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.
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
Inactivation Mutation in coding region Moonlighting and Pleiotropism
Proteins that can moonlight Enzymes Receptors Ion 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
Differential localization The Escherichia coli PutA protein In Plasma membrane prolinedehydrogenase and pyrroline-5- carboxylatedehydrogenaseactivity when associated with PM. In Cytoplasm- lacks enzymatic activity and binds DNA as a transcriptional repressor of put genes.
Inside and outside the cell Phosphoglucoseisomerase Catalyzes 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 differentiationof 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, SemaphorinIII, 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 dUTPduring DNA synthesis or deamination of cytosine residues.
Complex Formation E. coli thioredoxin deoxyribonucleotidesynthesis: it helps to reduce ribonucleosidediphosphatesto deoxyribonucleosidediphosphates. recruited by T7 phage, in which it functions as a subunit of a heterodimericDNA polymerase.
PutA Aconitase Fe-dependent enzyme that has catalytic activity only when cellular iron concentrations are high.
When the iron concentration decreases - Iron Responsive Element Binding Protein (IRE-BP).
By binding to a stem-loop, the IRE, in the 5’-untranslated region of ferritin mRNA, the IRE-BP prevents the synthesis of ferritin.
IREs in the 3’-untranslated region of the transferrinreceptor mRNA and controls the degradation of transferrinreceptor mRNA.
not both simultaneously.
Ligand /Substrate Concentration
Binding Site The E. coli aspartatereceptor, which functions in bacterial chemotaxis, is also a Maltose Binding Protein (MBP) Receptor. different but overlapping binding sites for aspartate and MBP Aconitaseactive site is the surface used for RNA binding when the protein functions as the IRE-BP.
Recently Crystallized Moonlight Protein-I-AniImaturase Group I intronsplicing factor and a homing endonucleaseThe protein is encoded in an intron of the Aspergillusnidulansmitochondrial apo-cytochromeb gene. homing endonuclease- I-AniIinitiates transfer of the intron by cleaving a DNA target sequence within a homologous allele that lacks the intronsequence. Splicing of the intron RNA during post-transcriptional excision from precursor RNA.
I-AniImaturase.. 2.6 A ° Resolution X-ray crystal structure of the I-AniImaturase in complex with a 31 base pair duplex DNA fragment containing the native homing site
I-AniImaturase.. 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-AniImaturase.. 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
239 Wild-type I-AniI Arg Mutant I-AniI Glu Tenfold slower RNA splicing and lower affinity for RNA Mutational analysis
I-AniImaturase.. 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 Immunohistochemistryor 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 ThymidylateSynthase- 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 FtsH Sometimes 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. DihydrolipoamideDehydrogenase (DLD) - Mitochondrial Enzyme component of at least 5 different multi-enzyme complexes critical for energy metabolism and redoxbalance deficiencies in DLD activity are associated with severe disorders in infancy, such as an inability to thrive, hypotonia and metabolic disorders.
Moonlighting-Proteins in Bacterial World…. 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 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 ingenuity It 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 derKlei 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