Our Interests Molecular and structural basis of protein action Interactions of proteins in cellular communications URL of our group:

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

N. SrinivasanMolecular Biophysics UnitIndian Institute of Science

MBU

Prediction of protein-protein interactions between a host

and a pathogen

O. Krishnadev

Smita Mohanty

K. Anamika

Supported by CSIR, DBT and Wellcome Trust

Plasmodium falciparum – Protozoan malarial parasite

d) e)

g)

f)

Protein-protein interactions network, biological pathways & processes in P. falciparum are dynamic

Mohanty, Pandit, Srinivasan (2008)In Protein Interaction Networks and Biological Data Mining (in press)

Exon1 Exon5Exon4Exon3Exon2 DNA

Mature mRNA

Protein

PFB0815c: Plasmodium falciparum Calcium dependent protein kinase (CDPK)

Zhao et al, Mol Biochem Parasitol (1994)

Host erythrocytic membrane proteins

Phosphorylation

Anamika, Srinivasan, Krupa Proteins, 2005Anamika & Srinivasan, Prot. Pept. Lett. 2007

Is it possible to predict protein-protein interactions acrossHuman and P. falciparum?

An Approach for detecting Protein – Protein interactions

across host and pathogen organisms

Krishnadev & Srinivasan (2008)In Silico Biology

DIP, iPFam, Y2H P-P interaction datasets

Host genome Guest genomeHomology

searches using variety of methods

Putative Interaction set

Refined Set with compatible localization

Final set with compatible expression and localization

Manual analysis on significance to pathogenesis

subcellular localization of host proteins subcellular localization of guest proteins

expression data from microarray/mass specexpression data from microarray/mass spec

Anand et.al., Bioinformatics (2005)Gowri et al Nucl. Acids Res. (2006)Gowri et al Proteins (2007)

An approach for remote homology detection and fold recognition from sequence

Gowri, Anand, Krishnadev, Sairam Swamy, Gracelin, Smita Mohanty

Searches in database of constant multiple profiles

Query

RPS-BLAST

Database ofsingle profiles

Hit profiles

Query

RPS-BLAST

Database ofMultiple profiles

Hit profiles

Hit profiles

OrdinaryReverseBlast

MulPSSM

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

URL: http://pauling.mbu.iisc.ernet.in/~pali

Balaji et al (2001) Nucl. Acids. Res.Sujatha et al (2001) BioinformaticsGowri et al (2003) Nucl. Acids. Res.

Log(E)

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Gowri, Krishnadev,Anand, SrinivasanBioinformatics 2005Proteins 2007Nucl. Acids Res. 2007

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

homologues

Interactions of distantly related homologues of protein modules are NOT conserved (Rekha et al

2005 Proteins)

Distantly (superfamily)

related - bindingto unrelated

domains

Unrelated

Closely (family)related

Pathway Ain organism X

Pathway Bin organism Y

Distant homologues act as turning points in otherwise similar signalling and metabolic pathways

Rekha et al (2005)Proteins










in vitro interactions

in vivo interactions

Gowri, Krishnadev,Anand, SrinivasanBioinformatics 2005Proteins 2007Nucl. Acids Res. 2007

Rekha et alProteins2005

Host – Pathogen pairs studied

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.

Human – P.falciparum interactions Statistics

StageTotal number

of proteins

PPI with compatible expression

PPI with compatible expression and

localization

Sporozoite (liver)

2147 27884 85

Merozoite (RBC)

2115 26121 116

Ring (RBC) 2866 30527 125

Trophozoite (RBC)

3493 33074 157

Schizont (RBC)

3722 22032 1226

Known InteractionTRAP/SSP2 protein with human Collagen and ICAMs(Thrombouspondin Related Anonymous Protein / Sporozoite Surface Protein 2)

Examples of PPI in Human – P.falciparum

• 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

Recognition of InteractionsTRAP/SSP2 protein with human Collagen and ICAMs

Pf SSP2 interacts withHuman ICAM

Plasmepsins with moesin/radixin/ezrin family proteins (ERM)

More newly recognized interactions

• 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.

Plasmepsin

Moesin

Radixin

Novel Interactions involving parasite proteins ofunknown function

PF14_0635

Conserved hypothetical protein (this includes an endonucleaseb domain)Which may or may not have endonucleqase fn)Expressed in ring and trophozoite stage

Actin

Myosin

Spectrin

Interacts with

PFL1480w

Conserved hypothetical protein

Expressed in ALL stages except gametocyte

Cop-coated vesicle membrane protein

emp24/gp25L/p24 family protein

VPS10 domain receptor

Interacts with

Pf protein ofunknown functioninteracts withHuman actin

114 Pf proteins ofyet unknown functionsare involved in interactionswith the host

“Holes” in metabolic pathways of Plasmodium falciparum- “Missing” enzymes

?

Triacylglycerol biosynthesis pathway

PhophatidylPhosphatase??

Number of g enes

2463

466

2434Unannotated

E nzymes

Annotated

P. falciparum genome sequence reveals a total of 5363 proteins. Nature 419:

498-511

• 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

Non-enzymes

“Holes” in Pf pathways have EC numbers

Sensitivity of prediction in P.falciparum

S. Mohanty & N. Srinivasan Protein & Peptide Letters (2008) (in press)

S.No.

Protein name Domain assignment made using MulPSSM Recognized fold using Phyre

1 *MAL13P1.275

[Hydrolase|SCOP:c.108.1.1] [1zrn|SCOP:c.108.1.1]

2 MAL7P1.156 [Abhydrolase_1|SCOP:c.69.1.-] [1orv|SCOP:c.69.1.241 tqh|SCOP:c.69.1.14]

3 MAL7P1.178 [Abhydrolase_1|SCOP:c.69.1.-] [1wm1|SCOP:c.69.1.4]

4 MAL8P1.154 [Amino_oxidase|SCOP:c.3.1.2] [1reo|SCOP:c.3.1.2]

5 MAL8P1.38 [Abhydrolase_1|SCOP:c.69.1.-] [1tqh|SCOP:c.69.1.29]

List of P. falciparum proteins identified in the present analysis as potentially involved in various metabolic pathways and their predicted functions.

….

.

58 *PFL0230w [EC:3.5.4.1|c.97.1.2|dCMP_cyt_deam_1] [1p6o|c.97.1.2]

59 PFA0120c [EC:3.1.1.23|c.69.1.-|Abhydrolase_1] [1tqh|c.69.1.29]

60 PFL2530w [EC:3.1.1.23|c.69.1.-|Abhydrolase_1] [1tqh|c.69.1.29]

S. Mohanty & N. Srinivasan Protein & Peptide Letters (2008) (in press)

Significant pathway holes filledP.falciparum Protein

identified

EC Number Pathway

MAL7P1_156 3.1.1.23 Glycerol biosynthesis

PF13_0048 5.3.3.2 Isoprenoid metabolism

PFl2285c 4.2.1.75 Porphyrin metabolism, Heme biosynthesis

PFL0230w 3.5.4.1 Pyrimidine ribonucleotide/ ribonucleoside metabolism

PF08_0109 2.4.1.13 Sucrose biosynthesis

PF11_0453 2.7.1.49 Thiamine Metabolism

PF11_0036 4.1.36 Pantothenate and coenzyme A biosynthesis

PFL1210W 3.1.3.4 Triacylglycerol biosynthesis, Glycerolipid metabolism, Glycerophospholipid metabolism

PFF1405C 2.3.1.4 Aminosugars metabolism

PFL0040C 2.7.1.100 Methionine recycling

Triacylglycerol biosynthesis

?

Phophatidyl phosphatase [E.C 3.1.3.4]

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

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.

Gene(Function)

SPO RIN TRO SCH MER GAM

PF13_0269(EC 2.7.1.30)

PF13_0100(EC 2.3.1.15)

PF14_0421(EC 3.1.3.4)

PFL1210w(EC 3.1.3.4)

PF14_0681(EC 2.7.1.107)

PFC0995c(EC 2.3.1.20)

Increase in PLP, DAG and TAG in Merozoites stage, essential for

parasitophorous vacuolar membrane synthesis. Microbiol Rev,1979, 43, 453-495

Application of prediction of host-pathogen protein-protein

interaction to

Mycobacterial organisms

Statistics of Protein-Protein Interactionsfor three organisms in Mycobacterium genus

Name of pathogen

# of pathogen proteins predicted to interact.

# of Human proteins predicted to interact.

Total predicted interactions.

PPIs with compatible sub-cellular localization.

M.Tuberculosis 1458 7328 62962 2156

M.Leprae 1605 6805 33491 1953

M.bovis 1431 7281 62585 2078

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


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


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.

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 ofPknB with predictedphosphorylation sites

3-D model of PknB-EmbR complex

In collaboration with Dr. Yogendra Singh, IGIB, DelhiSharma et al, 2006 FEBS J.

M.tb protein kinase PknD (gi:2078052) with Human proteinso 11 protein kinases detected in M.tb genome.o Some are membrane bound (PKNB, PKNF).o 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.

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 Immunology 168: 6358-6365.

Integration of cryoEM data and structural modelling

A B

S10

S19-2

S10-3

S10-2S10-1

S17-1

S17-2

S19-1

S12-1

S12-2

S12-3

S25

S24

C

Interactions between HCV RNA and human ribosomal proteins

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.

Acknowledgements

Smita Mohanty (P. falciparum pathways)

V.S. Gowri(Pf hypothetical proteins)

O. Krishnadev(Host-pathogen interactions)

MulPSSM teamV.S. GowriB. AnandTina GracelineO. KrishnadevSmita Mohanty

Supported by The Wellcome Trust, London

Department ofBiotechnology, New Delhi

Council of Scientific andIndustrial Research, New Delhi

K. Anamika(P. falciparum kinomics)

Nidhi Tyagi(Pf-specific protein families)

THANK YOU

Documents

Our Interests Molecular and structural basis of protein action Interactions of proteins in cellular communications URL of our group: