Rui AlvesCiencies Mèdiques Bàsiques

Universitat de Lleidaralves@cmb.udl.es

http://web.udl.es/usuaris/pg193845/Courses/Other%20Seminars/

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Network Reconstruction for Novel Pathways

Integrative in silico reconstruction of Fe-S biogenesis pathway in yeast.

Summary & Perspectives

Understanding pathway assembly and function is fundamental to the understanding of how a cell works.

In annotated genomes, network of cellular pathways is “known”. Mapping orthologues onto known maps (KEGG, BIOCYC, etc.). However, regulatory topology is organism specific.

Nevertheless, reconstructing the topology of new pathways can not be done by mapping. No maps available. How to reconstruct?

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Traditionally, identification & reconstruction of a pathway/circuit would entail painstaking, mostly blind, experimental work.

Currently, availability of “omics” data provides information to facilitate this task.

Computational Biology and Bioinformatics. Integrate information, predict systemic behavior and rank

hypothesis for experimental testing Facilitates a better understanding of how cellular systems

work.

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Develop and apply coherent yet flexible framework where different computational methods and data sets are integrated to predict the connectivity of biological pathways & circuits. Today: focus on the biology

and the reconstruction of FeS cluster biogenesis in yeast S. cerevisiae.

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Network Reconstruction for Novel Pathways

Integrative in silico reconstruction of Fe-S biogenesis pathway in yeast.

Summary & Perspectives

Iron-Sulfur Clusters are coordinated ions that participate in electron transfer.

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Protein Cysteine

Protein Cysteine

Fe FeS

S

e- e-

About 15 different mitochondrial proteins are known to be involved in yeast.

The assembly process is ill-understood.

It is unclear how most of the proteins assemble as a pathway and how the activity of this pathway is regulated.

All 15 proteins have one thing in common.

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WT

Fe Level

WT

FeSC Dependent Protein Activity

Fe Accumulates

FeSC dependent protein activity is impaired

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Fe S

Scaffold Scaffold

FeSCSynthesis

Transfer

RepairHolo-P

Damaged FeSC

Apo-PHolo-P

FeSC

Scaffold Scaffold

(S)

(T)

(R)

Grx5Isa1Isa2Isu1Isu2Nfu1Atm1Nfs1Arh1Yah1Yfh1Isd11Ssq1Jac1Mge1

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Process of

interest

1. Bibliometric analysis

Identify Genes

involved in process

2. Phylogenetic analysis

Identify additional

Genes involved in

process

Get protein structures

(PDB, models)

Genes with

similar co-evolution profiles

List of reported

Two-hybrid

interactions

List of predicted

interactions

2. Interrogate 2H databases

3. In silico protein docking

Human curation

Expert Knowledge

Derive alternative

network structures

Create mathematical models for

each alternative

network

No Valid Model 4. Simulation

and comparison to experimental

results

Validated

models

Falsified models

New Simulation experiments

Literature co-occurence of genes can be taken as a signal that they are functionaly related and maybe interact physically.

iHOP performes this type of analysis automatically.

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Proteins that are present and absent in the same set of genomes are likely to be involved in the same process and therefore interact.

Target Genome

Orthologue in Genome 1?

Orthologue in Genome 2?

…

Grx5BC…

YNY…

NYN…

…………

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Sequence (Grx5)

Protein id Grx5

Calculate coincidence

index.

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…SSQIE……SSQEE…

Sequence with known structure.

OPTIMIZE

DOCK

THREAD

Homologue sequence for structure prediction.

Differential scores for docking to

different targets.

Nfs1-SSG Nfs1

Grx5,…

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11 1 21 5 1... ...

hg gd NfsNfs Grx Nfs

dt

g<0 inhibits flux.

g=0 no influence on flux.

g>0 activates flux.

Use approximate formalism:•Power Law Formalism•No need for detailed mechanism.•Semi quantitative estimation of many parameter values.

Create models for alternative networks.

Normalize equations and scan parameters to see what happens when a gene is deleted from the model.

Compare simulations with known systemic behavior to validate or invalidate alternatives.

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Glutaredoxin: Mediates glutathionylation state of Cys residues. May mediate protein-protein disulfide bridge

reduction (Belli et al. 2002, Tamarit et al. 2003, JBC).

FeSC coordinate (mostly) with Cys residues.

Is Grx5 regulation of Cys reduction state in any specific protein(s) involved in FeSC biogenesis sufficient for phenotype?

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Grx5

Nfs1

Isa2

Isa1

Isu1Bibliography

Docking

Phylogeny+ Docking

Scaffolds

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Recovering Nfs1 and Scaffold

FeSC Dependent Protein Activity

Not recovering Nfs1 and Scaffold

Belli et al. 2002 MBC 13:1109

10000s of simulations

1

0.1

0.50.5

WT

1

0.1

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Fe Levels

WT Recovering Nfs1 and Scaffold

Not recovering Nfs1and Scaffold

Belli et al. 2002 MBC 13:1109

1 1

10000s of simulations

Grx5 modulates Nfs1 and Scaffold activity/Interactions.

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Possible Modes of action for

Grx5

9Reproducing experimental phenotype?

No 6

Yes 3 Nfs1-Scaffold

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Negative Controls

Grx5 Scaffold

Positive Control

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Alves et. al. 2004 Proteins 57:481

Vilella et. al. 2004 Comp. Func. Genomics 5:328

Alves et. al. 2004 Proteins 56:354

Alves & Sorribas 2007 BMC Systems Biology 1:10

Prediction Verified?

Grx5 modulates Nfs1 and Scaffold activity/Interactions

Detected interaction with scaffolds

Arh1-Yah1 act on S or ST Yes [PNAS 97:1050; JBC 276:1503]

Arh1-Yah1 interaction same as in mammals

No reported experiment

Yfh1 acts on S, T, or ST Yes [Science 305:242; EMBO Rep 4:906; JBC 281:12227; FEBS Lett

557:215]

Yfh1 storage of Fe not important for its role in biogenesis

Yes [EMBO Rep 5:1096]

Nfs1 acts in S, not necessarily in R No reported experiment

Chaperones act on Folding, Stability

Yes for Folding [JBC 281:7801]

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Network Reconstruction for Novel Pathways

Integrative in silico reconstruction of Fe-S biogenesis pathway in yeast.

Summary & Perspectives

Create a FLEXIBLE tool for other researchers. Automation of text search 75% done;

Phylogenetic profiling 75% done, Protein interactions 75% done, Automation of structural modeling & docking 0%.

Data sets very noise, human curation required & very important in the forseeable future.

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Process of

interest

1. Bibliometric analysis

Identify Genes

involved in process

2. Phylogenetic analysis

Identify additional

Genes involved in

process

Get protein structures

(PDB, models)

Genes with

similar co-evolution profiles

List of reported

Two-hybrid

interactions

List of predicted

interactions

2. Interrogate 2H databases

3. In silico protein docking

Human curation

Expert Knowledge

Derive alternative

network structures

Create mathematical models for

each alternative

network

No Valid Model Simulation and

comparison to experimental

results

Validated

models

Falsified models

New Simulation experiments

Add Genomics,

Proteomics, Metabolomics,

Fluxomics

Fe-S Human, chimp, coli, subtilis, xanthus, albicans. Fe-S biogenesis pathways shows variations in the

different organisms we are analyzing (coli, human, chimp, xanthus, subtilis). Design principles?

Signal transduction reconstruction in xanthus.

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Enric HerreroFelip VillelaAlbert Sorribas Ester VilaprinyoArmindo Salvador

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FCT (PORTUGAL)

MCyT (SPAIN)