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MASS SPECTROMETRY AND THE SEARCH FOR MOONLIGHTING PROTEINS Constance J. Jeffery* Laboratory for Molecular Biology, Department of Biological Sciences, MC567, University of Illinois, 900 S. Ashland Ave, Chicago, Illinois 60607 Received 12 April 2004; received (revised) 6 August 2004; accepted 17 August 2004 Published online 16 December 2004 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20041 Mass spectrometry has become one of the most important techniques in proteomics because of its use to identify the proteins found in different cell types, organelles, and multiprotein complexes. This information about protein location and binding partners can provide valuable clues to infer a protein’s function. However, more and more proteins are found that ‘‘moonlight,’’ or have more than one function, and the presence of moonlighting proteins can make more difficult the identification of protein function in those studies. This review discusses examples of moonlighting proteins and how their presence can affect the results of mass spectrometry studies that identify the locations, levels, and changes in protein expression. Although the presence of moonlighting proteins can complicate the results of those studies, mass spectrometry-derived proteinexpression profiles potentially provides a very powerful method to find additional moonlighting proteins because they do not require a prior hypothesis of the protein’s function. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:772–782, 2005 Keywords: moonlighting proteins; multifunctional; proteomics I. INTRODUCTION Mass spectrometry is widely used to identify proteins in complex mixtures, different cell types, organelles, or multiprotein complexes, and has become one of the most important techniques in large-scale proteomics projects to simultaneously identify, characterize, and determine the functions of thousands of proteins. However, the ability of a protein to moonlight, or to have more than one function, can complicate the results of those studies. Moonlighting proteins, also referred to as ‘‘gene sharing,’’ refer to a subset of multifunctional proteins in which two or more different functions are performed by one polypeptide chain. Moonlighting proteins do not include proteins that have multiple functions due to either gene fusions or multiple splice variants. In addition, they do not include proteins with the same function in multiple locations, or to protein families where different members have different functions, if each individual member has only ———— Contract grant sponsor: American Cancer Society and American Heart Association. *Correspondence to: Dr. Constance J. Jeffery, Laboratory for Molecular Biology, Dept. of Biological Sciences, MC567, University of Illinois, 900 S. Ashland Ave, Chicago, IL 60607. E-mail: [email protected] Mass Spectrometry Reviews, 2005, 24, 772– 782 # 2004 by Wiley Periodicals, Inc. one function. In this review, I discuss examples of moonlighting proteins and three reasons why moonlighting might be very common—the diverse types of proteins that moonlight, multiple ways in which they might benefit the cell, and how they are proposed to have evolved. I also discuss types of mass spectrometry studies where moonlighting proteins might be encountered; in particular, studies that involve the determination of protein locations, protein–protein interactions, and expression levels. Finally, I describe briefly the importance of moonlighting proteins in studies of disease progression and the development of novel therapeutics. Although the presence of moonlighting proteins can complicate the results of those studies, mass spectrometry has the potential to be a very powerful method to find additional moonlighting proteins. II. EXAMPLES OF MOONLIGHTING PROTEINS The several dozen proteins that have been found to moonlight (Table 1) include a wide variety of proteins from many different species, and those proteins have different combinations of functions. In some cases, the two functions are very different, as in PHGPx (glutathione peroxidase), a soluble enzyme that is also a sperm structural protein (Ursini et al., 1999). In other proteins, the two functions appear to be more closely related, such as the PMS2 DNA mismatch repair enzyme that also functions in hypermutation of antibody variable chains in immune cells (Cascalho et al., 1998). The known moonlighting proteins employ various methods to switch between functions (reviewed in Jeffery, 1999). Some proteins can have different functions when expressed in different locations within a cell. For example, several cytosolic or nuclear proteins are also found on the cell surface, where they function as receptors (Brix et al., 1998; Modun, Morrissey, & Williams, 2000). Other moonlighting proteins have different functions when they are expressed inside the cell and when they are located outside the cell. Phosphoglucose isomerase (PGI) and thymidine phosphorylase are two cytosolic enzymes that function as growth factors outside the cell (Gurney et al., 1986a,b; Furukawa et al., 1992; Watanabe et al., 1996; Xu et al., 1996; Benliname, Le, & Nabi, 1998). Other moonlighting proteins have different functions when expressed in different cell types. Still others switch function when they form multiprotein complexes, when they bind to a substrate, ligand, or product, or when they detect other changes in their environment. Paramyxovirus hemagglutinin-neuraminidase has different highand low-pH protein conformations. Movement of several amino MOONLIGHTING PROTEINS & TABLE 1. Examples of moonlighting proteins (Continued ) 773 & JEFFERY TABLE 1. (Continued ) acid sidechains and a loop in the active site may trigger a switch between the protein’s sialic acid binding and its hydrolysis functions (Crennell et al., 2000). Also, the crystal structures of a few moonlighting proteins have shown that some moonlighting proteins have multiple binding sites for different ligands. The methods for a moonlighting protein to switch between functions are not mutually exclusive, and, in some cases, a combination of factors determines protein function. III. WHY HAVE MOONLIGHTING PROTEINS? Why combine two functions in one protein? For many moonlighting proteins, the answer appears to lie in a combination of the benefits that moonlighting proteins can potentially provide to the cell or organism, and in the two proposed methods by which moonlighting proteins may have evolved. A. Benefits to the Organism In general, moonlighting proteins can benefit the organism by coordinating multiple cellular pathways or activities, providing a method to respond to changing conditions, or providing a feedback mechanism. The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is also a regulator of other channels (Stutts et al., 1995). It regulates the channel activity of another chloride channel, the outwardly rectifying chloride channel (ORCC), and a sodium channel (ENaC). Together, those two activities probably help to promote epithelial cell ion homeostasis. The enzyme lysyl hydroxylase 3 catalyzes two steps in collagen biosynthesis (Heikkinen et al., 2000). Combining the two catalytic activities in one protein might provide a way for the cell to coordinate two steps that are involved in collagen maturation. The enzymes delta-aminolevulinic acid dehydratase and Lon protease appear to switch between two different functions as a way for the cell to respond to a changing environment. Deltaaminolevulinic acid dehydratase, an enzyme in the heme biosynthesis pathway, is the same protein as the proteasome inhibitor CF-2 (Guo, Gu, & Etlinger, 1994). The combination of these two functions within one protein might provide a switch point between protein degradation and heme biosynthesis. Similarly, the Lon protease in eukaryotic mitochodria is a protease and a chaperone (Suzuki et al., 1997). The Lon protein 774 probably switches between degrading proteins and helping them to fold in response to changes in the cell’s needs. Several enzymes have a DNA- or RNA-binding activity as a second function that enables them to function in the regulation of transcription or translation. The cytosolic enzyme aconitase is the same protein as the iron-responsive element binding protein (IRE-BP). It loses cataltytic activity and binds to DNA to cause changes in transcription levels when the cell’s iron levels change (Kennedy et al., 1992). Thymidylate synthase 3, biotin synthetase (birA), and PutA proline dehydrogenase are three other enzymes that move to bind DNA or RNA and regulate transcription or translation in response to changing levels of substrate, product, or cofactor (Barker & Campbell, 1981; Chu et al., 1991; Ostrovsky de Spicer & Maloy, 1993). Although the examples above suggest that there is a benefit for a protein to have two functions, there is not always a clear connection between the two functions in some moonlighting proteins. For example, PGI is a cytosolic enzyme in glycolysis and an extracellular cytokine (Gurney et al., 1986a,b; Watanabe et al., 1996; Xu et al., 1996; Benliname, Le, & Nabi, 1998). Perhaps after the receptor-binding function evolved, there was simply no selective pressure to remove either function. B. Evolution There appears to be two general methods for proteins to have evolved a second function: modification of apparently unused solvent-exposed surface area, and the adoption into a new multiprotein complex without significant changes in protein structure. Both methods make use of general features of protein structure and could apply to many proteins. In many enzymes, the active site pocket is a relatively small part of the protein structure, and there is a lot of apparently unused solvent-exposed surface area that can undergo many changes without adversely affecting catalysis by the enzyme. PGI is one example of an enzyme that evolved a protein–protein interaction surface in addition to its active site pocket. PGI is a relatively large protein, a dimer with 557 amino acids in each subunit, but it has a relatively small active site pocket for binding a single monosaccharide (Jeffery et al., 1999). Because PGI is found in almost all species, it apparently first evolved over three billion years ago. Through all this time, its active site has been conserved, but its surface has undergone many changes (Fig. 1). A comparison of the crystal structures from rabbit PGI and MOONLIGHTING PROTEINS & FIGURE 1. A protein can evolve a second function through mutations of ‘‘unused’’ solvent-exposed surface area. PGI is an enzyme that catalyzes the second step in glycolysis in the cytoplasm of most cells. Outside the cell, PGI binds to a cell surface receptor that is found on some tumor cell types, pre-B cells, and some embryonal neurons. A: In a sketch of the PGI dimer crystal structure (black lines), the amino acids that make up the active site of phosphoglucose isomerase (red ovals) have been conserved throughout evolution. However, the surface of the protein has changed dramatically. Subunits of PGI from a crystal structure of the rabbit enzyme (B) and from Bacillus stearothermophilius (C) differ a great deal in the lengths of the C-terminus and in the lengths and composition of surface loops. The rabbit protein contains several additional surface helices, turns, and other features that are not found on the bacterial enzyme. One or more of these features on the rabbit enzyme might correspond to a receptor binding site. (Figures reproduced from Jeffery et al., 1999, with permission from American Chemical Society, copyright 1999.) bacterial PGI shows that many surface features have changed quite dramatically during evolution, with the addition or subtraction of entire helices, loop regions, and pockets. Because those surface features are all potential sites for a novel protein/ protein interaction function to evolve, it is quite possible that the random accumulation of mutations on PGI surface might have resulted in an additional binding site that enables PGI to serve as a growth factor outside of the cell. In general, as long as a second function, such as the receptor-binding function of PGI, does not adversely affect the protein’s first function, it could be retained during further evolution. In some cases where crystal structures of moonlighting proteins are available, the patterns of amino acid conservation and divergence can be used to identify the potential locations of additional functional sites within the protein structures. The enzyme 4a-carbinolamine dehydratase has evolved a protein–protein interaction function; it is the dimerization cofactor of the transcription factor hepatic nuclear factor 1a (DcoH) (Citron et al., 1992). Comparison of the protein structure and amino acid sequences from several species enabled the location of the protein–protein interaction surface. Interestingly, the new ligand-binding site does not have to be far from the original active site pocket. The enzyme prostaglandin H2 synthase-1 (PGHS) has two active sites, a hemedependent peroxidase site and a cyclooxygenase site, that are found adjacent to each other in the enzyme structure (Picot, Loll, & Garavito, 1994). The two active sites catalyze consecutive reactions in the synthesis of prostaglandin H2. Changes in protein structure or surface features are not always necessary for a protein to develop a second function. Several crystallins, which are expressed in high concentrations in 775 & JEFFERY the lens of the eye, are ubiquitous soluble cytosolic enzymes that were recruited for a second function when the eye evolved (Piatigorsky, 1998). E. coli thioredoxin was adopted by the T7 phage to become a subunit of its DNA polymerase (Mark & Richardson, 1976). Some ribosomal proteins have been found to be identical to cytoplasmic proteins and were probably recruited as the ribosome developed (Wool, 1996). In those cases, either the expression in a new cell type or the joining to a multiprotein complex were involved in the development of the new function. The variety of moonlighting proteins, the potential benefits that they can provide to the organism, and the two proposed methods of evolution of additional functions suggest that moonlighting proteins might be very common. The ability of a protein to have multiple functions might complicate studies of individual proteins, but moonlighting proteins might have a large impact on proteomics- the use of large-scale biochemical, genetic, or computational methods to identify, characterize, and determine the functions of many proteins simultaneously. IV. MOONLIGHTING PROTEINS AND MASS SPECTROMETRY PROTEOMICS METHODS A. Locations of Proteins Mass spectrometry is one of the most important techniques in proteomics studies because it enables the identification of proteins in complex mixtures and does not require any prior knowledge of protein function. The resulting protein-expression profiles for different cell types, organelles, or multiprotein complexes can be used to help predict protein functions. The results from those mass spectrometry studies can be affected by the presence of moonlighting proteins because, as described above, one of the general ways for a protein to moonlight is to be found in multiple locations: inside and outside of cells, within different cell types, in different locations within a cell, within different protein complexes, and with different binding partners (Fig. 2). However, although moonlighting proteins can complicate the interpretation of the results of those experiments, highthroughput mass spectrometry proteomics-level studies could provide an important way to find additional moonlighting proteins because the presence in an unexpected location or multiprotein complex can suggest that a protein has a second function. There are several examples of common cytosolic enzymes that have a second function outside of the cell. Phosphoglycerate kinase is a cytosolic enzyme in the glycolysis pathway that is secreted by some tumor cells (Lay et al., 2000). Outside the cell, it serves as a disulfide reductase. It reduces plasmin, and the reduced plasmin is cleaved to produce angiostatin, an angiogenesis inhibitor. As described above, another enzyme from glycolysis, PGI, also has a function outside the cell where it binds to cell surface receptors on target cells. Thymidine phosphorylase, the enzyme that dephosphorylates thymidine and deoxyuridine in the cytoplasm, is also an extracellular growth factor (Furukawa et al., 1992). It is the same protein as the platelet-derived endothelial cell growth factor, a growth factor that stimulates the growth and chemotaxis of endothelial cells. Thymosin beta 4 sulfoxide, an actin polymerization inhibitor in the cell cytoplasm, 776 FIGURE 2. Mass spectrometry can help to identify moonlighting proteins in which the protein function changes with the location or binding partners. A protein can have one function (for example, a catalytic function) in the cytosol of one cell type (represented as a gray oval with catalytic activity). The same protein can have a different function when it is outside of the cell, expressed in a different cell type, moves into an organelle, interacts with other proteins to form a multiprotein complex, or interacts with another protein to form a heterodimer. is secreted and serves as a modulator of the inflammation response to cell injury (Young et al., 1999). At least one nuclear protein also has a second function outside of the cell. The SMC3 protein (structural maintenance of chromosome 3) in mice is involved in sister chromatid cohesion in the nucleus, but is the same protein as bamacam, a component of the basement membrane (Wu & Couchman, 1997; Darwiche, Freeman, & Strunnikov, 1999). Moonlighting proteins can also perform different functions when expressed in different cell types. For example, neuropilin is a cell-surface receptor that binds different ligands on neurons and endothelial cells (Soker et al., 1998). In neurons, it binds semaphorin III and plays a role in axonal guidance. In endothelial cells, it binds vascular endothelial growth factor (VEGF) and is involved in the detection of the need for new blood cells. The ability of a protein to moonlight with two different functions in two different cell types can increase the difficulty of deducing protein functions from a comparison of expression profiles. In general, groups of proteins that function together in a biochemical pathway, multiprotein complex, or signaling pathway would be expected to have similar patterns of expression—an ‘‘all or none’’ pattern of expression—where all of the proteins are MOONLIGHTING PROTEINS FIGURE 3. Comparison of protein-expression profiles from different cell types could suggest that a protein moonlights. Parts A–C are schematic representations of mass spectrometry protein-expression profiles for three different cell types. Although many different proteins (vertical lines) are expressed in all three cell types, proteins A, B, C, D, and E work together in a biochemical pathway or multiprotein complex and are, therefore, either all expressed together (cell type A), or none is expressed (cell type B). However, protein D is a moonlighting protein that is also expressed in cell type C in the absence of proteins A, B, C, and E. expressed in some cell types and none is expressed in other cell types. A schematic diagram to show how this might be reflected in the results of a mass spectrometry experiment to compare protein expression in different cell types is shown in Figure 3. Proteins A, B, C, D, and E function together in a biochemical pathway or multiprotein complex, and are, therefore, all expressed together in the results in Figure 3A, but none of the five proteins is expressed in Figure 3B. However, if protein D has a moonlighting function, then it might have an unusual expression pattern. In Figure 3C, protein D is expressed in the absence of proteins A, B, C, or E. In other words, the expression of a protein in an unexpected cell type can suggest that the protein has a second function. Some moonlighting proteins are found in multiple cellular locations, or they move to different parts of the cell when they switch functions. The PutA protein in E. coli has enzymatic activity when it binds to the cell membrane, but it moonlights as a transcriptional repressor when it is in the cell cytoplasm (Ostrovsky de Spicer & Maloy, 1993). Histone H1 is best known as a nuclear protein in the interior of chromatin fibers, but it is also found on the extracellular surface of the cell membrane, where it is a receptor for thyroglobulin (Brix et al., 1998). Mass spectrometry can be used to identify the cellular location of different proteins by using purified organelle preparations, and unexpected locations or unusual combinations of locations can & suggest that a protein is moonlighting. For example, a mass spectrometry study of the Candida albicans cell wall proteome identified several cytoplasmic proteins, including glycolytic enzymes, as being part of the cell wall (Pitarch et al., 2002). Some of these proteins might have a moonlighting function in the cell wall in addition to their roles in the cytoplasm. However, additional experiments are needed to determine if the proteins perform their first function, catalysis, in the cell wall or if they have a second, moonlighting function. Mass spectrometry is also applied to even smaller groups of proteins–intracellular multiprotein complexes. Many proteins assemble into multiprotein complexes that serve as ‘‘molecular machines’’ with a particular defined function such as RNA splicing, or protein synthesis, transport, or degradation. Those complexes can often be purified, and mass spectrometry can identify the proteins that comprise the complex. When protein crosslinking steps are added before the mass spectrometry steps, analysis of pairs and triplets can help to identify any direct interactions within the complexes. Several moonlighting proteins are either found in more than one multiprotein complex, or have one function as a soluble protein and switch functions when they become part of a complex. There might be many more examples of moonlighting proteins of these categories because adopting a new binding site is one of the ways in which several different moonlighting proteins have apparently evolved. Several proteins in the ribosome have been found to be identical to cytoplasmic or nuclear proteins (Wool, 1996). The 240 kDa inhibitory component of the proteasome, a large multiprotein complex involved in the degradation of old proteins, is the same protein as deltaaminolevulinic acid dehydratase, an enzyme in the heme biosynthesis pathway (Guo, Gu, & Etlinger, 1994). Other proteasome proteins have a second function in transcription. Sug1/ Rpt6 and Sug2/Rpt4 are ATPase proteins in the proteasome base complex (part of the 19S particle) (Fig. 4). They are also found to have a role in RNA polIII transcription because they bind to the Gal4 transactivator protein and alter transcription levels from the GAL1-10 promoter (Gonzalez et al., 2002). Clf1p is a pre-mRNA splicing factor in S. cerevisiae, and it can be co-immunoprecipitated with the U5 and U6 small nuclear ribonucleoptrotein particles, pre-mRNA, and other components of in vitro splicing reactions. However, Clf1p is also found in the origin of replication complex (ORC) (Ben-Yehuda et al., 2000; Zhu et al., 2002) and functions in the initiation of DNA replication (Fig. 5). Mass spectrometry could be a powerful tool to identify additional proteins that moonlight by joining a multiprotein complex. For FIGURE 4. Proteasome proteins with a second function in gene transcription. When the concentration of glucose changes, proteins of the proteasome 19S particle base complex move to the nucleus to interact with Gal4 at the Gal1-10 promoter complex. That interaction causes a change in the level of expression from the Gal1-10 promoter. 777 & JEFFERY B. Unexpected Protein Expression Levels FIGURE 5. Clf1p has different functions when it is a part of different multiprotein complexes. Clf1p interacts with splicing factors to promote pre-mRNA splicing. It has a different function, in replication, when it interacts with other proteins at the origin of replication (ORC). example, a mass spectrometry study of bovine NADH:Ubiquinone Oxidoreductase (complex I of the mitochondrial electron transport chain) identified a homolog of GRIM-19, a cell death regulatory protein, as a new subunit of the complex (Fearnley et al., 2001). [However, since the cell death regulatory function has not been experimentally demonstrated for the bovine protein (it has been demonstrated for the human GRIM-19 homolog), it will be necessary to demonstrate the presence of the regulatory function before the bovine GRIM-19 protein can be called a moonlighting protein.] Many proteins that do not join into large multiprotein complexes can still have one or more binding partners, and mass spectrometry can be used to identify individual binding partners of a protein; those data can be used to suggest the protein’s function. If a protein is found to interact with multiple proteins that are known to be involved in different cellular activities, it might suggest that the protein moonlights. Other methods to identify protein function based on identification of protein binding partners, including yeast two-hybrid screens or peptide or protein microarrays, often result in ‘‘false positives’’; i.e., interactions that are unexpected based on the understood function of a protein. However, not all of those interactions might be ‘‘false’’—some of those unexpected protein interactions might be due to a second function of the protein. Mass spectrometry protein-expression profiles are likely to become a key method to identify more moonlighting proteins because, as described above, proteins that have different functions in different locations or within different multiprotein complexes might provide benefits to the cell (coordinate different cellular activities, provide a means to respond to changes in the environment, and provide a feedback mechanism), and therefore those kinds of moonlighting proteins might be common. When protein expression profiles identify proteins for which one function is already known in an unexpected cell type, organelle, or multiprotein complex, it is possible that the protein might have a moonlighting function. In the future, the results of mass spectrometry studies could be analyzed to specifically look for those proteins that give unexpected results. 778 Although the above examples emphasize studies of the presence or absence of a protein in different locations or complexes, an unusual amount of a particular protein might also suggest that the protein has a second function, and mass spectrometry can also be used to determine relative levels of protein expression. For example, many enzymes or signaling molecules are not needed at high concentrations because they are used repeatedly, are used in a very specialized pathway, or are involved in a cascade that amplifies a signal. However, sometimes one member of an enzyme pathway is found at unusually high levels (Fig. 6). For example, delta-aminolevulinic acid dehydratase is expressed at levels up to 1% of total soluble protein—far more than is needed to catalyze a step in heme biosynthesis. This unusually high level of expression makes more sense when we consider that it has a second function as a proteasome inhibitory subunit. In addition to differences in protein expression levels between cell types, mass spectrometry is often used to monitor changes in protein levels within one cell type. Protein expression levels can change in response to changes in the environment, such as the addition of a hormone or small molecule drug. Protein expression levels also vary at different stages in the cell cycle or during different developmental stages in an embryo. The addition of stable isotope-labeling methods enables the quantitation of differences in protein expression levels in different samples. Those methods can help to identify proteins that undergo an unusual or unexpected change in cellular concentration; those data might also suggest the protein is moonlighting. V. MOONLIGHTING PROTEINS IN DISEASE In addition to clarifying our understanding of basic physiological processes, proteomics methods, including mass spectrometry, FIGURE 6. Unusual protein expression levels can suggest that a protein is a moonlighting protein. In this schematic example, the ovals and rectangles represent six different enzymes that function in a biochemical pathway for the synthesis of molecule F. Five of the enzymes are expressed at moderate levels, but one enzyme, represented by the hexagon, is expressed at a much higher level. One explanation might be that enzyme also has a second function that is suggested here as a structural role in a multiprotein complex. MOONLIGHTING PROTEINS can help to identify proteins involved in disease. Proteinexpression profiles determined by mass spectrometry identify those proteins whose expression levels differ between healthy and diseased cells. That information can be important to understand disease development, and in the development of novel therapeutics. In addition, some specific proteins, whose concentrations change during progression of a disease, might serve as good biomarkers to diagnose or monitor the progression of the disease. However, if a protein moonlights, then its expression levels might vary in an unexpected fashion that could complicate an accurate understanding of the disease and the development of therapeutics. Moonlighting proteins are involved in tumor-cell motility, angiogenesis, DNA synthesis or repair, chromatin and cytoskeleton structure, extracellular matrix, metabolic pathways, and cystic fibrosis (reviewed in Jeffery, 2003). In general, much care must be taken in the choice of target proteins for the design of novel therapeutics, and moonlighting is one factor that can add to the difficulty of choosing a protein target (Searls, 2003). If a protein moonlights, then one or both of its functions might be involved in the disease. It is important that a drug that affects protein function affects the correct function. Problems could arise if the novel drug also affects additional functions that are not involved in the disease; that complexity could lead to deleterious side effects and to toxicity. Knowing whether or not a protein has moonlighting functions can also be the important choice of an appropriate biomarker. If a protein moonlights, then multiple factors might affect its expression level in addition to the presence or absence of disease; therefore, a moonlighting protein might not be a good biomarker for the disease. Moonlighting proteins might also obfuscate the understanding of the causes of genetic diseases because mutations in a moonlighting protein might lead to an unexpected combination of symptoms. Similarly, a moonlighting function might be one reason that experimental methods that alter a protein’s function (gene knockouts, RNA interference, antisense RNA, or protein overexpression) can result in unexpected phenotypes (Fig. 7). The observed phenotype caused by mutations or altered protein expression levels might be difficult to explain based on only one expected function of a protein. A second function might also be affected, and the effects on the second function might be responsible for the observed phenotype. For example, the tumor suppressor protein that causes inherited uterine fibroids, skin leiomyomata, and papillary renal cell cancer is fumarate hydratase—an enzyme from the Krebs citric acid cycle (Multiple Leiomyoma Consortium, 2002). It is possible that fumarate hydratase has a second, unknown function that, when altered, leads to the observed symptoms. In some cases, even if all of the functions of a protein are known, it might be unclear as to which function (or both) is affected by a disease-causing mutation. VI. A WORD OF CAUTION Although the above review focused on how the finding of a protein in multiple locations or complexes can suggest that a protein is moonlighting, it is important to also include a word of caution. The presence of a protein in an unexpected location, or the observation of unexpected results, can suggest that the protein & FIGURE 7. Moonlighting proteins might be one explanation for the unexpected results that are found in gene knockout experiments. A: Deletion of a gene that encodes a protein that functions in the adult mouse (dark circle) is expected to result in a mutant phenotype in the adult mouse, shown here as a change of coat color from gray to white. B: However, if the protein has one function in the adult mouse and also has a moonlighting function during development of the mouse embryo, then knocking out the gene might result in the death of the embryo. has a second function. However, it is not always the case that it does. Some single-function proteins are found in multiple locations because a single function might be used in both places (for example, a kinase activity), and a protein with the same function in two different locations is not a moonlighting function. Additional evidence that the protein truly performs two different functions in the two locations is needed before concluding that a protein is moonlighting. It is important that the observations of protein location be complemented with additional studies, such as further biochemical characterization, before a protein is determined to be moonlighting. The examples given in Table 1 were selected based on biochemical data or similar evidence that the one protein performs each of its two listed functions. In the example described earlier of the Candida albicans cell wall proteomics study, if the glycolytic enzymes found in the cell wall are using the same catalytic function that they use in their role in the cytosol, they are not moonlighting. They are only moonlighting if their second role involves a second molecular function, such as a structural function. Ideally, to clarify the function(s) of the many proteins being studied in proteomics projects, it would be helpful to combine the results from multiple methods, such as mass spectrometry data with gene knockout data and/or yeast two-hybrid data. Databases must be constructed to provide easier methods of combining data 779 & JEFFERY from different kinds of experiments. As the number of known moonlighting proteins increases, databases must be extended to include the description of the multiple functions; when, where, and how to switch between them; and the clarification of which species contain a form of the protein with multiple functions. Another note of caution regards the use of current proteinsequence databases to interpret the results of protein-expression profile experiments. Many proteins sequences are annotated with a suggested protein function that is based on amino acid sequence homology to another protein with a known function, often from another species. However, if one protein is a moonlighting protein, then it does not mean that all of its homologs would have both functions. For example, the E. coli aspartate receptor is also the receptor for the maltose binding protein, but the Salmonella typhimurium aspartate receptor does not bind to the maltose binding protein (Wolff & Parkinson, 1988; Mowbray & Koshland, 1990). Similarly, if a protein has multiple isoforms within one organism, then one isoform could have multiple functions, but other isoforms might each have one function. For example, three isoforms of lysyl hydroxylase (in collagen synthesis) share 60% overall amino acid sequence identity, and all three isoforms have lysyl hydroxylase activity. However, only isoform 3 (LH3) has galactosylhydroxylysyl glucosyltransferase activity. VII. CONCLUSIONS More and more proteins are being found that moonlight, and the diverse types of moonlighting proteins, methods to switch between functions, ways they might benefit the organism, and proposed methods of evolution suggest that many more moonlighting proteins might be found. Although moonlighting proteins can complicate the interpretation of the results of mass spectrometry projects, especially large-scale proteomics projects, those kinds of projects are likely to lead to the identification of many more moonlighting proteins because they do not require a prior estimate of protein function. Mass spectrometry has the potential of being very important in the future for identifying more candidates for moonlighting proteins, and specific experiments can be designed to do so by considering the methods by which moonlighting proteins can switch functions. The presence of moonlighting function can also complicate the use and annotation of protein sequence databases; however, in the future, novel methods to combine data from proteomics experiments with mass spectrometry, yeast-two-hybrid experiments, knockout experiments, and other methods could also aid in the identification of more moonlighting proteins. For now, the ability of many diverse proteins to moonlight could help to explain why humans, multicellular organisms with many different cell types and organ systems, have only approximately five times as many proteins as S. cerevisiae, a single-celled yeast. VIII. ABBREVIATIONS aiPLA2 CFTR 780 acidic Ca2þ phospholipase A2 cystic fibrosis transmembrane conductance regulator 1-cys DcoH LH3 NADH ORC PGI PMS2 SMC3 VEGF 1-cysteine dimerization cofactor of the transcription factor hepatic nuclear factor 1a lysyl hydroxylase 3 nicotinamide adenine dinucleotide origin of replication complex phosphoglucose isomerase mouse mismatch repair enzyme and homolog of the yeast ‘‘postmeiotic segregation increased 2’’ gene sister chromatin cohesion protein vascular endothelial growth factor ACKNOWLEDGMENTS Research on moonlighting proteins in the Jeffery lab is supported by grants from the American Cancer Society and the American Heart Association. 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Evidence that the pre-mRNA splicing factor Clf1p plays a role in DNA replication in Saccharomyces cerevisiae. Genetics 160:1319– 1333. 781 & JEFFERY Prof. Constance J. Jeffery received her B.S. degree in Biology from the Massachusetts Institute of Technology and her Ph.D. in Biochemistry from the University of California at Berkeley, where she worked on the bacterial aspartate receptor under the supervision of Daniel Koshland. She received a Cystic Fibrosis Foundation Postdoctoral Fellowship that allowed her to do postdoctoral studies with Greg Petsko and Dagmar Ringe at Brandeis University, where she studied protein X-ray crystallography. During that period, she worked on the crystal structure of phosphoglucose isomerase/autocrine motility factor, a cytosolic enzyme that is also an extracellular growth factor. Her work on phosphoglucose isomerase led her to coin the term moonlighting proteins. In 1999, she joined the faculty of the Department of Biological Sciences at the University of Illinois at Chicago. In addition to continuing studies of phosphoglucose isomerase and other moonlighting proteins, her laboratory’s research interests include the structures and molecular mechanisms of cell surface receptors, transporters, and other transmembrane proteins. 782