NATURE’S INGENUITY: PROTEIN MOONLIGHTING

Dr B.R. Ambedkar Center for Biomedical ResearchUniversity of Delhi

B Y - VANDANA GARG & VIMAL SINGH

Proteins can moonlight!!

What is Moonlighting ??

The idea of one gene-one protein-one function has become too old.

“Moonlighting-Old proteins learning new tricks”

Increasing number of proteins are found to perform two or more functions

Insight of Moonlighting..

Human genome encode few proteins, than expected from such a complex organism having a large genome.

One gene may encode for proteins with more than one function.

Various ways Alternative splicing DNA rearrangement Post translational modifications of proteins Gene Duplication with differential mutation Gene Fusion

Insight of Moonlighting….

Protein moonlighting (or gene sharing) is a phenomenon by which a protein can perform more than one function.

Excluding-• Splice variants• Gene fusions• Homologous but non identical protein• Post translational modifications• Same function -Different cellular location or substrates

Very special multifunctional proteins, Multiple autonomous, often unrelated, functions without

partitioning these functions into different protein domain

pleiotropismSingle gene-

multiple pathways

Multiple phenotypic

traits

Inactivation

Protein moonlighting

Single gene-protein with

multiple functions

Single function affected

Mutation in coding region

Moonlighting and Pleiotropism

Discovery

Crystallins

metabolism

Duck-lactate dehydrogenase Turtle-enolase

Eye

Structural role/vision

Proteins that can moonlight

EnzymesReceptorsIon channels Chaperones.

‘The most common primary function of moonlighting proteins is enzymatic catalysis, but these enzymes have

acquired secondary non-enzymatic roles.’ moonlighting functions secondary to catalysis include

signal transduction, transcriptional regulation, apoptosis, motility, and structural functions.

Examples of moonlighting-proteins from different kingdoms

1

2

3

Moonlighting Proteins: Molecular Mechanisms

Differential localizationDifferential expressionLigand/Substrate concentrationBinding sitesComplex formationOligomerization

Molecular mechanisms of Moonlighting

Differential localization

The Escherichia coli PutA protein

In Plasma membraneproline dehydrogenase and pyrroline-5- carboxylate dehydrogenase activity when associated with PM.

In Cytoplasm- lacks enzymatic activity and binds DNA as a transcriptional repressor of put genes.

Inside and outside the cell

Phosphoglucose isomeraseCatalyzes the second step in glycolysis, the inter-conversion of glucose 6-phosphate and fructose 6-phosphate .

Secreted by cells and plays at least 4 additional roles. Neuroleukin - both..

a cytokine - causes B cells to mature into antibody-secreting cells, a nerve growth factor that promotes the survival of some embryonic spinal neurons and some sensory nerves.

Autocrine Motility Factor (AMF) – a cytokine that stimulates cell migration .

A Differentiation and Maturation Mediator (DMM) – cause differentiation of human myeloid leukemia cells

Differential Expression

Neuropilin - Cell Surface Receptor on endothelial cells,

-detects vascular endothelial growth factor and indicates when new blood cells are needed.

Cell Surface Receptor in nerve axons, -detects a different ligand, Semaphorin III, and helps steer axons to their proper destinations.

Oligomerization.

Glyceraldehyde-3-phosphate dehydrogenase tetramer,

converts glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate.

monomer, it is a nuclear Uracil-DNA glycosylase-removing uracil that is present in DNA because of accidental use of dUTP during DNA synthesis or deamination of cytosine residues.

Complex Formation

E. coli thioredoxin deoxyribonucleotide synthesis: it helps to reduce

ribonucleoside diphosphates to deoxyribonucleoside diphosphates.

recruited by T7 phage, in which it functions as a subunit of a heterodimeric DNA polymerase.

PutAAconitase

Fe-dependent enzyme that has catalytic activity only when cellular iron concentrations are high.

o When the iron concentration decreases - Iron Responsive Element Binding Protein (IRE-BP).

o By binding to a stem-loop, the IRE, in the 5’-untranslated region of ferritin mRNA, the IRE-BP prevents the synthesis of ferritin.

o IREs in the 3’-untranslated region of the transferrin receptor mRNA and controls the degradation of transferrin receptor mRNA.

o not both simultaneously.

Ligand /Substrate Concentration

Binding Site

The E. coli aspartate receptor, which functions in bacterial chemotaxis, is also a Maltose Binding Protein (MBP) Receptor. different but overlapping binding sites for aspartate and MBP

Aconitase active site is the surface used for RNA binding when the protein functions as the IRE-BP.

Recently Crystallized Moonlight Protein-I-AniI maturase

Group I intron splicing factor and a homing endonuclease The protein is encoded in an intron of the Aspergillus nidulans mitochondrial apo-cytochrome b gene.

homing endonuclease-I-AniI initiates transfer of the intron by cleaving a DNA target sequence within a homologous allele that lacks the intron sequence.

Splicing of the intron RNA during post-transcriptional excision from precursor RNA.

2.6 A ° Resolution X-ray crystal structure of the I-AniI maturase in complex with a 31 base pair duplex DNA

fragment containing the native homing site

I-AniI maturase..

I-AniI maturase..

Although the crystal structure does not include RNA, several pieces of evidence indicate that the surface of I-AniI involved in RNA binding is distinct from the DNA-binding sites.

Two mutations that prevented DNA binding and cleavage did not affect RNA maturation activity

I-AniI maturase..

Further studies identified clusters of basic amino acids in the N- and C-terminal domains.

distant from the DNA binding sites.

A potential surface for interacting with the RNA

Arg

Glu

Wild-type I-AniI

Mutant I-AniI

239

Tenfold slower RNA splicing and lower affinity for RNA

Mutational analysis

I-AniI maturase..

Crystal Structure And Mutational Analysis-

strongly support the model of two independent functional

surfaces for the endonuclease and maturase activities.

good example of a protein in which a second activity, the

maturase activity, evolved from changes in an unused

solvent exposed surface area

How does one identify Protein-Moonlighting ??

Usually by chance from activity studies

Immuno histochemistry or Mass Spectrometry to

detect their sub-cellular location, tissues, cell-types.

By mutational analysis

Identification….

Determination by Structure Features-X-ray Crystallography, NMR, Large size of protein and

unused surface area may suggest for its multiple function.System Biology applications like Interactomics, to know with

what it interacts.Comparison with orthologous proteins with the same

primary function.With time, development of statistical, data-mining and

machine learning approaches will help to examine these proteins and making predictions.

Evolution

Moonlighting Proteins- Generally Highly Conserved, “Why moonlighting functions are so frequently identified in highly conserved proteins?”

Ubiquitous in all kingdoms, has been around for over a billion years of time – ample time for evolution

Example- Sugar(glycolytic) pathway enzymes- 7 out of 10 are moonlighting enzymes.

Moonlighting functions also seem to occur more often in proteins that are constitutively expressed at relatively high levels

Why Moonlighting-Proteins ever came into being?

To expand the functional capabilities of an organism without the burden of an expanding genome size . (contradicts the presence of large amount of Non-Coding DNA)

Tinkerer's way of Evolution – which means that there is no end goal in evolution and that novel functions only develop by adapting existing ones. If a particular novel function results in an advantage for the organism, this function will be selected during evolution.

Structural Features/Changes in Moonlighting-Proteins

Some enzymes are larger than necessity for their function, unused solvent exposed surface and many pockets on the protein surface could be modified to make the additional binding sites.

New use of existing binding sites or modifications of the unused regions.

Low specificity of the active site-mutations in active site or use of different regions surrounding it.

Benefits

Prokaryotes - Fewer proteins to synthesize, less DNA to replicate-Saves Energy

Coordinating Cellular activities- Some proteins with one activity regulate other proteins with similar activity-Regulation of Epithelial Sodium Ion Channel by CFTR Channel-maintains epithelial cell homoeostasis.

Self regulation of transcription and translation- Many biosynthetic or catabolic enzymes regulate their own synthesis Thymidylate Synthase- binds to stem loop structure at 5’-end of

mRNA Put A protein

Benefits..

Effective cellular response- Thrombin that trigger multiple pathways

Switches between pathways- several proteins are both proteases and chaperone activity- FtsH, yeast mitochondrial homolog of FtsH-(Afg3P, Rca1P)

Correlation between multiple functions – yeast FtsHSometimes no clear connection- band 3 protein in RBC’s

plasma membrane

Medical Relevance

The complex phenotypes of several disorders, may be

related to the involvement of moonlighting proteins.

Dihydrolipoamide Dehydrogenase (DLD) - Mitochondrial Enzyme component of at least 5 different multi-enzyme complexes critical for energy metabolism and redox balance deficiencies in DLD activity are associated with severe

disorders in infancy, such as an inability to thrive, hypotonia and metabolic disorders.

Virulence properties, particularly invasion

Mycobacterium tuberculosis glutamate racemase (MurI )cell wall (peptidoglycan) biosynthesis

MurI -DNA gyrase inhibitor, by reducing binding of gyrase to DNA

Prevents action of the ciprofloxacin because MurI inhibits binding of gyrase to DNA

Cytotoxic double-strand DNA breaks x

DNA replication inhibition X

Moonlighting-Proteins in Bacterial World….

Moonlighting Actions of Bacterial Metabolic Proteins

Moonlighting Actions of Bacterial Metabolic Proteins..

Concluding with tasks ahead..

There is a great deal of interaction between macromolecules, and the modern cell is a sophisticated and highly organized network.

It adds to the difficulty of interpreting genome sequences.

Many proteins can have multiple interactions, it could be difficult to ascertain who interacts with whom in the cell.

“Moonlighting functions create a whole new level of complexity in the cell. A moonlighting protein may link a metabolic pathway to a signalling pathway in a completely unexpected manner”Moonlighting is a phenomenon that

illustrates nature's ingenuityIt is a source of inspiration that

should remind scientists to always keep the unexpected in mind, even on

familiar ground

References

Jeffery CJ (January 1999). "Moonlighting proteins". Trends Biochem. Sci. 

Jeffery CJ (December 2004). "Molecular mechanisms for multitasking:

recent crystal structures of moonlighting proteins". Curr. Opin. Struct. Biol.

Huberts DH, van der Klei IJ (April 2010). "Moonlighting proteins: an

intriguing mode of multitasking” Journal of Biochemistry and Biophysics

Bolduc JM, Spiegel PC, Chatterjee P, Brady KL, Downing ME, Caprara MC,

Waring RB, Stoddard BL: Structural and biochemical analysis of DNA and

RNA binding by a bifunctional homing endonuclease and group I intron

splicing factor. Genes Dev 2003