a central metabolic pathway. It also has been proposed to play at least four other roles: a signaling molecule that aids B-cell maturation, a nerve growth factor, a stimulator of differentiation in myeloid leukemia cells, and a migratory mediator for cancers and some other cells.10 "Perhaps blocking that function could be one way of inhibiting metastasis," says Jeffery, whose laboratory is studying the protein, but "it would be important not to adversely affect its catalytic activity." Cystic fibrosis is another disease involving a multifunctional protein. The cystic fibrosis transmembrane conductance regulator is both a channel for chloride ions and a regulator of a sodium ion channel in the lungs. Some researchers are modeling cystic fibrosis as the result of the combined effect of defects in these functions.11 CATALYTIC CHAMELEONS Many moonlighting proteins aren't known for involvement in disease, but are notable for how central they are to biochemical pathways. Some proteins, including the well-studied cytochrome C, serve as part of the electron-transport mechanism for generating energy, and are involved in apoptosis.7 Various components of ribosomes also have functions in DNA repair. Fumarate hydratase is an enzyme in the Krebs cycle, a central metabolic pathway; it is also a tumor suppressor.4 Some proteins may conduct their multiple functions by using different domains or binding sites, like molecular Swiss Army knives. Others switch among their roles by appearing in different locations in an organism, or as parts of different complexes, or in conjunction with different concentrations of substrates, ligands, or cofactors.10 The discovery of widespread moonlighting has implications for researchers using protein function prediction programs. The programs usually indicate several potential functions for a protein based on comparisons with other proteins and sequences. Researchers tend to ignore all except the top-scored hit, but this may be unwise, says Enrique Querol, professor of biochemistry at the Universitat Autonoma de Barcelona. Researchers also may want to consider whether a particular program can predict multiple functions successfully. Querol and colleagues assessed about a dozen leading sequence-analysis algorithms for their ability to disclose multiple functions, using 45 known moonlighter proteins as tests. The team found that two programs, PSI-BLAST and PRODOM, performed best.5 Researchers uncover moonlighters through various means, often without intending to, Jeffery says. "People are finding the protein they're studying is the same as a protein someone else is studying" in a different role, she notes. "The pace of research being done, the number of assays, and the number of sequences available are all increasing. So the number of examples known of moonlighting is increasing." Jack Lucentini (jekluc@aol.com) is a freelance writer in New York City. References 1. J.M. Derry et al., "Isolation of a novel gene mutated in Wiskott-Aldrich syndrome," Cell, 78:635-44, 1994. 2. S. Nonoyama, "Wiskott-Aldrich syndrome (role of WASP)," J Med Dent Sci, 48:1-6, 2001. 3. B.A. Citron et al., "Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins," Proc Natl Acad Sci, 89:11891-4, 1992. 4. C.J. Jeffery, "Moonlighting proteins: old proteins learning new tricks," Trends Genet, 19:415-7, August. 2003. 5. A. Gomez et al., "Do current sequence analysis algorithms disclose multifunctional (moonlighting) proteins?" Bioinformatics, 19:895-6, May 1, 2003. 6. C.J. Jeffery, "Multifunctional proteins: examples of gene sharing," Ann Med, 35:28-35, 2003. 7. S.D. Copley, "Enzymes with extra talents: moonlighting functions and catalytic promiscuity," Curr Opin Chem Biol, 7:265-72, April 2003. 8. M. Cascalho, "Mismatch repair co-opted by hypermutation," Science, 279:1207-10, 1998. 9. J. Bachl et al., "Enhancers of hypermutation," Immunogenetics, 45:59-64, 1996. 10. C.J. Jeffery, "Moonlighting proteins," Trends Biochem Sci, 24:8-11, 1999. 11. R.C. Boucher, "An overview of the pathogenesis of cystic fibrosis lung disease," Adv Drug Deliv Rev, 54:1359-71, 2002. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Enzymatic Alter-Egos Unmasked

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