Protein moonlighting


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  • Protein moonlighting

    1. 1. NATURE’S INGENUITY: PROTEIN MOONLIGHTING<br />Dr B.R. Ambedkar Center for Biomedical ResearchUniversity of Delhi<br /> B Y - VANDANA GARG & VIMAL SINGH<br />
    2. 2. Proteins can moonlight!!<br />
    3. 3. What is Moonlighting ??<br />The idea of one gene-one protein-one function has become too old.<br /> “Moonlighting-Old proteins learning new tricks”<br />Increasing number of proteins are found to perform two or more functions<br />
    4. 4. Insight of Moonlighting..<br />Human genome encode few proteins, than expected from such a complex organism having a large genome.<br />One gene may encode for proteins with more than one function.<br />Various ways <br />Alternative splicing<br />DNA rearrangement<br />Post translational modifications of proteins<br />Gene Duplication with differential mutation<br />Gene Fusion<br />
    5. 5. Insight of Moonlighting….<br /><ul><li>Protein moonlighting (or gene sharing) is a phenomenon by which a protein can perform more than one function.</li></ul>Excluding-<br /><ul><li>Splice variants
    6. 6. Gene fusions
    7. 7. Homologous but non identical protein
    8. 8. Post translational modifications
    9. 9. Same function -Different cellular location or substrates
    10. 10. Very special multifunctional proteins,
    11. 11. Multiple autonomous, often unrelated, functions without partitioning these functions into different protein domain</li></li></ul><li>Inactivation<br />Mutation in coding region<br />Moonlighting and Pleiotropism<br />
    12. 12. Discovery<br />
    13. 13. Proteins that can moonlight<br />Enzymes<br />Receptors<br />Ion channels <br />Chaperones. <br /> ‘The most common primary function of moonlighting proteins is enzymatic catalysis, but these enzymes have acquired secondary non-enzymatic roles.’<br /><ul><li>moonlighting functions secondary to catalysis include </li></ul>signal transduction, transcriptional regulation, apoptosis, motility, and structural functions.<br />
    14. 14. Examples of moonlighting-proteins from different kingdoms<br />1<br />
    15. 15. 2<br />
    16. 16. 3<br />
    17. 17. Moonlighting Proteins: Molecular Mechanisms<br />Differential localization<br />Differential expression<br />Ligand/Substrate concentration<br />Binding sites<br />Complex formation<br />Oligomerization<br />
    18. 18. Molecular mechanisms of Moonlighting<br />
    19. 19. Differential localization<br />The Escherichia coli PutA protein <br />In Plasma membrane<br />prolinedehydrogenase and pyrroline-5- carboxylatedehydrogenaseactivity when associated with PM.<br />In Cytoplasm- <br /> lacks enzymatic activity and binds DNA as a transcriptional repressor of put genes.<br />
    20. 20. Inside and outside the cell<br />Phosphoglucoseisomerase<br />Catalyzes the second step in glycolysis, the inter-conversion of glucose 6-phosphate and fructose 6-phosphate .<br />Secreted by cells and plays at least 4 additional roles. <br /><ul><li>Neuroleukin - both..</li></ul> a cytokine - causes B cells to mature into antibody-secreting cells, <br /> a nerve growth factor that promotes the survival of some embryonic spinal neurons and some sensory nerves.<br /><ul><li>Autocrine Motility Factor (AMF) – </li></ul> a cytokine that stimulates cell migration .<br /><ul><li>A Differentiation and Maturation Mediator (DMM) – </li></ul> cause differentiationof human myeloid leukemia cells<br />
    21. 21. Differential Expression <br />Neuropilin -<br /><ul><li>Cell Surface Receptor on endothelial cells, </li></ul> -detects vascular endothelial growth factor and indicates when new blood cells are needed. <br /><ul><li>Cell Surface Receptor in nerve axons, </li></ul> -detects a different ligand, SemaphorinIII, and helps steer axons to their proper destinations.<br />
    22. 22. Oligomerization.<br />Glyceraldehyde-3-phosphate dehydrogenase<br />tetramer, <br /> converts glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. <br />monomer, <br /> it is a nuclear Uracil-DNA glycosylase-<br /> removing uracil that is present in DNA because of accidental use of dUTPduring DNA synthesis or deamination of cytosine residues.<br />
    23. 23. Complex Formation<br />E. coli thioredoxin<br />deoxyribonucleotidesynthesis: it helps to reduce ribonucleosidediphosphatesto deoxyribonucleosidediphosphates.<br />recruited by T7 phage, in which it functions as a subunit of a heterodimericDNA polymerase.<br />
    24. 24. PutA<br />Aconitase<br /> Fe-dependent enzyme that has catalytic activity only when cellular iron concentrations are high. <br /><ul><li>When the iron concentration decreases - Iron Responsive Element Binding Protein (IRE-BP).
    25. 25. By binding to a stem-loop, the IRE, in the 5’-untranslated region of ferritin mRNA, the IRE-BP prevents the synthesis of ferritin.
    26. 26. IREs in the 3’-untranslated region of the transferrinreceptor mRNA and controls the degradation of transferrinreceptor mRNA.
    27. 27. not both simultaneously.</li></ul>Ligand /Substrate Concentration<br />
    28. 28. Binding Site<br />The E. coli aspartatereceptor, which functions in bacterial chemotaxis, is also a Maltose Binding Protein (MBP) Receptor. <br />different but overlapping binding sites for aspartate and MBP<br />Aconitaseactive site is the surface used for RNA binding when the protein functions as the IRE-BP.<br />
    29. 29. Recently Crystallized Moonlight Protein-I-AniImaturase<br />Group I intronsplicing factor and a homing endonucleaseThe protein is encoded in an intron of the Aspergillusnidulansmitochondrial apo-cytochromeb gene.<br />homing endonuclease-<br />I-AniIinitiates transfer of the intron by cleaving a DNA target sequence within a homologous allele that lacks the intronsequence.<br />Splicing of the intron RNA during post-transcriptional excision from precursor RNA.<br />
    30. 30. I-AniImaturase..<br /> 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<br />
    31. 31. I-AniImaturase..<br />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. <br />Two mutations that prevented DNA binding and cleavage did not affect RNA maturation activity<br />
    32. 32. I-AniImaturase..<br />Further studies identified clusters of basic amino acids in the N- and C-terminal domains. <br />distant from the DNA binding sites.<br />A potential surface for interacting with the RNA <br />
    33. 33. 239<br />Wild-type I-AniI<br />Arg<br />Mutant I-AniI<br />Glu<br />Tenfold slower RNA splicing and lower affinity for RNA<br />Mutational analysis<br />
    34. 34. I-AniImaturase..<br />Crystal Structure And Mutational Analysis-<br />strongly support the model of two independent functional surfaces for the endonuclease and maturase activities.<br />good example of a protein in which a second activity, the maturase activity, evolved from changes in an unused solvent exposed surface area<br />
    35. 35. How does one identify Protein-Moonlighting ??<br />Usually by chance from activity studies<br />Immunohistochemistryor Mass Spectrometry to detect their sub-cellular location, tissues, cell-types.<br />By mutational analysis<br />
    36. 36. Identification….<br />Determination by Structure Features-<br />X-ray Crystallography, NMR, Large size of protein and unused surface area may suggest for its multiple function.<br />System Biology applications like Interactomics, to know with what it interacts.<br />Comparison with orthologous proteins with the same primary function.<br />With time, development of statistical, data-mining and machine learning approaches will help to examine these proteins and making predictions.<br />
    37. 37. Evolution<br />Moonlighting Proteins- Generally Highly Conserved, <br />“Why moonlighting functions are so frequently identified in highly conserved proteins?”<br />Ubiquitous in all kingdoms, has been around for over a billion years of time – ample time for evolution<br />Example- Sugar(glycolytic) pathway enzymes- 7 out of 10 are moonlighting enzymes.<br />Moonlighting functions also seem to occur more often in proteins that are constitutively expressed at relatively high levels<br />
    38. 38. Why Moonlighting-Proteins ever came into being?<br />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)<br />Tinkerer's way of Evolution – <br /> which means that there is no end goal in evolution and that novel functions only develop by adapting existing ones. <br />If a particular novel function results in an advantage for the organism, this function will be selected during evolution.<br />
    39. 39. Structural Features/Changes in Moonlighting-Proteins<br />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.<br />New use of existing binding sites or modifications of the unused regions.<br />Low specificity of the active site-mutations in active site or use of different regions surrounding it.<br />
    40. 40. Benefits<br />Prokaryotes - Fewer proteins to synthesize, less DNA to replicate-Saves Energy<br />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.<br />Self regulation of transcription and translation- Many biosynthetic or catabolic enzymes regulate their own synthesis<br />ThymidylateSynthase- binds to stem loop structure at 5’-end of mRNA<br />Put A protein<br />
    41. 41. Benefits..<br />Effective cellular response- Thrombin that trigger multiple pathways <br />Switches between pathways- several proteins are both proteases and chaperone activity- FtsH, yeast mitochondrial homolog of FtsH-(Afg3P, Rca1P)<br />Correlation between multiple functions– yeast FtsH<br />Sometimes no clear connection-band 3 protein in RBC’s plasma membrane<br />
    42. 42. Medical Relevance<br />The complex phenotypes of several disorders, may be related to the involvement of moonlighting proteins.<br />DihydrolipoamideDehydrogenase (DLD) - <br />Mitochondrial Enzyme <br />component of at least 5 different multi-enzyme complexes <br />critical for energy metabolism and redoxbalance <br />deficiencies in DLD activity are associated with severe disorders in infancy, such as an inability to thrive, hypotonia and metabolic disorders. <br />
    43. 43. Moonlighting-Proteins in Bacterial World….<br />Virulence properties, particularly invasion<br />Mycobacterium tuberculosis glutamate racemase (MurI )<br /> cell wall (peptidoglycan) biosynthesis <br />MurI -DNA gyrase inhibitor, by reducing binding of gyrase to DNA<br />Prevents action of the ciprofloxacin because MurI inhibits binding of gyrase to DNA <br />Cytotoxic double-strand DNA breaks x<br />DNA replication inhibition X<br />
    44. 44. Moonlighting Actions of Bacterial Metabolic Proteins<br />
    45. 45. Moonlighting Actions of Bacterial Metabolic Proteins..<br />
    46. 46. Concluding with tasks ahead..<br />There is a great deal of interaction between macromolecules, and the modern cell is a sophisticated and highly organized network.<br />It adds to the difficulty of interpreting genome sequences.<br />Many proteins can have multiple interactions, it could be difficult to ascertain who interacts with whom in the cell.<br />
    47. 47. “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”<br />Moonlighting is a phenomenon that illustrates nature's ingenuity<br />It is a source of inspiration that should remind scientists to always keep the unexpected in mind, even on familiar ground<br />
    48. 48. References<br />Jeffery CJ (January 1999). "Moonlighting proteins". Trends Biochem. Sci. <br />Jeffery CJ (December 2004). "Molecular mechanisms for multitasking: recent crystal structures of moonlighting proteins". Curr. Opin. Struct. Biol.<br />Huberts DH, van derKlei IJ (April 2010). "Moonlighting proteins: an intriguing mode of multitasking” Journal of Biochemistry and Biophysics<br />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<br />