Protein moonlighting: Difference between revisions
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A number of the currently known moonlighting proteins are evolutionarily derived from highly [[conserved sequence|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.<ref name="Huberts_2010"/> 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.<ref name="Sriram_2005"/> |
A number of the currently known moonlighting proteins are evolutionarily derived from highly [[conserved sequence|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.<ref name="Huberts_2010"/> 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.<ref name="Sriram_2005"/> |
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An example of a moonlighting enzyme is [[pyruvate carboxylase]]. This catalyzes the carboxylation of [[pyruvic acid|pyruvate]] into [[oxaloacetic acid|oxaloacetate]], thereby replenishing the [[citric acid cycle|tricarboxylic acid cycle]]. Surprisingly, in yeast species such as ''[[Hansenula polymorpha|H. polymorpha]]'' and ''[[Pichia pastoris|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 [[flavin group|flavoenzyme]]. In wild type cells, this enzyme is present as enzymatically active AO octamers in the [[peroxisome|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.<ref name="Huberts_2010"/> |
An example of a moonlighting enzyme is [[pyruvate carboxylase]]. This enzyme catalyzes the carboxylation of [[pyruvic acid|pyruvate]] into [[oxaloacetic acid|oxaloacetate]], thereby replenishing the [[citric acid cycle|tricarboxylic acid cycle]]. Surprisingly, in yeast species such as ''[[Hansenula polymorpha|H. polymorpha]]'' and ''[[Pichia pastoris|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 [[flavin group|flavoenzyme]]. In wild type cells, this enzyme is present as enzymatically active AO octamers in the [[peroxisome|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.<ref name="Huberts_2010"/> |
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The ''[[Escherichia coli|E. coli]]'' anti-oxidant [[thioredoxin]] protein is another example of a moonlighting protein. Upon infection with the [[T7 phage|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.<ref name="Huberts_2010"/> |
The ''[[Escherichia coli|E. coli]]'' anti-oxidant [[thioredoxin]] protein is another example of a moonlighting protein. Upon infection with the [[T7 phage|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.<ref name="Huberts_2010"/> |
Revision as of 00:10, 11 May 2011
A moonlighting protein (or gene sharing protein) is a protein that can perform more than one function.[2] 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.[3]
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.[4]
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."[5] Originally Piatigorsky called these proteins "gene sharing" proteins, but the colloquial description moonlighting was subsequently applied to proteins by Constance Jeffery in 1999[6] to draw a similarity between multitasking proteins and people who work two jobs.[7] 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.[7]
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.[4] Many moonlighting proteins are the result of gene fusion of two single function genes.[8] 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.[6] However there is no universally agreed upon theory that explains why proteins with multiple roles evolved.[7][6] 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.[7]
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.[7] 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.[3]
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.[7]
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.[7]
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 |
| |
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
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".[10] 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.[4] 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.[10]
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.[3] Hence when a mutation takes place that inactivates a function of a moonlighting proteins, the other function(s) are not necessarily affected.[7]
The crystal structures of several moonlighting proteins, such as I-AniI homing endonuclease / maturase[11] and the PutA proline dehydrogenase / transcription factor,[12] have been determined.[13] 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.[13] 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.[13]
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.[7]
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.[10] 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.[3]
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[14] 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,[3] 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.[3]
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.[7][4] Specifically, M. tuberculosis gains antibiotic resistance against ciprofloxacin from overexpression of Glutamate racemase in vivo.[4]
References
- ^ PDB: 3EL3; Zhao B, Lei L, Vassylyev DG, Lin X, Cane DE, Kelly SL, Yuan H, Lamb DC, Waterman MR (2009). "Crystal structure of albaflavenone monooxygenase containing a moonlighting terpene synthase active site". J. Biol. Chem. 284 (52): 36711–9. doi:10.1074/jbc.M109.064683. PMC 2794785. PMID 19858213.
{{cite journal}}
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ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link) - ^ Jeffery CJ (2003). "Moonlighting proteins: old proteins learning new tricks". Trends Genet. 19 (8): 415–7. PMID 12902157.
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ignored (help) - ^ a b c d e f Sriram G, Martinez JA, McCabe ER, Liao JC, Dipple KM (2005). "Single-gene disorders: what role could moonlighting enzymes play?". Am. J. Hum. Genet. 76 (6): 911–24. doi:10.1086/430799. PMC 1196451. PMID 15877277.
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ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b c d e Sengupta S, Ghosh S, Nagaraja V (2008). "Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme". Microbiology (Reading, Engl.). 154 (Pt 9): 2796–803. doi:10.1099/mic.0.2008/020933-0. PMID 18757813.
{{cite journal}}
: Unknown parameter|month=
ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link) - ^ Piatigorsky J, O'Brien WE, Norman BL, Kalumuck K, Wistow GJ, Borras T, Nickerson JM, Wawrousek EF (1988). "Gene sharing by delta-crystallin and argininosuccinate lyase". Proc. Natl. Acad. Sci. U.S.A. 85 (10): 3479–83. PMC 280235. PMID 3368457.
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ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b c Jeffery CJ (1999). "Moonlighting proteins". Trends Biochem. Sci. 24 (1): 8–11. PMID 10087914.
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ignored (help) - ^ a b c d e f g h i j k Huberts DH, van der Klei IJ (2010). "Moonlighting proteins: an intriguing mode of multitasking". Biochim. Biophys. Acta. 1803 (4): 520–5. doi:10.1016/j.bbamcr.2010.01.022. PMID 20144902.
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ignored (help) - ^ Gancedo C, Flores CL (2008). "Moonlighting proteins in yeasts". Microbiol. Mol. Biol. Rev. 72 (1): 197–210, table of contents. doi:10.1128/MMBR.00036-07. PMC 2268286. PMID 18322039.
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ignored (help) - ^ Lauble H, Kennedy MC, Beinert H, Stout CD (1994). "Crystal structures of aconitase with trans-aconitate and nitrocitrate bound". J. Mol. Biol. 237 (4): 437–51. doi:10.1006/jmbi.1994.1246. PMID 8151704.
{{cite journal}}
: Unknown parameter|month=
ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b c Jeffery CJ (2005). "Mass spectrometry and the search for moonlighting proteins". Mass Spectrom. Rev. 24 (6): 772–82. doi:10.1002/mas.20041. PMID 15605385.
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: Unknown parameter|month=
ignored (help) - ^ PDB: 1P8K; Bolduc JM, Spiegel PC, Chatterjee P, Brady KL, Downing ME, Caprara MG, Waring RB, Stoddard BL (2003). "Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor". Genes Dev. 17 (23): 2875–88. doi:10.1101/gad.1109003. PMC 289148. PMID 14633971.
{{cite journal}}
: Unknown parameter|month=
ignored (help)CS1 maint: multiple names: authors list (link) - ^ PDB: 1K87; Lee YH, Nadaraia S, Gu D, Becker DF, Tanner JJ (2003). "Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein". Nat. Struct. Biol. 10 (2): 109–14. doi:10.1038/nsb885. PMID 12514740.
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: Unknown parameter|month=
ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b c Jeffery CJ (2004). "Molecular mechanisms for multitasking: recent crystal structures of moonlighting proteins". Curr. Opin. Struct. Biol. 14 (6): 663–8. doi:10.1016/j.sbi.2004.10.001. PMID 15582389.
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ignored (help) - ^ Sriram G, Parr LS, Rahib L, Liao JC, Dipple KM (2010). "Moonlighting function of glycerol kinase causes systems-level changes in rat hepatoma cells". Metab. Eng. 12 (4): 332–40. doi:10.1016/j.ymben.2010.04.001. PMID 20399282.
{{cite journal}}
: Unknown parameter|month=
ignored (help)CS1 maint: multiple names: authors list (link)