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Our Interests Molecular and structural basis of protein action

N. Srinivasan Molecular Biophysics Unit Indian Institute of Science. MBU. Our Interests Molecular and structural basis of protein action. Interactions of proteins in cellular communications URL of our group: http://pauling.mbu.iisc.ernet.in

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Our Interests Molecular and structural basis of protein action

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  1. N. Srinivasan Molecular Biophysics Unit Indian Institute of Science MBU Our Interests Molecular and structural basis of protein action Interactions of proteins in cellular communications URL of our group: http://pauling.mbu.iisc.ernet.in Support:The Wellcome Trust, London; DBT and CSIR, Delhi

  2. Prediction of protein-protein interactions between a host and a pathogen O. Krishnadev Smita Mohanty K. Anamika Supported by CSIR, DBT and Wellcome Trust

  3. Plasmodium falciparum – Protozoan malarial parasite

  4. Protein-protein interactions network, biological pathways & processes in P. falciparum are dynamic d) e) f) g) Mohanty, Pandit, Srinivasan (2008) In Protein Interaction Networks and Biological Data Mining (in press)

  5. PFB0815c:Plasmodium falciparum Calcium dependent protein kinase (CDPK) DNA Exon1 Exon2 Exon3 Exon4 Exon5 Mature mRNA Protein Phosphorylation Host erythrocytic membrane proteins Anamika, Srinivasan, Krupa Proteins, 2005 Anamika & Srinivasan, Prot. Pept. Lett. 2007 Zhao et al, Mol Biochem Parasitol (1994)

  6. Is it possible to predict protein-protein interactions across Human and P. falciparum?

  7. An Approach for detecting Protein – Protein interactionsacross host and pathogen organisms Krishnadev & Srinivasan (2008) In Silico Biology

  8. DIP, iPFam, Y2H P-P interaction datasets Host genome Guest genome Homology searches using variety of methods Putative Interaction set subcellular localization of host proteins subcellular localization of guest proteins Refined Set with compatible localization expression data from microarray/mass spec expression data from microarray/mass spec Final set with compatible expression and localization Manual analysis on significance to pathogenesis

  9. An approach for remote homology detection and fold recognition from sequence Gowri, Anand, Krishnadev, Sairam Swamy, Gracelin, Smita Mohanty Anand et.al., Bioinformatics (2005) Gowri et al Nucl. Acids Res. (2006) Gowri et al Proteins (2007)

  10. RPS-BLAST RPS-BLAST Query Query Hit profiles Hit profiles Database of Multiple profiles Database of single profiles Searches in database of constant multiple profiles Hit profiles Ordinary Reverse Blast MulPSSM

  11. PALI- Database of Phylogeny and ALIgnment of homologous protein structures. S.Balaji, V.S.Gowri, G.Agarwal, S. Pandit, S.Sujatha, C.S. Swamy, O.Krishnadev Balaji et al (2001) Nucl. Acids. Res. Sujatha et al (2001) Bioinformatics Gowri et al (2003) Nucl. Acids. Res. URL: http://pauling.mbu.iisc.ernet.in/~pali

  12. Sensitivity Specificity Error rate

  13. DIP, iPFam, Y2H P-P interaction datasets Host genome Guest genome Homology searches using variety of methods Gowri, Krishnadev, Anand, Srinivasan Bioinformatics 2005 Proteins 2007 Nucl. Acids Res. 2007 Putative Interaction set subcellular localization of host proteins subcellular localization of guest proteins Refined Set with compatible localization expression data from microarray/mass spec expression data from microarray/mass spec Final set with compatible expression and localization Manual analysis on significance to pathogenesis

  14. Conservation with a purpose: Protein-protein interactions in closely related homologues

  15. Interactions of distantly related homologues of protein modules are NOT conserved (Rekha et al 2005 Proteins)

  16. Distant homologues act as turning points in otherwise similar signalling and metabolic pathways Pathway B in organism Y Pathway A in organism X Closely (family) related Distantly (superfamily) related - binding to unrelated domains Rekha et al (2005) Proteins Unrelated

  17. DIP, iPFam, Y2H P-P interaction datasets Host genome Guest genome Homology searches using variety of methods Gowri, Krishnadev, Anand, Srinivasan Bioinformatics 2005 Proteins 2007 Nucl. Acids Res. 2007 in vitro interactions Putative Interaction set Rekha et al Proteins 2005 subcellular localization of host proteins subcellular localization of guest proteins Refined Set with compatible localization expression data from microarray/mass spec expression data from microarray/mass spec in vivo interactions Final set with compatible expression and localization Manual analysis on significance to pathogenesis

  18. Host – Pathogen pairs studied

  19. Our approach has a good success rate -Example, herpes virus and human P-P interactions • 12 experimentally studied interactions using two-hybrid and co-immunoprecipitation. • 11 predicted’ by our method. • Novel predictions • Viral ribonucleotide reductase small subunit with human p53 protein. This can point to a mechanism of regulating apoptosis which occurs upon viral infection. • Known PPI in other organisms • Viral uracil DNA glycosylase with human PCNA. PCNA important for replication. Viral UDG shown to localize to DNA replisome.

  20. Human – P.falciparum interactions Statistics

  21. Examples of PPI in Human – P.falciparum Known Interaction TRAP/SSP2 protein with human Collagen and ICAMs (Thrombouspondin Related Anonymous Protein / Sporozoite Surface Protein 2)

  22. Recognition of Interactions TRAP/SSP2 protein with human Collagen and ICAMs • TRAP/SSP2 activity disruption leads to a marked decrease in the ability to invade the human hepatocytes. • TRAP/SSP2 shown to be present on the sporozoite membrane. • Experimental evidence points out a mechanism of gliding motility in apicomplexans which involves non-specific binding of pathogen surface proteins to human surface proteins. • Presence of A-domain which is known to interact with different human cell surface proteins

  23. Pf SSP2 interacts with Human ICAM

  24. More newly recognized interactions Plasmepsins with moesin/radixin/ezrin family proteins (ERM) Plasmepsin • Plasmepsins are hemoglobin degrading enzymes required for the survival of the pathogen. • Plasmepsin II shown to be localized to erythrocyte membrane. • Protease inhibitors lead to abrogation of host membrane rupture and leads to the trapping of mature merozoites inside the erythrocytes. • Moesin/radixin/ezrin family proteins involved in maintaining cyto-skeleton integrity. • The interaction thus is quite interesting and provides new avenues to explore experimentally. Moesin Radixin

  25. Novel Interactions involving parasite proteins of unknown function Actin Interacts with PF14_0635 Myosin Conserved hypothetical protein (this includes an endonucleaseb domain) Which may or may not have endonucleqase fn) Expressed in ring and trophozoite stage Spectrin Cop-coated vesicle membrane protein Interacts with emp24/gp25L/p24 family protein PFL1480w Conserved hypothetical protein Expressed in ALL stages except gametocyte VPS10 domain receptor

  26. Pf protein of unknown function interacts with Human actin

  27. 114 Pf proteins of yet unknown functions are involved in interactions with the host

  28. “Holes” in metabolic pathways of Plasmodium falciparum - “Missing” enzymes Triacylglycerol biosynthesis pathway Phophatidyl Phosphatase?? ?

  29. P. falciparum genome sequence reveals a total of 5363 proteins. Nature 419: 498-511 Non-enzymes • 75 different physiological processes are characterized encompassing 696 proteins. • Several enzymes involved in metabolic pathways are not yet identified. • Approximately 57 enzymatic steps are still undetected

  30. “Holes” in Pf pathways have EC numbers

  31. Sensitivity of prediction in P.falciparum S. Mohanty & N. Srinivasan Protein & Peptide Letters (2008) (in press)

  32. List of P. falciparum proteins identified in the present analysis as potentially involved in various metabolic pathways and their predicted functions. ….. S. Mohanty & N. Srinivasan Protein & Peptide Letters (2008) (in press)

  33. Significant pathway holes filled

  34. Triacylglycerol biosynthesis ? Phophatidyl phosphatase [E.C 3.1.3.4]

  35. Phophatidyl phosphatase [E.C 3.1.3.4] • Catalyzes hydrolysis of L-phosphatidate to1, 2-diacylglycerol and phosphate. • Involved in: • Glycerophospholipid metabolism • Glycerolipid metabolism • Triacylglycerol biosyntheisis • Essential for membrane biogenesis and also in production of energy. • Identified PAP2 superfamily related protein (PFL1210w, Id=15%, E-value=: 2e-08 in MulPSSM search ). Members known to act as phophatidyl phosphatases

  36. Structurally equivalent modelled active site of Pf protein PFL1210w (blue) superimposed on homologous Phophatidyl phosphatase of known structure (purple). The core conserved active site residues are H (316)-H (275)-R (314)-D (320) in PF1210w as highlighted.

  37. Increase in PLP, DAG and TAG in Merozoites stage, essential for parasitophorous vacuolar membrane synthesis. Microbiol Rev,1979, 43, 453-495

  38. Application of prediction of host-pathogen protein-protein interaction to Mycobacterial organisms

  39. Statistics of Protein-Protein Interactions for three organisms in Mycobacterium genus

  40. Examples of M.tuberculosis interactions with human proteins M.tb membrane protein (gi:3261503) with Human toll-like receptor 8 (gi:20302166) • Toll-like receptor pathway is known to be important for tuberculosis development. • Toll-like receptors are predicted to interact with a few membrane proteins. • Some of the proteins predicted to interact are proteases. Intracellular signalling cascades regulating innate immune responses to Mycobacteria: branching out from Toll-like receptors (2007). Cell Microbiol. 9(5):1087-98

  41. Examples of M.tuberculosis interactions with human proteins M.tb 10 kDa antigen (gi:1449369)with Human TNF receptor-associated protein 1 (gi:7706485) • 10 kDa antigen known to be important for pathogenesis from various experiments. • M.tuberculosis interferes with TNF signalling which is important for immune evasion. • Interaction predicted using DIP interaction between two proteins from Yeast. • Predicted interaction can help in understanding TNF signalling in infected macrophages. Effect of Mycobacterium tuberculosis-Specific 10-Kilodalton Antigen on Macrophage Release of Tumor Necrosis Factor Alpha and Nitric Oxide. (2002)Infection and Immunity 70(12):6558-6566

  42. Examples of M.tuberculosis interactions with human proteins M.tb Probable respiratory nitrogen reductase (narG, gi:2117199) with Human apoptosis-inducing factor (gi : 21389617) • M.tb infected macrophages show abnormal apoptosis. • The mechanisms by which M.tb triggers this transformation is unknown. • narG known to be important for survival under in vitro hypoxic conditions. • Reports suggest narG deletion mutants can survive in vivo conditions. • Predicted interaction can point to a novel function for narG in the pathogen. • Is narG involved in the apoptosis regulation in infected macrophages ? Apoptosis-Inducing Factor Participation in Bovine Macrophage Mycobacterium bovis-Induced Caspase-Independent Cell Death.(2007) Infect Immun. 75(3):1223–1228.

  43. It was predicted, on the basis of co-evolution analysis and structural modelling, that PknB of M.tubercolosis phosphorylates EmbR. This was later confirmed by experimentation. Domain structure of PknB with predicted phosphorylation sites In collaboration with Dr. Yogendra Singh, IGIB, Delhi Sharma et al, 2006 FEBS J. 3-D model of PknB-EmbR complex

  44. Examples of M.tuberculosis interactions with human proteins M.tb protein kinase PknD (gi:2078052) with Human proteins • 11 protein kinases detected in M.tb genome. • Some are membrane bound (PKNB, PKNF)‏. • Predicted interacting partners for PknD include many proteins such as: • HLA-B associated transcript 8 (gi:18426879) • Vascular cell adhesion molecule 1(gi:18201909) Figure showing the dimer of PknD from M.tuberculosis. The interaction of an eukaryotic kinase modelled on the template of PknD shows good conservation of interacting residues.

  45. M.tuberculosis interactions with human proteins – Novel examples M.tb probable export protein (gi:1314027) with Human proteins • Many pathogen proteins are exported to host cytoplasm. • Exported proteins play an important role in pathophysiology. • Function of many of the proteins exported is not known. • Predicted interaction between an export protein and actin is thus interesting. • Predicted interactions include many phosphatases in human. M.tb conserved hypothetical protein (gi:2104343) with Bcl-XL interacting protein (gi:20070384) • M.tb arrests the apoptosis of infected macriopahges. • Mechanism is not completely understood. • Bcl-XL protein accessory protein involved in apoptosis. • Predicted interaction could thus help us in understanding apoptosis signalling in TB. Mycobacterium tuberculosis Promotes Apoptosis in Human Neutrophils by Activating Caspase-3 and Altering Expression of Bax/Bcl-xL Via an Oxygen-Dependent Pathway (2002) The Journal of Immunology168: 6358-6365.

  46. Integration of cryoEM data and structural modelling C A B S19-2 S17-2 S25 S19-1 S17-1 S10 S10-2 S10-1 S12-2 S12-1 S10-3 S24 S12-3 Interactions between HCV RNA and human ribosomal proteins

  47. Conclusions • Within organism interactions can be used to detect across organisms interactions. • Combining physiological data from different sources leads to recognition of potential interactions during pathogenesis. • Previously unknown human - parasite protein-protein interactions were predicted involving parasite proteins known to be important for pathogenesis of P.falciparum and M.tubercolosis • We could detect interactions involving parasite proteins of yet unknown function. This could improve our understanding of the pathogenesis process • Our approach is general so it could be applied to any pair of host-guest organisms. This is helpful as currently no large-scale experimental approach is available for identification of comprehensive list of host-pathogen protein-protein interactions.

  48. Acknowledgements K. Anamika (P. falciparum kinomics) Nidhi Tyagi (Pf-specific protein families) Smita Mohanty (P. falciparum pathways) V.S. Gowri (Pf hypothetical proteins) O. Krishnadev (Host-pathogen interactions) MulPSSM team V.S. Gowri B. Anand Tina Graceline O. Krishnadev Smita Mohanty Supported by The Wellcome Trust, London Department of Biotechnology, New Delhi Council of Scientific and Industrial Research, New Delhi

  49. THANK YOU

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