Download Mass spectrometry and the search for moonlighting proteins

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. The author thanks Crystal Deberry and Sarah
Goldrick for assistance in manuscript preparation.
REFERENCES
Barker DF, Campbell AM. 1981. Genetic and biochemical characterization of
the birA gene and its product: Evidence for a direct role of biotin
holoenzyme synthetase in repression of the biotin operon in Escherichia
coli. J Mol Biol 146:469–492.
Ben-Yehuda S, Dix I, Russel CS, McGarvey M, Beggs JD, Kupiec M. 2000.
Genetic and physical interactions between factors involved in both cell
cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae. Genetics 156:1503–1517.
Benliname N, Le PU, Nabi IR. 1998. Localization of autocrine motility factor
receptor to caveolae and clathrin-independent internalization of its
ligand to smooth endoplasmic reticulum. Mol Biol Cell 9:1773–1786.
Bolduc JM, Spiegel PC, Chatterjee P, Brady KL, Downing ME, Caprara MC,
Waring RB, Stoddard , BL. 2003. Structural and biochemical analysis of
DNA and RNA binding by a bifunctional homing endonuclease and
group I intron splicing factor. Genes Dev 17:2875–2888.
Brix K, Summa W, Lottspeich F, Herzog V. 1998. Extracellularly occurring
histone H1 mediates the binding of thyroglobulin to the cell surface of
mouse macrophages. J Clin Invest 102:283–293.
Caprara MG, Mohr G, Lambowitz AM. 1996. A tyrosyl-tRNA synthetase
protein induces tertiary folding of the group I intron catalytic core. J Mol
Biol 257:512–531.
Cascalho M, Wong J, Steinberg C, Wabl M. 1998. Mismatch repair co-opted
by hypermutation. Science 279:1207–1210.
Chen J-W, Dodia C, Feinstein SI, Jain MK, Fisher AB. 2000. 1-Cys
peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and
phospholipase A2 activities. J Biol Chem 275:28421–28427.
Chu E, Koeller DM, Casey JL, Drake JC, Chabner BA, Elwood PC, Zinn S,
Allegra CJ. 1991. Autoregulation of human thymidylate synthase
messenger RNA translation by thymidylate synthase. Proc Natl Acad
Sci USA 88:8977–8981.
Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR,
Kaufman S. 1992. Identity of 4a-carbinolamine dehydratase, a
component of the phenylalanine hydroxylation system, and DCoH, a
transregulator of homeodomain proteins. Proc Natl Acad Sci USA
89:11891–11894.
Crennell S, Takimoto T, Portner A, Taylor G. 2000. Crystal structure of the
multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat
Struct Biol 7:1068–1074.
MOONLIGHTING PROTEINS
Darwiche N, Freeman LA, Strunnikov A. 1999. Characterization of the
components of the putative mammalian sister chromatid cohesion
complex. Gene 233:39–47.
Fearnley IM, Carroll J, Shannon RJ, Runswick MJ, Walker JE, Hirst J. 2001.
GRIM-19, a cell death regulatory gene product, is a subunit of bovine
mitochondrial NADH:Ubiquinone oxidoreductase (complex I). J Biol
Chem 276:38345–38348.
Furukawa T, Yoshimura A, Sumizawa T, Haraguchi M, Akiyama S-I. 1992.
Angiogenic factor. Nature 356:668.
Gonzalez F, Delahodde A, Kodadek T, Johnstom SA. 2002. Recruitment of a
19S proteasome subcomplex to an activated promoter. Science 296:
548–550.
Guo GG, Gu M, Etlinger JD. 1994. 240-kDa proteasome inhibitor (CF-2) is
identical to delta-aminolevulinic acid dehydratase. J Biol Chem 269:
12399–12402.
Gurney ME, Apatoff BR, Spear GT, Baumel MJ, Antel JP, Bania MB, Reder
AT. 1986a. Neuroleukin: A lymphokine product of lectin-stimulated T
cells. Science 234:574–581.
Gurney ME, Heinrich SP, Lee MR, Yin HS. 1986b. Molecular cloning and
expression of neuroleukin, neurotrophic factor for spinal and sensory
neurons. Science 234:566–74.
Heikkinen J, Risteli M, Chunguag W, Latvala J, Rossi M, Valtavaara M,
Myllyla R. 2000. Lysyl hydroxylase 3 is a multifunctional protein
posessing collagen glucosyltransferase activity. J Biol Chem 275:
36158–36163.
Jan Y, Matter M, Pai J, Chen Y-L, Pilch J, Komatsu M, Ong E, Fukuda M,
Ruoslahti E. 2004. A mitchondrial protein, bit1, mediates apoptosis
regulated by integrins and groucho/TLE corepressorss. Cell 116:751–
762.
Jeffery CJ. 1999. Moonlighting proteins. Trends Biochem Sci 24:8–11.
Jeffery CJ. 2003. Multifunctional proteins: Examples of gene sharing. Ann
Med 35:28–35.
Jeffery CJ, Bahnson BJ, Chien W, Ringe D, Petsko GA. 1999. Crystal
structure of rabbit phosphoglucose isomerase, a glycolytic enzyme that
moonlights as neuroleukin, autocrine motility factor, and differentiation
mediator. Biochemistry 39:955–964.
Kennedy MC, Mende-Mueller L, Blondin GA, Beiner H. 1992. Purification
and characterization of cytosolic aconitase from beef liver and its
relationship to the iron-responsive element binding protein. Proc Natl
Acad Sci USA 89:11730–11734.
Lay AJ, Jiang X-M, Kisker O, Flynn E, Underwood A, Condron R, Hogg PJ.
2000. Phosphoglycerase kinase acts in tumour angiogenesis as a
disulphide reductase. Nature 408:869–873.
Mark DF, Richardson CC. 1976. Escherichia coli thioredoxin: A subunit of
bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 73:780–
784.
Modun B, Morrissey J, Williams P. 2000. The staphylococcal transferrin
receptor: A glycolytic enzyme with novel functions. Trends Microbiol.
8:231–237.
Mowbray SL, Koshland DE, Jr. 1990. Mutations in the aspartate receptor of
Escherichia coli which affect aspartate binding. J Biol Chem 265:
15638–15643.
Multiple Leiomyoma Consortium 2002. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and
papillary renal cell cancer. Nature Genetics 30:406–410.
Numata O. 1996. Multifunctional proteins in Tetrahymena: 14-nm filament
protein/citrate synthase and translation elongation factor-1 alpha. Int
Rev Cytol 164:1–35.
&
Ostrovsky de Spicer P, Maloy S. 1993. PutA protein, a membrane-associated
flavin dehydrogenase, acts as a redox-dependent transcriptional
regulator. Proc Natl Acad Sci USA 90:4295–4298.
Piatigorsky J. 1998. Multifunctional lens crystallins and corneal enzymes.
More than meets the eye. Ann NY Acad Sci 842:7–15.
Picot D, Loll PJ, Garavito RM. 1994. The X-ray crystal structure of the
membrane protein prostaglandin H2 synthase-1. Nature 367:243–
249.
Pitarch A, Sanchez M, Nombela C, Gil C. 2002. Sequential fractionation and
two-dimensional gel analysis unravels the complexity of the dimorphic
fungus Candida albicans cell wall proteome. Mol Cell Proteomics 1:
967–982.
Russell CS, Ben-Yehuda S, Dix I, Kupiec M, Beggs JD. 2000. Functional
analyses of interacting factors involved in both pre-mRNA splicing and
cell cycle progression in Saccharomyces cerevisiae. RNA 6:1565–
1572.
Searls DB. 2003. Pharmacophylogenomics: Genes, evolution and drug
targets. Nat Rev Drug Discov 2:613–623.
Soker S, Takashim S, Miao HQ, Neufeld G, Klagsbrun M. 1998. Neuropilin-1
is expressed by endothelial and tumor cells as an isoform-specific
receptor for vascular endothelial growth factor. Cell 92:735–745.
Stallmeyer B, Schwarz G, Schulze J, Nerlich A, Reiss J, Kirsch J,
Mendel RR. 1999. The neurotransmitter receptor-anchoring protein
gephyrin reconstitutes molybdenum cofactor biosynthesis in
bacteria, plants, and mammalian cells. Proc Natl Acad Sci USA 96:
1333–1338.
Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC,
Boucher RC. 1995. CFTR as a cAMP-dependent regulator of sodium
channels. Science 269:847–850.
Suzuki CK, Rep M, Maarten van Dijl J, Suda K, Grivell LA, Schatz G. 1997.
ATP-dependent proteases that also chaperone protein biogenesis.
Trends Biochem Sci 22:118–123.
Ursini F, Heim S, Kiess M, Maiorino M, Roveri A, Wissing J, Flohe L. 1999.
Dual function of the selenoprotein PHGPx during sperm maturation.
Science 285:1393–1396.
Watanabe H, Takehana K, Date M, Shinozaki T, Raz A. 1996. Tumor cell
autocrine motility factor is the neuroleukin/phosphohexose isomerase
polypeptide. Cancer Res 56:2960–2963.
Wolff C, Parkinson JS. 1988. Aspartate taxis mutants of the Escherichia coli
tar chemoreceptor. J Bacteriol 170:4509–4515.
Wool IG. 1996. Extraribosomal functions of ribosomal proteins. Trends
Biochem Sci 21:164–165.
Wu RR, Couchman JR. 1997. cDNA cloning of the basement membrane
chondroitin sulfate proteoglycan core protein, bamacan: A five
domain structure including coiled-coil motifs. J Cell Biol 136:433–
444.
Xu W, Seiter K, Feldman E, Ahmed T, Chiao JW. 1996. The differentiation
and maturation mediator for human myeloid leukemia cells shares
homology with neuroleukin or phosphoglucose isomerase. Blood 87:
4502–4506.
Young JD, Lawrence AJ, Maclean AG, Leung BP, McInnes IB, Canas B,
Pappin DJC, Stevenson RD. 1999. Thymosin beta 4 sulfoxide is an antiinflammatory agent generated by monocytes in the presence of
glucocorticoids. Nat Med 5:1424–1427.
Zhu W, Rainville IR, Ding M, Bolus M, Heintz NH, Pederson DS.
2002. 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