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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)
-5 -4 -3 -2 -1 0
% S
ensi
tivi
ty
0
20
40
60
80
100
SPAMPAHMM
Log(E)
-5 -4 -3 -2 -1 0
Sp
ecif
icit
y(%
)
0
20
40
60
80
100
120
SPAMPAHMM
Specificity
Log(E)
-5 -4 -3 -2 -1 0
Err
or
rate
(%
)
0
20
40
60
80
100
120
HMMMPASPA
Sensitivity
Error rate
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
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
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
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
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
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.
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)
Examples of M.tuberculosis interactions with human proteins
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