In silico pathway reconstruction: Iron-sulfur cluster biogenesis in Saccharomyces cerevisiae

Abstract

Background: Current advances in genomics, proteomics and other areas of molecular biology make the identification and reconstruction of novel pathways an emerging area of great interest. One such class of pathways is involved in the biogenesis of Iron-Sulfur Clusters (ISC).

Results: Our goal is the development of a new approach based on the use and combination of mathematical, theoretical and computational methods to identify the topology of a target network. In this approach, mathematical models play a central role for the evaluation of the alternative network structures that arise from literature data-mining, phylogenetic profiling, structural methods, and human curation. As a test case, we reconstruct the topology of the reaction and regulatory network for the mitochondrial ISC biogenesis pathway in S. cerevisiae. Predictions regarding how proteins act in ISC biogenesis are validated by comparison with published experimental results. For example, the predicted role of Arh1 and Yah1 and some of the interactions we predict for Grx5 both matches experimental evidence. A putative role for frataxin in directly regulating mitochondrial iron import is discarded from our analysis, which agrees with also published experimental results. Additionally, we propose a number of experiments for testing other predictions and further improve the identification of the network structure.

Conclusion: We propose and apply an iterative in silico procedure for predictive reconstruction of the network topology of metabolic pathways. The procedure combines structural bioinformatics tools and mathematical modeling techniques that allow the reconstruction of biochemical networks. Using the Iron Sulfur cluster biogenesis in S. cerevisiae as a test case we indicate how this procedure can be used to analyze and validate the network model against experimental results. Critical evaluation of the obtained results through this procedure allows devising new wet lab experiments to confirm its predictions or provide alternative explanations for further improving the models.

Figure 1

Network of protein and gene interactions as derived from different theoretical and computational approaches. Light blue lines connect genes that co-occur in papers, as determined using iHOP. Mauve lines connect genes that have a significant phylogenetic coincidence, as measured by both the mutual information and the transformed Hamming distance index we use. Yellow lines connect proteins that have either been experimentally reported to interact or have been predicted in this work via in silico protein docking as being more likely to interact. Dark blue lines connect genes that co-occur in the literature and have a significant Phylogenetic coincidence. Green lines connect genes/proteins that co-occur in the literature and have been predicted to interact by our in silico docking experiments. Red lines connect genes/proteins that have a significant Phylogenetic coincidence and are predicted to interact by our docking experiments. Black lines connect genes/proteins that co-occur in the literature, have a significant Phylogenetic conservation and have been predicted to interact by the docking experiments.

Figure 2

Network model for ISC biogenesis in S. cerevisiae. The letters in the grey circles stand for the different stage of the Biogenesis. A stands for the recovery of Arh1 by Grx5. D stands for the recovery of the dead end complex between Nfs1 and the scaffold by Grx5. F stands for folding. FI stands for Fe import. I stands for the recovery of the scaffolds by Grx5. N stands for Nfs1 recovery by Grx5. R stands for the repair of the clusters. S stands for the synthesis of ISC. St stands for stabilization of the ISC assembled on the scaffolds. T stands for the transfer of the ISC to apo-proteins. The species Apo-P and P in the transfer of a 2Fe2S cluster reaction represent the pairs Apo P1 and P1, Apo P2 and Fe2S2P2 or Fe2S2P2 and P2, respectively. See text and supplementary materials for details and for kinetic representation of the reactions. Dotted arrows represent reactions that have been observed to occur experimentally. Dashed arrows represent the alternative modes of regulatory action for the different proteins. Light grey arrows represent the network interactions that are not likely to exist in the pathway, according to our analysis.

Figure 3

Comparison of the phenotypes in each of the alternative networks upon depletion of a given protein to the corresponding phenotype observed experimentally. Within each panel, we present the overall shapes of the curves that were obtained in our in silico simulations upon depletion of the target protein and points are presented to represent the curves of published experiments. Only in two cases were there more than two measurements made along the depletion curve. The curves are connected to the alternative networks according to the following labels. R: network where deleted protein acts only on repair. F: network where deleted protein acts only on folding. S: network where deleted protein acts only on synthesis. St: network where deleted protein acts only on stability of clusters. A: network where Grx5 acts on regulating glutathionylation state of Arh1. Any combination of labels indicates that the deleted protein in the network acts on more that one process. For example RS means that said protein acts on repair and synthesis of clusters. Panels A, C, D, E, G and I compare the accumulation of Fe in the network to that in the experiments. The Y-axis represents the level of free Fe. Panels B, D, F, H and J compare the evolution of ISC dependent protein activity in the alternative networks to that observed experimentally upon depletion of the protein. The Y-axis represents normalized ISC dependent protein activity. Panels A and B – Experimental data from [74] and [73]. Panels C and D – Experimental data from [88] and [87]. Panels E and F – Experimental data from [36]. Panels G and H – Experimental data from [125] and [126]. Panels I and J – Experimental data from [77, 127, 128].

Figure 4

A simplified flow chart of the procedure suggested in the discussion. See discussion for details.