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Rui AlvesCiencies Mèdiques Bàsiques
Universitat de [email protected]
http://web.udl.es/usuaris/pg193845/Courses/Other%20Seminars/
04/21/23 2
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?
04/21/23 3
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]
04/21/23 25
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.
04/21/23 26
04/21/23 27
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)