Reconstruction modeling of mapkinase signaling pathway
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Reconstruction & Modeling of MAPKinase Signaling Pathway. Sonia Chothani (IAS-INSA-NASI Summer research fellow) Department of Biotechnology IIT Madras. Biochemical Pathways. Molecular interaction network of biological processes in a cell. The major types of pathways we are looking into:

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Reconstruction & Modeling of MAPKinase Signaling Pathway

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Reconstruction modeling of mapkinase signaling pathway

Reconstruction & Modeling of MAPKinase Signaling Pathway

Sonia Chothani

(IAS-INSA-NASI Summer research fellow)

Department of Biotechnology

IIT Madras


Biochemical pathways

Biochemical Pathways

  • Molecular interaction network of biological processes in a cell.

  • The major types of pathways we are looking into:

    • Metabolic

    • Signaling


Metabolic pathways

Metabolic Pathways

  • In simple words it is a step by step modification of the initial molecule to shape it into another product

Substrate 1

Enzyme 1

Product 1 Substrate 2

Enzyme 2

Product 2 Substrate 3

Enzyme 3

Product3 Substrate 4

Enzyme 4

Product


Signaling pathways

Signaling pathways

  • A mechanism that converts an extracellular signal to a cell into a specific cellular response

Stimulus

Receptor

2nd messengers

Cellular Response


Why do we study them

Why do we study them?

  • To understand the biochemical processes involved in the cell

  • Identify difference of mechanism between species

  • Molecular pathological studies, as in most of the diseases there is some change in these normal pathways

  • Our study is concentrated on the MAPK pathway


Mitogen activated protein kinase pathway mapk

Mitogen Activated Protein Kinase pathway (MAPK)

  • Mitogens - Chemical substances that triggers mitosis

  • Ser/Thr specific protein kinases

  • Occurs in almost all kinds of cells

  • Responses like Proliferation, differentiation

  • Specific Mutations cause uncontrolled proliferation  Cancer

  • Hence studying this pathway can help understanding the progression of the disease

Stimulus

MAPKKK

MAPKK

MAPK

Cellular response


Our work

Objective

Our Work

To study MAPK pathway and hence identify important molecules that are involved in Cancer

  • Reconstruction of MAPK Pathway

  • Mathematical Modeling of the reconstructed pathway

  • Identification of optimal intervention points (targets) and alternative paths

  • Connecting to other pathways related to cancer


Why do we need reconstruction

Why do we need reconstruction?

  • Numerous signaling databases present online

  • Inconsistent & Incomplete data available on different databases.

  • We studied 13 databases for MAPK signaling pathway and cross-checked with >70 published papers


Protein lounge vs kegg

Protein Lounge vs KEGG


Reconstruction modeling of mapkinase signaling pathway

ERK-MAPK Pathway

JNK Pathway

P38 Pathway

MAPKK

MAPK

MAPKKK

ERK5 Pathway


Logical steady state analysis lssa

Logical Steady State Analysis (LSSA)

  • Analogous to Flux Balance Analysis

    • Differential Equations Logical Equation (AND, OR, NOT operators)

    • Stoichiometry Matrix Interaction matrix

  • We used CellNetAnalyzer, a MATLAB supported software for the Logical Steady State Analysis of MAPK pathway


Cellnetanalyzer

CellNetAnalyzer

  • Designed for structural & functional analysis of biochemical networks

  • Facilitates Logical Steady State Analysis


Standardization of software

Standardization of software

  • Signaling Toy Network was simulated in CNA

  • Results were verified with published literature

    Steffen Klamt, Julio Saez-Rodriguez, Jonathan A Lindquist, Luca Simeoniand Ernst D Gilles, “A methodology for the structural and functional analysis of signaling and regulatory networks” BMC Bioinformatics 2006, 7:56

  • Then we proceed to model MAPK pathway in CNA


Procedure we followed

Procedure we followed


Logical equations interaction graphs

Logical Equations & Interaction Graphs

2

1

3

PI3K

Grb2-SOS: Growth factor receptor-bound protein – Son of Sevenless (GEF) complex

PKC: Protein Kinase C

Gap1m: RAS GTP-ase activating protein (GTP hydrolysis)

Ras: GTP-ases

4

Grb2-SOS

PKC

Activators = Grb2-SOS + PKC

RAS

PI3K: Phosphoinositide 3 kinase

Gap1m

Interaction Graph

Interaction Hyper-graph

RAS = Activators.!Gap1m


Validation of model

Validation of model

  • Published Literature for verification

  • Example: PTEN is a tumor suppressor

    Akira Suzuki, José Luis de la Pompa, Vuk Stambolic, Andrew J. Elia “High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice” Current Biology 1998, 8:1169–1178

  • EGFR an oncogene is kept on

  • 1) PTEN (tumor suppressor) is kept off

    => Uncontrolled Proliferation

  • 2) PTEN (tumor suppressor) is kept on

    => Controlled proliferation & Apoptosis


Effect on transcription factors varying pten

Effect on Transcription factors varying PTEN

X axis: Pathway/Response

Y Axis: No. of Transcription factors

EGFR on PTEN off

Uncontrolled proliferation

EGFR on PTEN on

Normal cellular processes


Interaction matrix

Interaction Matrix

YL axis: Species

X Axis: Reactions

YR axis: (g/r/b)

i has no effect on j

i has an activating influence on j

i has an inhibiting influence on j

i is activated by j


Dependency matrix shortest path analysis

No influence of i on j

i has activating and inhibiting effect on j

i is a pure inhibitor of j

i is a pure activator of j

i is an independent inhibitor of j

i is an independent activator of j

Dependency Matrix & Shortest Path Analysis

X Axis: Species

Y Axis: Species


Identification of key species

Identification of Key Species

  • Interactions with more number of molecules

  • Influencing low number but crucial molecules

  • Transcription factors leading to important pathways/cellular responses

  • Published literature


Reconstruction modeling of mapkinase signaling pathway

Ambivalent Effect

Inhibiting Effect

Activating Effect

Totally Inhibiting Effect

Totally Activating Effect

Surface Molecules, Growth Factors, Ion channels

‘y’ no. of molecules are influenced by ‘x’

X Axis: Molecule Y Axis: No. of molecules

‘y’ no. of molecules influence ‘x’


Reconstruction modeling of mapkinase signaling pathway

Ambivalent Effect

Inhibiting Effect

Activating Effect

Totally Inhibiting Effect

Totally Activating Effect

Transcription factors, Output Molecules

‘y’ no. of molecules are influenced by ‘x’

X Axis: Molecule Y Axis: No. of molecules

‘y’ no. of molecules influence ‘x’


Reconstruction modeling of mapkinase signaling pathway

Ambivalent Effect

Inhibiting Effect

Activating Effect

Totally Inhibiting Effect

Totally Activating Effect

Intermediate Molecules

‘y’ no. of molecules are influenced by ‘x’

X Axis: Molecule Y Axis: No. of molecules

‘y’ no. of molecules influence ‘x’


Transcription factors leading to significant effects on other pathways

Transcription factors leading to significant effects on other pathways

X axis: Pathway/Response

Y Axis: No. of Transcription factors


Reconstruction modeling of mapkinase signaling pathway

No Influence Totally Activating Effect

Ambivalent Effect Totally Inhibiting Effect

Activating Effect Inhibiting Effect

X Axis: Molecule Y Axis: No. of molecules

Influence on other species

i.e.; ‘y’ no. of molecules get Influenced by ‘x’

Transcription Factors, Outputs

Growth Factors, Surface Proteins, Inputs

Intermediate Molecules

Influenced by other species

i.e.; ‘y’ no. of molecules Influence ‘x’


Reconstruction modeling of mapkinase signaling pathway

Perturbation Study

IL-1/TNF-alpha

Caspase-3

Apoptosis

Independent pathway to apoptosis

Need to prevent this inhibition

JNK pathway

Proliferation and Apoptosis

PKB/AKT

P38 pathway

B-raf

Uncontrolled Proliferation

NKX-3

Better targets because stops uncontrolled proliferation

Just negatively regulates so not a beneficial reaction to target

B-Raf

MEK1

ERK

Proliferation


Linking with metabolic pathways

Linking with metabolic pathways

  • Transcription factors lead to cellular responses but undergo other processes which might regulate the response

  • TNFR, MEKK1, TPI2, TAK1 have an activating influence and PKB/AKT has an inhibiting influence on NF-KB

  • NF-KB (TF) is one of the regulators for IDH1(Isocitrate dehydrogenase-1)

  • IDH1 decarboxylates isocitrate to 2-oxoglutarate (TCA cycle)

  • Hence we can further see effects on this metabolic pathway


Conclusion

Conclusion

  • This kind of a study is important to identify important molecules and related sub-pathways for further experimental study

  • Identifies possible alternative pathways hence identifies optimal intervention points (targets)

Further Work

  • Transcription factors need not be the output molecules, we need to consider detailed downstream paths.

  • We would like to further combine this pathway with other cancer related pathways (even some metabolic) to be able to confirm our conclusions and similarly identify more targets.


Reconstruction modeling of mapkinase signaling pathway

Dr. Ram Rup Sarkar and Dr. Somdatta Sinha for the continuous guidance and all the patience.

I would also like to thank Dr. C Suguna and all other lab members for all the support and discussions.

Last but not the least I would like to thank CCMB and IAS-NASI-INSA for giving me this great opportunity.


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