Protein moonlighting

A moonlighting protein (or gene sharing protein) is a protein that can perform more than one function. Many proteins that moonlight are enzymes; others are receptors, transmembrane channels or chaperones. Some proteins obtain the distinction of moonlighting because they contribute to a cell's structure in some way in addition to its normal enzymatic catalysis. Some examples of functions of protein include signal transduction events, such as transcriptional regulation and apoptosis; to growth and motility; to structural functions, such as those of lens crystallins.

History

The first implication of a moonlighting protein was brought about in the 1980s by Joram Piatigorsky and Graeme Wistow during their research of crystallin enzymes. Originally, Piatigorsky called these proteins 'gene sharing' proteins, but the common name moonlighting is used to draw a similarity between these metabolic enzymes and people who work two jobs.

It is believed that moonlighting proteins have existed for over a billion years and came about by means of evolution. Many moonlighting proteins are the result of a fusion of genes which originally coded for proteins with a single function. Proteins are quite large compared to the active site of a protein, meaning a large majority of the protein is inactive. Through alterations of the active site or areas around the active site, it is believed that these uni-functional proteins now had the ability to perform multiple functions. These moonlighting proteins became evolutionarily favorable for a cell; the cell can produce less proteins if a single protein can do the job of multiple proteins. As a result, the DNA needed for a cell is consolidated making the cell more efficient.

Techniques used to determine a protein's functions

Mass spectrometry

One system a scientist may use to classify a protein that appears in a biological setting, such as a cell or an organelle, is mass spectrometry. Mass spectrometry calibrates the mass-to-charge ratio of an ion, and from that, verifies some of its physical and chemical traits. When attempting to identify single proteins using the mass spectrometry process, scientists find moonlighting proteins annoying, as they make it difficult to differentiate between proteins, but the technique has proven to be quite useful in discovering new moonlighting proteins. The manner in which scientists have been able to identify moonlighting proteins using mass spectrometry is through two principle indicators. The first of which is the presence of a protein in an unexpected location in the cell, in an unexpected cell type, or in an unexpected multiprotein complex. The second is whether the 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.

Comparing normal and moonlighting proteins

Structure

Moonlighting enzymes may have two active sites, whereas a normal enzyme has just one active site.

Many enzymes of the sugar metabolism pathways including glycolysis, the archetype of a universal metabolic pathway exhibit moonlighting. 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. Some proteins exhibit a crystal structure, such as I-Amil maturase and PutA. By analyzing these crystal structures, scientists have been able to determine that moonlighting proteins can either be able to perform multiple functions at the same time, or alternate between the various functions, depending on the bending, folding, or other conditions of the protein at a given time. 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.

Functions

The most natural function of an enzyme is to catalyze a chemical reaction.

Many of the currently known moonlighting proteins are highly conserved enzymes, also called ancient enzymes. They are frequently identified in the moonlighting function yet are very speculative. Possibly this could related to the fact that highly conserved proteins are present in many different organisms and hence create a higher chance in moonlighting.

An example of a moonlighting enzyme is pyruvate carboxylase. This catalyzes the carboxylation of pyruvate into oxaloacetate, thereby replenishing the tricarboxyllic acid cycle. Surprisingly, in yeast species such as Hansenula polymorpha and Pichia 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 Eschericia coli anti-oxidant 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	Functions
Animal
	Aconitase	H. sapiens	TCA cycle enzyme	Iron homeostasis
	ATF2	H. sapiens	Transcription factor	DNA damage response
	Crystallins	Various	Lens structural protein	Various enzymes
	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

How they moonlight

In the case of a protein whose functions specifically include a structural role in a cell, this structure is a result of a combination of its secondary, tertiary, and quaternary structures. In many cases, the functionality of a protein not only depends on the structure, but also the location of the particular protein within a cell. For example, a single protein may have one function when found in the cytoplasm of a cell, and a completely different function when interacting with a membrane. This property of moonlighting proteins is known as "Differential Localization". Furthermore, moonlighting proteins can exhibit different behaviors not only as a result of location within a cell, but also whether or not the protein is in a cell and the type of cell the protein exists in. Other methods through which proteins may moonlight are by changing its oligomeric state, different concentrations of the ligand or substrate, different binding sites, and phosphorylation. Proteins may moonlight through changing their oligomeric state by adding additional proteins to a chain. For example, "Pyruvate kinase exhibits metabolic activity as a tetramer and thyroid hormone–binding activity as a monomer." Different concentrations of ligands or substrates, such as in the example of the effects of iron levels on aconitase, can affect how the protein functions, either as an enzyme or as an IREBP. Proteins may also perform separate functions through the use of multiple binding sites that perform different tasks when bound to a substrate. 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 is likely to sometimes cause switching between the functions of a moonlighting protein. An example of this case is the phosphorylation of PGI at Ser185 by protein kinase CK2 which causes it to cease functioning as an enzyme, but remain functioning as an autocrine motility factor. When a mutation takes place that inactivates a function of a moonlighting proteins, the other function(s) are not necessarily affected.

Scientific relevance

The complex phenotypes of several disorders are due to the involvement of moonlighting proteins. 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. The bacterium M. tuberculosis has a moonlighting protein in which one of its functions counteracts the effects of antibiotics.

The moonlighting functions of proteins have usually been found by chance because there is no clear procedure to identify moonlighting functions. Despite such difficulties, scientists are quickly identifying more and more moonlighting proteins. Such large numbers suggest that they are abundant in all kingdoms of life. Further research will allow scientist to uncover mysteries about how proteins evolve and how proteins perform their specific functions.

Engineering proteins

Main article: Protein engineering

Moonlighting proteins are of particular interest in protein engineering, the study of proteins to better understand their functions, importance, and how we can modify them to better or lessen their effects.