Protein moonlighting

Crystallographic structure of cytochrome P450 from the bacteria S. coelicolor (rainbow colored cartoon, N-terminus = blue, C-terminus = red) complexed with heme cofactor (magenta spheres) and two molecules of its endogenous substrate epi-isozizaene as orange and cyan spheres respectively. The orange colored substrate resides in the monooxygenase site while the cyan colored substrate occupies the substrate entrance site. An unoccupied moonlighting terpene synthase site is designated by the orange arrow.

A moonlighting protein (or gene sharing protein) is a protein that can perform more than one function. Ancestral moonlighting proteins originally possessed a single function but through evolution, acquired additional functions. Many proteins that moonlight are enzymes; others are receptors, ion channels or chaperones. The most common primary function of moonlighting proteins is enzymatic catalysis, but these enyzmes have acquired secondary non-enyzmatic roles. Some examples of functions of moonlighting proteins secondary to catalysis include signal transduction, transcriptional regulation, apoptosis, motility, and structural.

Various technics have been used to reveal moonlighting functions in proteins. The detection of a protein in unusual locations within cells, cell types, or tissues may suggest that a protein has a moonlighting function. Furthermore sequence or structure homology of a protein may be used to infer both primary function as well as secondary moonlighting functions of a protein.

Inappropriate moonlighting is a contributing factor in some genetic diseases while moonlighting provides a possible mechanism by which bacteria may become resistant to antibiotics.

Discovery

The first observation of a moonlighting protein was made in the late 1980s by Joram Piatigorsky and Graeme Wistow during their research of crystallin enzymes. Piatigorsk determined that the answer to the question why "lens crystallins are both highly conserved and very diverse in number ... is that they have vital functions outside the lens." Originally Piatigorsky called these proteins "gene sharing" proteins, but the colloquial description moonlighting was subsequently applied to proteins by Constance Jeffery in 1999 to draw a similarity between multitasking proteins and people who work two jobs. The phrase "gene sharing" is ambiguous since it is also used to describe horizontal gene transfer, hence the phrase "protein moonlighting" has become the preferred description for proteins with more than one function.

Evolution

It is believed that moonlighting proteins came about by means of evolution through which uni-functional proteins gained the ability to perform multiple functions. With alterations, much of the protein's unused space can provide new functions. Many moonlighting proteins are the result of gene fusion of two single function genes. Alternatively a single gene can acquire a second function since the active site of the encoded protein typically is small compared to the overall size of the protein leaving considerable room to accommodate a second functional site. In yet a third alternative, the same active site can acquire a second function through mutations of the active site.

The development of moonlighting proteins may be evolutionary favorable to the organism since a single protein can do the job of multiple proteins conserving amino acids and energy required to synthesize these proteins. However there is no universally agreed upon theory that explains why proteins with multiple roles evolved. While using one protein to perform multiple roles seems advantageous because it keeps the genome small, we can conclude that this is probably not the reason for moonlighting because of the large of amount of noncoding DNA.

Functions

Many proteins catalyze a chemical reaction. Other proteins fulfill structural, transport, or signaling roles. Furthermore, numerous proteins have the ability to aggregate into supramolecular assemblies. For example, a ribosome is made up of 90 proteins and RNA.

A number of the currently known moonlighting proteins are evolutionarily derived from highly conserved enzymes, also called ancient enzymes. These enzymes are frequently speculated to have evolved moonlighting functions. Since highly conserved proteins are present in many different organisms, this increases the chance that they would develop secondary moonlighting functions. A high fraction of enzymes involved in glycolysis, an ancient universal metabolic pathway, exhibit exhibit moonlighting behavior. Furthermore it has been suggested that as many as 7 out of 10 proteins in glycolysis and 7 out of 8 enzymes of the tricarboxylic acid cycle exhibit moonlighting behavior.

An example of a moonlighting enzyme is pyruvate carboxylase. This enzyme catalyzes the carboxylation of pyruvate into oxaloacetate, thereby replenishing the tricarboxylic acid cycle. Surprisingly, in yeast species such as H. polymorpha and P. pastoris, pyruvate carboylase is also essential for proper targeting and assembly of the peroxisomal protein alcohol oxidase (AO). AO, the first enzyme of methanol metabolism, is a homo-octameric flavoenzyme. In wild type cells, this enzyme is present as enzymatically active AO octamers in the peroxisomal matrix. However, in cells lacking pruvate carboxylase, AO monomers accumulate in the cytosol, indicating that pyruvate carboxylase has a second fully unrelated function in assembly and import. The function in AO import/assembly is fully independent of the enzyme activity of pyruvate carboxylase, because amino acid substitutions can be introduced that fully inactive the enzyme activity of pryuvate carboxylase, without affecting its function in AO assembly and import. Conversely, mutations are known that block the function of this enzyme in import and assembly of AO, but have no effect on the enzymatic activity of the protein.

The E. coli anti-oxidant thioredoxin protein is another example of a moonlighting protein. Upon infection with the bacteriophage T7, E. coli thioredoxin forms a complex with T7 DNA polymerase, which results in enhanced T7 DNA replication, a crucial step for successful T7 infection. Thioredoxin binds to a loop in T7 DNA polymerase to bind more strongly to the DNA. The anti-oxidant function of thioredoxin is fully autonomous and fully independent of T7 DNA replication, in which the protein most likely fulfills the functional role.

Examples of Moonlighting Proteins

Kingdom	Protein	Organism	Function
			primary	moonlighting
Animal
	Aconitase	H. sapiens	TCA cycle enzyme	Iron homeostasis
	ATF2	H. sapiens	Transcription factor	DNA damage respons
	Crystallins	Various	Lens structural protein	Various enzyme
	Cytochrome c	Various	Energy metabolism	Apoptosis
	DLD	H. sapiens	Energy metabolism	Protease
	ERK2	H. sapiens	MAP kinase	Transcriptional repressor
	ESCRT-II complex	D. melanogaster	Endosomal protein sorting	Biocoid mRNA localization
	STAT3	M. musculus	Transcription factor	Electron transport chain
Plant
	Hexokinase	A. thaliana	Glucose metabolism	Glucose signaling
	Presenilin	P. patens	γ-secretase	Cystoskeletal function
Fungus
	Aconitase	S. cerevisiae	TCA cycle enzyme	mtDNA stability
	Aldolase	S. cerevisiae	Glycolytic enzyme	V-ATPase assembly
	Arg5,6	S. cerevisiae	Arginine biosynthesis	Transcriptional control
	Enolase	S. cerevisiae	Glycolytic enzyme	Homotypic vacuole fusion Mitochondrial tRNA import
	Galactokinase	K. lactis	Galactose catabolism enyzme	Induction galactose genes
	Hal3	S. cerevisiae	Halotolerance determinant	Coenzyme A biosynthesis
	HSP60	S. cerevisiae	Mitochondrial chaperone	Stabilization active DNA ori's
	Phosphofructokinase	P. pastoris	Glycolytic enzyme	Autophagy peroxisomes
	Pyruvate carboxylase	H. polymorpha	Anaplerotic enzyme	Assembly of alcohol oxidase
	Vhs3	S. cerevisiae	Halotolerance determinant	Coenzyme A biosynthesis
Prokaryotes
	Aconitase	M. tuberculosis	TCA cycle enzyme	Iron-responsive protein
	CYP170A1	S. coelicolor	Albaflavenone synthase	Terpene synthase
	Enolase	S. pneumoniae	Glycolytic enzyme	Plasminogen binding
	GroEL	E. aerogenes	Chaperone	Insect toxin
	Glutamate racemase (MurI)	E. coli	cell wall biosynthesis	gyrase inhibition
	Thioredoxin	E. coli	Anti-oxidant	T7 DNA polymerase subunit
Protist
	Aldolase	P. vivax	Glycolytic enzyme	Host-cell invasion

Mechanisms

Crystallographic structure of aconitase

In many cases, the functionality of a protein not only depends on its structure, but also its location. For example, a single protein may have one function when found in the cytoplasm of a cell, a different function when interacting with a membrane, and yet a third function if excreted from the cell. This property of moonlighting proteins is known as "differential localization". For example, in higher temperatures Degp (HtrA) will function as a protease by the directed degradation of proteins and in lower temperatures as a chaperone by assisting the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures. Furthermore, moonlighting proteins may exhibit different behaviors not only as a result of its location within a cell, but also the type of cell that the protein is expressed in.

Other methods through which proteins may moonlight are by changing their oligomeric state, altering concentrations of the protein's ligand or substrate, use of alternative binding sites, or finally through phosphorylation. An example of a protein that displays different function in different oligomeric states is pyruvate kinase which exhibits metabolic activity as a tetramer and thyroid hormone–binding activity as a monomer. Changes in the concentrations of ligands or substrates may cause a switch in protein a protein's function. For example, in the presence of low iron concentrations, aconitase functions as an enzyme while at high iron concentration, aconitase functions as an iron-responsive element-binding protein (IREBP). Proteins may also perform separate functions through the use of alternative binding sites that perform different tasks. An example of this is ceruloplasmin, a protein that functions as an oxidase in copper metabolism and moonlights as a copper-independent glutathione peroxidase. Lastly, phosphorylation may sometimes cause a switch in the function of a moonlighting protein. For example, phosphorylation of phosphoglucose isomerase (PGI) at Ser-185 by protein kinase CK2 causes it to stop functioning as an enzyme, while retaining its function as an autocrine motility factor. Hence when a mutation takes place that inactivates a function of a moonlighting proteins, the other function(s) are not necessarily affected.

The crystal structures of several moonlighting proteins, such as I-AniI homing endonuclease / maturase and the PutA proline dehydrogenase / transcription factor, have been determined. An analysis of these crystal structures has demonstrated that moonlighting proteins can either perform both functions at the same time, or through conformational changes, alternate between two states, each of which is able to perform a separate function. For example, the protein DegP plays a role in proteolysis with higher temperatures and is involved in refolding functions at lower temperatures. Lastly, these crystal structures have shown that the second function may negatively affect the first function in some moonlighting proteins. As seen in ƞ-crystallin, the second function of a protein can alter the structure, decreasing the flexibility, which in turn can impair enzymatic activity somewhat.

Identification methods

Moonlighting proteins have usually been identified by chance because there is no clear procedure to identify secondary moonlighting functions. Despite such difficulties, the number of moonlighting proteins that have been discovered is rapidly increasing. Furthermore, moonlighting proteins appear to be abundant in all kingdoms of life.

Various methods have been employed to determine a protein's function including secondary moonlighting functions. For example, the tissue, cellular, or subcellular distribution of a protein may provide hints as to the function. Real-time PCR is used to quantify mRNA and hence infer the presence or absence of a particular protein which is encoded by the mRNA within different cell types. Alternatively immunohistochemistry or mass spectrometry can be used to directly detect the presence of proteins and determine in which subcellular locations, cell types, and tissues a particular protein is expressed.

Mass spectrometry may be used to detect proteins based on their mass-to-charge ratio. Because of alternative splicing and posttranslational modification, identification of proteins based on the mass of the parent ion alone is very difficult. However tandem mass spectrometry in which each of the parent peaks is in turn fragmented can be used to unambiguously identify proteins. Hence tandem mass spectrometry is one of the tools used in proteomics to identify the presence of proteins in different cell types or subcellular locations. While the presence of a moonlighting protein in an unexpected location may complicate routine analyses, at the same time, the detection of a protein in unexpected multiprotein complexes or locations suggests that protein may have a moonlighting function. Furthermore, mass spectrometry may be used to determine if a protein has high expression levels that do not correlate to the enzyme's measured metabolic activity. These expression levels may signify that the protein is performing a different function than previously known.

The structure of a protein can also help determine its functions. Protein structure in turn may be elucidated with various techniques including X-ray crystallography or NMR. Dual polarization interferometry may be used to measure changes in protein structure which may also give hints to the protein's function. Finally, application of systems biology approaches such as interactomics give clues to a proteins function based on what it interacts with.

Clinical significance

The multiple roles of moonlighting proteins complicates the determination of phenotype from genotype, hampering the study of inherited metabolic disorders.

The complex phenotypes of several disorders are suspected to be caused by the involvement of moonlighting proteins. The protein GAPDH has at least 11 documented functions, one of which includes apoptosis. Excessive apoptosis is involved in many neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's as well as in brain ischemia. In one case, GAPDH was found in the degenerated neurons of individuals who had Alzheimer's disease.

Although there is insufficient evidence for definite conclusions, there are well documented examples of moonlighting proteins that play a role in disease. One such disease is tuberculosis. One moonlighting protein in the bacterium M. tuberculosis has a function which counteracts the effects of antibiotics. Specifically, M. tuberculosis gains antibiotic resistance against ciprofloxacin from overexpression of Glutamate racemase in vivo.