Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins.

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Exploiting bacterial operons to illuminate human iron-sulfur proteins Claudia Andreini, Lucia Banci, and Antonio Rosato J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00045 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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Journal of Proteome Research

Exploiting bacterial operons to illuminate human iron-sulfur proteins

Claudia Andreini1,2, Lucia Banci1,2,*, Antonio Rosato1,2

1

Magnetic Resonance Center, University of Florence, 50019 Sesto Fiorentino, Italy.

2

Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Italy.

Corresponding Author: Prof. Lucia Banci Magnetic Resonance Center University of Florence Via Luigi Sacconi 6 50019 Sesto Fiorentino (Italy) Tel.: +39 055 4574273 Fax: +39 055 4574253 E-mail: [email protected]

Running title: The portfolio of human iron-sulfur proteins

Abbreviations FeS: Iron-sulfur cluster FeS-P: Iron-sulfur cluster-binding protein CIA machinery: Cytosolic Iron-sulfur Assembly machinery ISC machinery: Iron-Sulfur Cluster assembly machinery TCA: TriCarboxylic Acid cycle

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Abstract Organisms from all kingdoms of life use iron-sulfur proteins (FeS-Ps) in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of FeS-Ps. 61% of human FeS-Ps bind Fe4S4 clusters whereas 39% bind Fe2S2 clusters. However, this relative ratio varies significantly depending on the specific cellular compartment. We compared the portfolio of human FeS-Ps to 12 other eukaryotes and to about 700 prokaryotes. The comparative analysis of the organization of the prokaryotic homologs of human FeS-Ps within operons allowed us to reconstruct the human functional networks involving the conserved FeS-Ps common to prokaryotes and eukaryotes. These functional networks have been maintained during evolution and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps. Purine metabolism is another process including several FeS-Ps, in which BOLA proteins possibly have a regulatory role. The analysis of the co-occurrence of human FeS-Ps with other proteins highlighted numerous links between the ISC machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of ROS within the biogenesis and degradation of FeS-Ps.

Keywords Bioinorganic chemistry; iron; mitochondrion; respiration; biogenesis; Fe2S2; 2Fe-2S; Fe4S4; 4Fe-4S


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INTRODUCTION Iron-sulfur (FeS) clusters are among the most ancient and versatile inorganic cofactors. They are involved in an a plethora of functional processes, including aerobic as well as anaerobic respiration, regulation of gene expression, amino acid and nucleotide metabolism, DNA modification and repair and tRNA modification

1,2

. Metalloproteins containing FeS clusters (FeS-

Ps, for FeS-binding Proteins) can be identified in organisms from all kingdoms of life 3. In these systems, the formation of FeS clusters in vitro is often spontaneous, when the apo-protein is exposed to inorganic iron and sulfur sources 4. Structural studies showed that the presence of the FeS cluster is a crucial factor to achieve proper folding of small FeS-Ps 5. The formation of FeS clusters in vivo requires complex protein machineries, namely the so-called FeS biogenesis systems. Several such systems have been identified across all kingdoms of life, with different specificities for the types of FeS-Ps that are eventually assembled. The most common biogenesis systems are the nitrogen fixation (NIF), sulfur utilization factor (SUF), the iron-sulfur cluster (ISC) and the cytosolic iron-sulfur protein assembly (CIA) pathways assembly of holo-nitrogenase

6-11

. The NIF system is devoted to the

10,12,13

. Instead, the other systems are involved in the biogenesis of all

FeS-Ps in the cell. Homologs of the SUF and ISC systems are present both in prokaryotes and in eukaryotes, whereas the CIA system appears to be specific of eukaryotes. Eukaryotes contain protein homologs of some components of the ISC system in mitochondria, while they have homologs of components of the SUF system in chloroplasts. In the early reducing atmosphere, ancient organisms took advantage of the redox properties of FeS clusters and used them as redox centers

14

. Even in the present-day oxidizing atmosphere,

FeS-Ps have a crucial role as electron carriers. Indeed, the combined chemical versatility of iron and sulfur generates ideal devices for accepting, donating, shifting, and storing electrons 15. In the redox complexes of aerobic and anaerobic respiratory systems, multiple FeS clusters can occur in a single multimeric hetero-enzyme to build up electron transfer chains that span several tens of Å, such as in respiratory complex I

16

. In addition, FeS-Ps can play other functional roles, not of an

oxidoreductive nature. FeS clusters can stabilize specific 3D structures by connecting protein structural elements, such as for eukaryotic DNA polymerases

17

. FeS-Ps are members of several

classes of enzymes that interact with nucleic acids, including helicases, nucleases and polymeases 18

. A redox role for the FeS clusters in endonucleases was excluded based on its reduction potential

19

. It has been proposed that electron transfer between FeS clusters of different DNA-bound FeS-Ps

via DNA-mediated charge transfer provides a signaling mechanism that would explain the importance of the cluster 20. Finally, another crucial aspect of the versatile chemistry of FeS clusters is their structural plasticity, which permits the sensing of intracellular levels of gaseous molecules 3 ACS Paragon Plus Environment


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such as O2 or NO 21. Modification or loss of the FeS cluster in eukaryotic aconitases is a crucial step in the cell response to oxidative stress or ROS damage22. In this work, we exploited the extensive amount of information made available by genome sequencing projects to predict the occurrence of FeS-Ps as well as of FeS biogenesis systems in a large variety of organisms. To this end, we relied on the protocols that we previously developed for the bioinformatics investigation of metalloproteins across the kingdoms of life 23-25. These protocols are based on the identification of protein domains associated to the binding of inorganic cofactors, such as FeS clusters in the present work, filtered by the presence of conserved binding patterns in order to enhance the reliability of the predictions. The co-occurence in genomes of functionally related components is important to support the assignment of a metabolic/biosynthetic capability to an organism 26,27. Further insight is obtained for prokaryotes by inspecting the genomic organization of the genes of interest, such as their operon structure. The present analysis addressed more than 700 organisms from all kingdoms of life. As a result, we obtained a global view of the distribution of FeS-Ps among living organisms, providing insight into the physiological roles of these proteins and highlighting potential new functional interactions occurring in human cells.

EXPERIMENTAL SECTION Using the approach described by Andreini et al. 23 as implemented in the RDGB program 26, we identified the iron-sulfur proteome in 12 Eukaryotes and 701 Prokaryotes (82 Archaea, 619 Bacteria). Our search started from 113 Pfam

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profiles, with or without 3D structure. The 3D

structure of a metalloprotein can be used to define both the type of FeS cluster bound by the domain and the pattern of amino acids that are involved in the interaction of the protein with the metal cofactor. Such a pattern, which is called the metal binding pattern (MBP) 29, is a regular expression defining the identity and spacing of the metal-binding residues, e.g. CX(4)CX(20)H, where X is any amino acid. For the profiles with a 3D structure, the MBP can be used as a filter to remove false positives

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and the corresponding FeS cluster type is assigned as the potential cofactor. We

matched the 113 profiles mentioned above to 1131 3D structures of iron-sulfur proteins available from the Protein Data Bank, corresponding to 293 distinct binding patterns of the cofactors. The human FeS-Ps were manually analyzed to check whether they were assigned the correct type of FeS cluster and to assign them, when possible, to a specific process by using as starting point the work by Lill and coworkers 2. We identified six main processes involving human FeS-Ps, i.e. the CIA machinery, the mitochondrial ISC machinery, DNA maintenance, respiratory chain, ribosome function and tRNA 4 ACS Paragon Plus Environment

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modification, and amino acid and nucleotide metabolisms. Eukaryotic and prokaryotic FeS proteomes were then compared to the human FeS proteome using BLAST with an E-value cutoff of 10-5 30. To define homology relationships, we retained only the best BLAST hit of each FeS-P in the entire human FeS proteome. The identification of the ISC machinery in prokaryotes was not simply based on the search of homologues because these organisms may possess additional machineries 9,11

(i.e. SUF and NIF machineries

) that share some components with ISC 8. To identify

unambiguously the ISC machinery, we firstly searched all the bacterial organisms for the presence of the members of all three bacterial machineries. Then, we clustered the retrieved proteins in potential operons by grouping together all the proteins close in the genome (i.e. whose genes are separated in the genomic sequence by no more than three other genes). Finally, we assigned each group of neighboring proteins as representing one of the three machineries based on its overall composition. We analyzed the operons of a subset of 80 organisms by exploiting the ProOpDB database 31. For this purpose, we took into account only bacteria that possess the ISC machinery or the respiratory chain, or both. We focused on these two machineries because they are the most conserved among prokaryotes. For the analysis, we extracted all operons that contained the homologs of at least two human proteins, of which at least one is a FeS-P. For all the pairs of human proteins present in these operons, we then identified those that occurred in at least eight operons from different organisms (corresponding to at least 10% of the organisms analyzed). All the resulting pairs were merged into virtual operons by joining pairs having one protein in common. The 3D structural models of the AIFM3 and RFESD putative Rieske domains were built using MODELLER v.9.2

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and energy-refined using the AMBER web server provided by the WeNMR

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platform . The quality of models was assessed with the QMEAN server 34. The intracellular location as well as the identity of the cellular process(es) involving each protein was taken from UniProt

35

. We used BioGrid

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to obtain interactomics data on selected

human proteins. The annotation of bacterial organisms was extracted from the Golden Database (https://gold.jgi-psf.org/ 37).

RESULTS Human FeS proteins The search in the human genome for Iron-Sulfur Proteins (FeS-Ps hereafter) mapped 70 unique genes (i.e. 0.35% of the human genes). These are listed in Table 1. Experimental verification of their ability to bind an iron-sulfur cofactor is available for most of these proteins, either directly or by similarity to some close homolog. As the list includes all the experimentally validated human 5 ACS Paragon Plus Environment


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Iron-Sulfur Proteins (FeS-Ps hereafter) 2, it can be regarded as a minimum inventory of such systems. The two main iron-sulfur cluster types in biological systems are Fe2S2 and Fe4S4 clusters. In our predictions, we assigned respectively 37 and 55 proteins binding either type of cluster. Note that the sum is greater than the 70 genes because the same protein can contain more than one site type. In the human proteome, Fe4S4 sites are thus significantly more common than the simpler Fe2S2 clusters. Figure 1 shows that almost 70% of the human FeS-Ps are involved in just 6 main broad cellular processes 2. The single process that involves the largest number of FeS-Ps (i.e. 13 FeS-Ps) is DNA maintenance, owing to the binding of iron-sulfur cofactors by DNA polymerases and some helicases. Essentially all of these proteins are nuclear. The second more populated process is respiration (9 FeS-Ps), which mainly includes FeS-Ps that belong to complex I, thus localized in the inner membrane of the mitochondrion. Other relevant processes are amino acid and nucleotide metabolism (6 FeS-Ps) and ribosome function/tRNA modification (5 FeS-Ps) which are mainly occurring in cytosol. The biogenesis of iron-sulfur cofactors, which in humans involves two different yet connected mechanisms, cumulatively involves 14 FeS-Ps. 8 FeS-Ps take part in the socalled mitochondrial machinery (ISC machinery, hereafter), while 5 FeS-Ps are in the so-called cytosolic machinery (CIA machinery, hereafter). However, it should be kept in mind that the localization of these proteins is not exclusive to one cellular compartment. Furthermore, FeS-Ps involved in the biogenesis process often bind iron-sulfur clusters only transiently, on the way for delivery of the assembled cofactor to their final protein target(s). Many steps along this process involve the formation of clusters at a protein-protein interface, thus having cluster ligands from two molecules. This makes the identification of the genes encoding these proteins more difficult because of the reduced set of ligands from each protein. Indeed, the single protein pattern would not be able to bind a cluster and it is only the protein dimerization that allows cluster binding. This was one of the main motivations that led us to manually review the prediction of human FeS-Ps in this work. The distribution within cellular compartments of human FeS-Ps binding Fe2S2 and Fe4S4 clusters is shown in Figure 2: 60% of Fe2S2-binding FeS-Ps have mitochondrial localization (not necessarily in an exclusive manner), 29% are in the cytoplasm and 11% are in the nucleus, whereas Fe4S4-binding FeS-Ps are more evenly distributed among the cytoplasm (33%), mitochondrion (33%) and nucleus (34%). Thus, mitochondrial localization is nearly two times more likely for a Fe2S2-binding than a Fe4S4-binding FeS-P. Instead, nuclear localization is significantly more likely for Fe4S4-binding FeS-Ps. In absolute terms, the number of mitochondrial FeS-Ps binding the Fe2S2 or Fe4S4 cofactors are very similar (Figure 2). Within this cellular location, the two main physiological processes involving FeS-Ps are respiration and biogenesis of FeS-Ps. Instead, there is 6 ACS Paragon Plus Environment

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a higher number of proteins binding the Fe4S4 cofactor in the cytoplasm, where the ratio of Fe4S4:Fe2S2 cluster-binding proteins is close to 2:1, and, to an even higher degree, in the nucleus, where the corresponding ratio is nearly 5:1. The cytoplasm is the cellular compartment where the machinery for cytosolic iron-sulfur assembly predominantly operates. In addition, cytoplasmic FeSPs, binding either Fe2S2 or Fe4S4 clusters, are involved in various metabolic processes, including amino acid and nucleotide metabolism, and ribosome function and tRNA modification. Finally, nuclear FeS-Ps are almost exclusively of the Fe4S4 type and mainly act in DNA maintenance. It has been proposed that specific short sequence motifs can steer substrate discrimination and guide delivery of FeS clusters from proteins of the assembly machineries to specific subsets of target FeS-Ps

38

. These motifs consist of three consecutive amino acids and are present in the

sequence of the target FeS-P. Their functional role is to facilitate recognition by and the interaction with the appropriate biogenesis protein. We analyzed all putative human FeS-Ps for tripeptide motifs containing non-polar residues (as Ile, Leu, Pro or Val) at the first position, a Phe or Tyr aromatic hydrophobic residue at the second position and positively charged Arg or Lys residues at the third position

39,40

. The first motif to be experimentally characterized was in fact the LYR

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tripeptide . The last column of Table 1 reports the results of our analysis. About 55% of the human target FeS-Ps contain a tripeptide motif complying with the above rule. Among the 14 FeS-Ps of the CIA and ISC machineries, only three contained a motif (Table 1). Within human target FeS-Ps, tripeptide motifs are found in 81% of nuclear FeS-Ps, and about 50% of both mitochondrial and cytoplasmic FeS-Ps. Moreover, the motif occurs more commonly in Fe4S4-binding (62%) than in Fe2S2-binding (43%) proteins. The most common motif is LFR (11 target FeS-Ps in Table 1), followed by LFK, VFK and VFR (7 FeS-Ps each). The LYR motif was found in 6 target FeS-Ps. Finally, we compared the occurrence of the tripeptide motifs in the human FeS-proteome to their occurrence in the entire human proteome. We observed that only the LFR motif is significantly more common in FeS-Ps, being two-fold enriched. Within our inventory of human FeS-Ps, there are eight proteins known to bind a Fe2S2 cluster but could not be firmly assigned to a specific cellular process because their function is substantially uncharacterized. These included BOLA1, BOLA2 and BOLA3, three paralogs some of which are potentially involved in the regulation of the mitochondrial thiol redox potential 41 or more generally in redox regulation mechanisms 42. Then there are RFESD, UQCRFS1P1 and AIFM3, all proteins that contain a Rieske-domain. The sequences of RFESD and AIFM3 encompass a LYR-type tripeptide motif. The function of RFESD and UQCRFS1P1 is completely uncharacterized. UQCRFS1P1 has 98% sequence identity to a Rieske-subunit of the cytochrome b-c1 complex (UQCRFS1 in Table 1); its expression in human heart cells has been detected by proteomics using 7 ACS Paragon Plus Environment


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2D gel electrophoresis and tandem MS 43. AIFM3 is a mitochondrial protein that has been shown to induce apoptosis through a caspase dependent pathway 44. NARF (or IOP2) is a nuclear paralog of one of the proteins involved in the CIA machinery, called NARFL (or IOP1). Finally, CISD3 is the least characterized member of the NEET family, a group of proteins which have the capability to transfer their FeS cluster, and which are involved in the regulation of ROS and iron homeostasis 45. To validate the cluster-binding capability of the potential FeS-Ps containing a Rieske domain, we searched the PDB database for templates suitable for structural modelling. We could identify suitable templates for the Rieske domains of AIFM3 and RFESD, with sequence identity levels of 33% and 90% respectively. Notably, also all other structural templates identified by the BLAST search tool, which we discarded because of their lower sequence identity to our target proteins, contained a Fe2S2 cluster. The models generated by homology modeling are of good quality (i.e. |Z| < 1), as defined by their QMEAN

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scores. The structural models display a canonical Rieske site,

with the proper configuration of proteins ligands around the Fe2S2 cofactor (Figure 3). In the article reporting the RFESD template structure, solved for a mouse protein, it is stated that human RFESD has the same UV-Vis spectrum, thus confirming the presence of the Fe2S2 cluster, but no data are shown and no further protein characterization has been performed 46. We then identified a novel candidate FeS-P in the human proteome. GRXCR1 is a cysteine-rich protein that contains a glutaredoxin domain. While glutaredoxin domains can bind iron-sulfur clusters at the interface with other proteins in protein:protein adducts

47-49

, the cysteine-rich region

of GRXCR1 may actually allow this protein to bind its cofactor(s) without involving a protein partner.

Eukaryotic FeS-Ps We extended our analysis to 12 additional eukaryotes (Table S-1) in order to identify possible organism-specific variations of the FeS-P repertoire. With the exception of the simpler S. cerevisiae and C. thermopium organisms (48 and 53 FeS-Ps, respectively) as well as of the plant A. thaliana (162 FeS-Ps), the number of FeS-P-coding genes ranges between 62 and 82. Of these, the percentage of proteins homologous to human FeS-Ps is between 73% (D. rerio, which is also the organism with the largest number of FeS-Ps) and 94% (mouse). Interestingly, we detected the enzyme glutamate synthase, which binds a Fe3S4 cluster in bacteria 50, only in invertebrates and in A. thaliana. In all these cases, the cluster-binding site is maintained. Four out of five FeS-Ps that are central to the human CIA machinery are present in all the analyzed eukaryotes, namely ABCB7, NUPB1, IOP1, and CIAPIN1. NUBP1 interacts with NUBP2 8 ACS Paragon Plus Environment

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to form a complex binding a bridging Fe4S4 cluster; a NUBP2 homolog was found in 83% of the organisms (it is absent in A. thaliana and C. elegans). The NUBP1 proteins of A. thaliana and C. elegans are similar to one another (sequence identity 55%); for comparison, the sequence identity between human NUBP1 and NUBP2 is 52%. We did not analyze non-FeS-proteins even if part of the CIA machinery. The FeS-Ps in the ISC machinery are also highly conserved, with four proteins present in all the organisms considered (GLRX5, ISCA1, ISCU, NFU1), two present in 11 organisms out of 12 (FDX2, absent in chicken, and ISCA2, absent in C. thermopium) and NUBPL lacking in S. cerevisiae and C. elegans. Ferredoxin FDX1, whose role in FeS-P biosynthesis is debated 51, is less widespread than FDX2, being present in 67% of the organisms analyzed. Overall, the average conservation of the FeS-P biosynthesis mechanisms is 94% over the 12 organisms examined, with respect to the human genome. The FeS-Ps that constitute elements of the respiration chain showed high levels of conservation. More specifically, we identified nine such FeS-Ps in the human proteome, all of which were present in 83% to 100% of the organisms investigated. The most relevant exception is complex I, which is lacking altogether in S. cerevisiae. One of these proteins is absent also in the pig proteome.

Prokaryotic FeS-Ps homologous to human FeS-Ps We then analyzed the genomes of 701 prokaryotic organisms and predicted the FeS-Ps there present. Among these, we performed a deeper analysis mainly on the occurrence of possible homologs to human FeS-Ps. This constituted the basis to exploit the organization of prokaryotic genomes into operons for identifying possible other players in the cellular processes involving FeSPs as well as for obtaining hints (to be then verified experimentally) on the function of the less characterized human FeS-Ps. In line with the great diversity of prokaryotic organisms, the FeS-P portfolio of prokaryotes is also quite variable. For the aim of the present work, it is thus not useful to analyze individual organisms in detail. Instead, we reasoned that it would be more instructive to aggregate the data at the level of phyla, except for proteobacteria. This allowed us to observe broader trends and qualitatively evaluate their relevance. Another useful aggregation was to analyze the results at the level of whole processes. Figure 4 shows the distribution of FeS-Ps by cellular process except for iron-sulfur biogenesis, which is addressed separately below. Each process comprises six to fourteen proteins in humans. For aerobic respiration, organisms from various different phyla encode in their genomes homologs to more than 60% of human FeS-Ps involved in the process (Figure 4). Alpha and betaproteobacteria show the highest fraction of FeS-Ps common to those in the human respiratory chain 9 ACS Paragon Plus Environment


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(between 76% and 83%). Gamma- and delta/epsilon-proteobacteria have a 52-54% degree of conservation. Other phyla that encode homologs for a significant fraction of the FeS-Ps in the human respiratory process are Acidobacteria (65%) and Aquificae (68%). DNA maintenance appears to involve FeS-Ps primarily in eukaryotes. For FeS-Ps involved in amino acid and nucleotide metabolism as well as in ribosome function and tRNA modification, the fraction of human proteins with homologs in bacteria rarely exceeds 40% in all phyla (Figure 4). Among these, the variability of this percentage is lower in ribosome function and tRNA modification, indicating that 30-40% of the human FeS-Ps in this process have homologs in most prokaryotes. A further level of detail is achieved by inspecting the human FeS-Ps that have homologs in at least one third of the prokaryotes studied (Table 2). Globally, five out of nine FeS-Ps in the human respiratory chain have homologs in 60% to 72% of prokaryotic organisms. These involve proteins from complex I and complex II. For amino acid and nucleotide metabolism, three out of six human FeS-Ps appear in Table 2; however, two of them have an average occurrence below 50%. This explains the overall average as well as the variability observed in Figure 4 for this process. On the other hand, the occurrence of two out of six human FeS-Ps in ribosome function and tRNA modification is 83% and 62% respectively. DNA maintenance involves as many fourteen proteins in human cells, but only two of these often occur also in bacteria, leading to the low fractions observed in Figure 4. Thus, at the level of the proteins in each process, the respiratory chain features five proteins conserved from prokaryotes to humans, whereas all other processes (with the exception of the ISC machinery, discussed in the next section) only feature two or three.

The prokaryotic ISC machinery for iron-sulfur cluster biogenesis There are three known FeS-P assembly systems in prokaryotes (ISC, NIF and SUF)

8,11,52

. Of

these three, the SUF machinery is the most ancient and the most widespread in prokaryotes, but there is not a eukaryotic homolog system. The ISC system is common also to eukaryotes, being the main mechanism for the maturation of mitochondrial FeS-Ps. In order to build a systematic comparison between the human and prokaryotic ISC machineries, we searched complete prokaryotic genomes for ISC operons, i.e. for neighboring genes in the genome sequence encoding proteins that have sequence similarity to known ISC proteins. Note that not all proteins in the ISC machinery are FeS-Ps (Table 3). We retained only operons that comprised at least three different putative ISC proteins. We thus identified the ISC machinery in 130 prokaryotic organisms, i.e. 18.5% of all organisms analyzed. ISC operons are present in various Acidobacteria, 4 out of 60 Bacteroidetes, 2 out of 48 Clostridia, 14 out of 75 alpha-proteobacteria, nearly all 52 beta10 ACS Paragon Plus Environment

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proteobacteria, 5 out of 39 delta/epsilon-proteobacteria, the majority of 101 gamma-proteobacteria, 2 out of 3 Nitrospira. Of the 130 operons identified, 82.0% contained 6 or 7 different ISC proteins, whereas 12.5% contained the required minimum of three different ISC proteins. Operons with only 4 or 5 different ISC proteins were thus relatively uncommon (about 5% and 1%, respectively). The most common protein in ISC operons is a homolog of the IscS (human NFS1) cysteine desulfurase, which is found in 96% of the cases. IscA, IscU, HscA and HscB are also very common, all being present in 90% or more of the operons. The bacterial ferredoxin (Fdx or Yah1) that donates electrons to the desulfurase is slightly less common than the HscA:HscB complex (84% of the operons), whereas IscX, a potential iron chaperone for the system, is not very widespread, as we detected it in only 50% of the operons. The more common six-protein operon encodes homologs of IscS, IscU, IscA, HscA, HscB and Fdx (note that we did not seek homologs of the IscR regulator as it is not present in the human proteome). This is the entire operon required for production of Fe2S2 and Fe4S4 clusters 8,53. Seven-gene operons, such as that of Escherichia coli, additionally encode the IscX protein, which is a potential donor of iron(II) ions and a regulator of IscS activity

54

. The

shorter three and four-gene operons lack, as expected based on the above data, any IscX and Fdx homologs. Occurrence of the HscA:HscB complex is also less probable in these systems. Six out of eight proteins in the human ISC machinery have homologs in more than one third of prokaryotes (Table 2), with fractional occurrences between 34% and 53%, with the exception of NUBPL that has a degree of occurrence of 72%. The higher occurrence of the latter protein can be due to the similarity of its domain structure to that of other molecular chaperones

55

. This result

differs from the observation that 18.5% of the prokaryotes analyzed contain a potential ISC operon, because some proteins of the human ISC machinery are homologous also to proteins of the SUF machinery, which is much more widespread. Indeed, if the search is performed only for homologs of individual proteins on the basis of sequence similarity and the operon structure is not taken into account, the picture would have been quite different, with homologs of IscU occurring in 64% of the organisms and homologs of NFS1 occurring in 98% of the organisms. By taking into account the distribution of proteins in prokaryotic operons, a much higher level of confidence in the proposed homologies is achieved 56.

Investigating bacterial operons to reconstruct cellular processes involving FeS-Ps Co-occurrence of proteins in operons is associated with an increased likelihood of functional or physical interaction among them. This can be exploited to obtain a broad view of cellular processes as networks of such interactions, as well as to identify unprecedented potential players in the processes

57,58

. To this end, we created virtual operons for the human proteome through the 11 ACS Paragon Plus Environment


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definition of networks linking human protein pairs whose homologs co-occurred in a single prokaryotic operon in at least 10% of the analyzed organisms. Such virtual operons included both FeS-Ps and non-FeS-Ps. We identified nine virtual operons involving at least two proteins (Figure 5), of which at least one FeS-P. Note that our approach allowed us to reconstruct only processes that are common to both eukaryotes and prokaryotes. Prokaryotic genes can have multiple homologs in humans. Consequently, virtual operons may connect different human processes occurring in different compartments if these processes involve various paralogs of the same prokaryotic gene with different cellular locations or if the same human protein can localize to different compartments. The two virtual operons that involve the largest number of human FeS-Ps are the ISC machinery (described in the next paragraphs) and the respiratory chain. Out of the entire respiratory chain, we actually extracted only the proteins in complexes I, II and III, as complex IV does not contain FeSPs. Complex II (i.e. succinate dehydrogenase or succinate-coenzyme Q reductase) is also involved in the tricarboxylic acid (TCA) cycle of which it catalyzes the sixth step. Besides complex II, the other FeS-P in the TCA cycle is aconitase (ACON). The TCA cycle consumes acetyl-CoA that is produced by the pyruvate dehydrogenase complex starting from pyruvate. The decarboxylation of pyruvate occurs along with the reductive acetylation of lipoic acid, which is covalently bound to a component of the complex called dihydrolipoamide acetyltransferase (DLAT). Lipoic acid is produced via a radical SAM mechanism, by lipoyl synthase (LIAS), another FeS-P (Figure 5). In prokaryotes, LIAS can occur in operons containing LIPT2 or in operons encoding the subunits of pyruvate dehydrogenase 59. The lypoate synthesis pathway is essential in mitochondrial metabolism 60

. This interplay of functional relationships links process #2 to #5 in Figure 5. A separate virtual

operon (#6 in Figure 5) is determined by the overlap of four prokaryotic operons (Figure 6). Functionally,

one

of

the

operons

is

linked

to

purine

biosynthesis

and

involves

amidophosphoribosyltransferase (PPAT), which is an FeS-P. The bacterial operons containing homologs of human glutaredoxins and/or BOLA proteins include additional proteins in this virtual operon, such as PAICS and PFAS, both of which are enzymes involved in the biosynthesis of inosine monophosphate. Interactomics studies indicated that human GLRX5 and PAICS physically interact

61,62

. However, the functional relationship among all the proteins in the virtual operon is

unclear (see also Table 4); a further complication in this respect is that most of these proteins are cytosolic, while some are mitochondrial. Notably, human glutaredoxins and BOLA proteins are present in both cellular compartments 63 (Table 1). The ISC machinery is composed by one virtual operon that includes most of the known human proteins in the process as well as some other proteins not previously reported to be related to iron12 ACS Paragon Plus Environment

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sulfur biogenesis (Table 4). In the absence of experimental data, it appears that most likely the latter proteins form functional interactions with one or two ISC proteins bridging this process to other cellular processes. Indeed, the additional proteins are not in the operons encoding the prokaryotic ISC machinery but are associated to single ISC paralogs in other operons. In total, we identified three proteins as related to the ISC process not previously reported to play a role in it. DNAJA3 (also called TID1) is a zinc-binding mitochondrial protein 64, whose prokaryotic homolog is found in three-gene operons containing homologs of human mtHsp70 (also called mortalin or HspA9 in mammals, or HscA in prokaryotes) and GRPE. Both mtHsp70 and GRPE are known members of the ISC machinery that do not bind iron-sulfur clusters. AIFM3 is an FeS-P that contains a Rieske domain fused to an oxidoreductase domain. Various proteobacteria have operons that encode a homolog of AIFM3 next to a paralog of the Fdx ferredoxin of the ISC machinery. Furthermore, the Rieske domain of AIFM3 displays homology to Rieske-type ferredoxins in bacterial SUF operons (not shown), suggesting a direct involvement in FeS-P biogenesis. Finally, APEX1 (DNAapurinic/apyrimidinic site lyase) has a prokaryotic homolog that neighbors ISCA2 in a two-gene operon in alpha-proteobacteria.



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DISCUSSION In this work, we obtained an extensive overview of the occurrence of FeS-Ps in prokaryotes and some eukaryotes, and specifically in humans, as a way to broaden our understanding of their relevance for many cellular roles. Furthermore, we compared the operon structure of the prokaryotic homologs of human FeS-Ps to identify recurring cases of protein (including also nonFeS-Ps) co-occurrence. Through the identification of the human homologs of commonly cooccurring proteins, this investigation provided hints for a more comprehensive appreciation of human cellular processes involving FeS-Ps. The human genome contains 70 genes that encode FeS-Ps (Table 1), the large majority of which has been experimentally validated. This portfolio of FeS-Ps includes one potential human member lacking experimental validation, GRXCR1, a cysteine-rich protein that contains a glutaredoxin domain. Physiologically, GRXCR1 is involved in cross-linking and/or bundling Factin in stereocilia

65

and mutations in the Grxcr1 gene have been shown to cause hearing

impairment and other dysfunctions

66,67

. No atomic- or molecular-level biochemical studies are

available for this protein. In 59% of the cases, human FeS-Ps bind at least one Fe4S4 cluster, as opposed to a 41% occurrence of Fe2S2 cluster. This includes also clusters formed at protein-protein interface, e.g. as it happens in the assembly of the clusters themselves or during their transfer to the final protein targets. This different occurrence of Fe4S4 vs. Fe2S2 clusters is the average at the level of the whole cell. The situation within each cellular compartment can be quite different (Figure 2). Indeed, Fe4S4 clusters are largely predominant in the nucleus, as cofactors of several proteins involved in DNA maintenance, and are also preferred in the cytoplasm, where the Fe4S4: Fe2S2 ratio is close to 2:1. On the other hand, the number of FeS-Ps binding either cluster type is similar within mitochondria, thus corresponding to an enrichment in Fe2S2 clusters in mitochondria with respect to the global average in the cell. Such enrichment is the result of the use of Fe2S2 clusters in respiration as well as in the ISC machinery. The large majority of nuclear FeS-Ps contain a LYRtype tripeptide motif. This motif is used to recruit the complex HSCB:ISCU:HSPA9 of the ISC assembly machinery to a specific target protein

38

. The present observation may thus support the

involvement of ISC proteins, which are traditionally regarded as mitochondrial proteins, in the maturation of nuclear FeS-Ps 39. 24 FeS-Ps are widespread as they have homologs in at least one third of all the prokaryotes investigated as well as in eukaryotes (Table 2). 20 of these are involved in the virtual operons of Figure 5, which in total includes 29 FeS-Ps. We can thus speculate that the physiological processes 14 ACS Paragon Plus Environment

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identified in Figure 5 constitute a core set of cellular processes depending on FeS-Ps. Probably, these processes have appeared relatively early in the course of evolution, although not necessarily all at the same time, and have been maintained also in extant higher organisms. Our analysis focused on FeS-Ps that are encoded in the human genome, leaving open the possibility that other evolutionarily ancient processes are maintained in extant organisms but only in the prokaryotic kingdom. The 20 FeS-Ps mentioned above constitute the majority of the 34 mitochondrial FeS-Ps that we identified, i.e. 59%. As we will discuss in more detail in the next paragraphs, most of the conserved FeS-Ps are part of the respiratory chain or of the ISC machinery for the biogenesis of FeS-Ps. Also the metabolism of purines, both biodegradation and biosynthesis, involves several conserved FeS-Ps. Interestingly, this observation provides a hint for the functional role of at least some of the human proteins of the BOLA family, for which we may hypothesize a regulatory role in purine biosynthesis. E. coli strains overexpressing bolA display transcriptomic activation of genes related to purine and pyrimidine metabolism 68. A significant fraction of the human FeS-Ps involved in aerobic respiration have homologs in various phyla, especially proteobacteria and acidobacteria. This is presumably due to the prokaryotic origin of the mitochondrion so that these proteins have a relatively short evolutionary distance. The fraction of human FeS-Ps that have prokaryotic homologs in the respiratory process is somewhat higher for alpha- and beta-proteobacteria than for gamma- and delta/epsilonproteobacteria (76%-83% vs. 52-54%). The alpha lineage occupies a central position in “phylum” proteobacteria, because it originated after the separation of the predominantly anaerobic delta and epsilon branches but before the split of the predominantly facultatively anaerobic gamma and beta branches

69

. Indeed, the ancestral mitochondrion has been often proposed to be an alpha-

proteobacterium 70. The other process that has significant similarities between its proteins in human and those in prokaryotic cells is the ISC process. FeS-Ps of the ISC machinery are responsible for the initial assembly of the iron-sulfur cluster, as well as for its subsequent transfer to the final recipients of the cofactor. An ISC operon was present in 18.5% of the organisms that we analyzed. These organisms were prevalently aerobic or facultatively anaerobic. We observed that the most common structure of the prokaryotic ISC operon involves six proteins (IscS, IscU, IscA, HscA, HscB and Fdx), all of which have human homologs. The human ISC machinery has multiple links to proteins involved in cell apoptosis as well as in the regulation of response to oxidative stress. mtHsp70 performs an anti-apoptotic role, also by maintaining normal ROS levels 71. Within the ISC machinery, mtHsp70 facilitates the transfer of a Fe2S2 cluster from ISCU to GLRX5. This transfer is accompanied by the hydrolysis of an ATP 15 ACS Paragon Plus Environment


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molecule, leaving the protein in the ADP-bound state in a ternary protein complex with the ISCU and GLRX5 72. The nucleotide exchange factor GRPE mediates the release of ADP and the binding of a new ATP molecule to mtHsp70 73, which in turn triggers the dissociation of the ternary protein complex thus completing the transfer reaction. An additional physiological partner of mtHsp70, as shown also by interactomics studies, is DNAJA3. The latter is a modulator of apoptosis with contrasting roles. It has two splice variants, of which the longer one increases apoptosis triggered by both TNFα and the DNA-damaging agent mitomycin c (MMC), whereas the shorter variant is able to suppress apoptosis

74

. Expression of both forms affects cytochrome c release from the

mitochondria and caspase 3 activation. DNAJA3 and mtHsp70 concur in affecting mitochondrial morphology. In particular, the interaction between the two proteins can alter the activity of mtHsp70. Whether this interaction also affects the biosynthesis of FeS-Ps in mitochondria is unclear. Also the interaction of DNAJA3 and mtHsp70 depends on the nucleotide exchange factor GRPE

75

, and indeed interactomics studies demonstrated a physical interaction between DNAJA3

and GRPE 76. Notably, we identified a three-gene operon encoding homologs of mtHsp70, GRPE, and DNAJA3 in 65% of all the 700 prokaryotic organisms analyzed. Several of these organisms had an operon for the full ISC machinery typically encoding an additional mtHsp70/HscA paralog. In these cases, the human mtHsp70 featured a higher sequence similarity to its homolog in the threegene operon than to HscA in the prokaryotic ISC operon. In bacteria this system is involved in the assembly of protein complexes, refolding of stress-denatured proteins, and transport of newly synthesized peptides across membranes

77

. In human cells, it has been proposed that the

mtHsp70:DNAJA3 complex, with the assistance of GRPE, may scavenge toxic protein aggregates in stressed, diseased, or aging human mitochondria 78. Our virtual operon analysis (Figure 5) suggested that AIFM3 is another human protein with a role in apoptosis and a link to some FeS-Ps involved in the ICS machinery, namely FDX2 and ISCU. This link is beyond the fact that the ISC machinery presumably has a role in the maturation of AIFM3. Indeed, co-occurrence of homologs of AIFM3 with homologs of FDX2 and/or ISCU in genomic proximity points to these proteins serving a common physiological role. There is no experimental evidence of physical interactions between AIFM3 and these proteins. AIFM3 induces apoptosis in a caspase-dependent manner when overexpressed

44

. Deletion of the related

mitochondrial AIF2 protein in the ascomycete Podospora anserina leads to a significantly increased ROS tolerance and a prolonged lifespan, as a probable consequence of delayed cell death response to ROS

79

. Interactomics indicated that in Drosophila melanogaster AIFM3 has a physical

interaction with CISD1

80

. CISD1 is located to the outer mitochondrial membrane, with the FeS-

binding domain oriented toward the cytoplasm

81

. AIFM3 is located predominantly to the inner


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membrane, but a low abundance in the outer membrane was also observed 44. CISD1 is a FeS-P that regulates bioenergetics in cells as a function of oxidative stress and of the presence of ROS. In addition, it plays a role in the repair of other FeS-Ps that suffered oxidative damage 82. CISD1 and CISD2 have a role in the regulation of cellular iron, calcium and ROS homeostasis 45. For example, concurrent exposure of hepatocytes to fructose and ethanol induces CISD1 overexpression. The subsequent treatment of such hepatocytes with tumor necrosis factor α (TNFα) triggers a series of molecular events that promote the release of the Fe2S2 cluster of CISD1, eventually leading to accumulation of mitochondrial iron and increased formation of ROS 83. In turn, this brings to cell death in a caspase-independent manner. Another new functional link suggested by the virtual operon analysis includes APEX1 and ISCA2 (Figure 5). APEX1 is a DNA repair zinc-protein involved in regulating cell response to oxidative stress

84

. Interestingly, translocation of APEX1

from the cytoplasm to the mitochondria is mediated by ROS signaling

85

. APEX1 serves as the

molecular switch to engage both repair pathways and death pathways that determine cell fate in response to certain types of DNA damage. In particular, the inhibition of the repair function of APEX1 enhances caspase-3 activity and apoptotic cell death, thus pointing APEX1 as a regulator of cell death initiation 86. In summary, our virtual operon analysis and the available experimental data on the physiological role of mtHsp70 (mortalin) and DNAJA3 concur to support a tight interplay of the ISC machinery and the response of cells, including programmed cell death, to stress factors. The main factors at stake are oxidative stress and ROS production, consistently with the role of iron ions in the catalysis of ROS production. Leakage of iron ions and/or electrons from the ISC process as well as damage of FeS-Ps leading to cluster disassembly are all factors that can enhance the production of ROS. Accordingly, the functional interactions that our analysis pinpointed engage proteins with a role in damage repair and modulation of damage response, thereby directly linking FeS-P biogenesis in mitochondria to the response of cells to environmental stress.

Acknowledgements This work was supported by Ente Cassa di Risparmio di Firenze (Grant ID no.2013/7201) and the Ministero

dell'Istruzione,

dell'Università

e

della

Ricerca

CTN01_00177_962865).


(Grant

ID

number:


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glutaredoxin-ISU

[2Fe-2S]

cluster

exchange

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Clark, G. W.; Wu, G. C.; Wodak, S. J.; Tillier, E. R.; Paccanaro, A.; Marcotte, E. M.; Emili, A. A census of human soluble protein complexes Cell 2012, 150, 1068-1081. (62) Wan, C.; Borgeson, B.; Phanse, S.; Tu, F.; Drew, K.; Clark, G.; Xiong, X.; Kagan, O.; Kwan, J.; Bezginov, A.; Chessman, K.; Pal, S.; Cromar, G.; Papoulas, O.; Ni, Z.; Boutz, D. R.; Stoilova, S.; Havugimana, P. C.; Guo, X.; Malty, R. H.; Sarov, M.; Greenblatt, J.; Babu, M.; Derry, W. B.; Tillier, E. R.; Wallingford, J. B.; Parkinson, J.; Marcotte, E. M.; Emili, A. Panorama of ancient metazoan macromolecular complexes Nature 2015, 525, 339-344. (63) Banci, L.; Camponeschi, F.; Ciofi-Baffoni, S.; Muzzioli, R. Elucidating the molecular function of human BOLA2in GRX3-Dependent anamorsin maturation pathway J.Am.Chem.Soc. 2015, 137, 16133-16134. (64) Cheng, H.; Cenciarelli, C.; Shao, Z.; Vidal, M.; Parks, W. P.; Pagano, M.; Cheng-Mayer, C. Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70 Curr.Biol. 2001, 11, 1771-1775. (65) Drummond, M. C.; Belyantseva, I. A.; Friderici, K. H.; Friedman, T. B. Actin in hair cells and hearing loss Hear.Res. 2012, 288, 89-99. (66) Odeh, H.; Hunker, K. L.; Belyantseva, I. A.; Azaiez, H.; Avenarius, M. R.; Zheng, L.; Peters, L. M.; Gagnon, L. H.; Hagiwara, N.; Skynner, M. J.; Brilliant, M. H.; Allen, N. D.; Riazuddin, S.; Johnson, K. R.; Raphael, Y.; Najmabadi, H.; Friedman, T. B.; Bartles, J. R.; Smith, R. J.; Kohrman, D. C. Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse Am.J.Hum.Genet. 2010, 86, 148-160. (67) Schraders, M.; Lee, K.; Oostrik, J.; Huygen, P. L.; Ali, G.; Hoefsloot, L. H.; Veltman, J. A.; Cremers, F. P.; Basit, S.; Ansar, M.; Cremers, C. W.; Kunst, H. P.; Ahmad, W.; Admiraal, R. J.; Leal, S. M.; Kremer, H. Homozygosity mapping reveals mutations of GRXCR1 as a cause of autosomalrecessive nonsyndromic hearing impairment Am.J.Hum.Genet. 2010, 86, 138-147. (68) Dressaire, C.; Moreira, R. N.; Barahona, S.; Alves de Matos, A. P.; Arraiano, C. M. BolA is a transcriptional switch that turns off motility and turns on biofilm development MBio. 2015, 6, e02352-14. (69) Battistuzzi, F. U.; Feijao, A.; Hedges, S. B. A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land BMC.Evol.Biol. 2004, 4, 44. (70) Degli, E. M. Bioenergetic evolution in proteobacteria and mitochondria Genome Biol.Evol. 2014, 6, 3238-3251. (71) Tai-Nagara, I.; Matsuoka, S.; Ariga, H.; Suda, T. Mortalin and DJ-1 coordinately regulate hematopoietic stem cell function through the control of oxidative stress Blood 2014, 123, 41-50. (72) Uzarska, M. A.; Dutkiewicz, R.; Freibert, S. A.; Lill, R.; Muhlenhoff, U. The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation Mol.Biol.Cell 2013, 24, 1830-1841. (73) Dutkiewicz, R.; Marszalek, J.; Schilke, B.; Craig, E. A.; Lill, R.; Muhlenhoff, U. The Hsp70 chaperone Ssq1p is dispensable for iron-sulfur cluster formation on the scaffold protein Isu1p J.Biol Chem 2006, 281, 7801-7808.


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(74) Syken, J.; De-Medina, T.; Munger, K. TID1, a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions Proc.Natl.Acad.Sci.U.S.A 1999, 96, 8499-8504. (75) Mapa, K.; Sikor, M.; Kudryavtsev, V.; Waegemann, K.; Kalinin, S.; Seidel, C. A.; Neupert, W.; Lamb, D. C.; Mokranjac, D. The conformational dynamics of the mitochondrial Hsp70 chaperone Mol.Cell 2010, 38, 89-100. (76) Goswami, A. V.; Samaddar, M.; Sinha, D.; Purushotham, J.; D'Silva, P. Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease Hum.Mol.Genet. 2012, 21, 3317-3332. (77) Sharma, S. K.; De los, R. P.; Christen, P.; Lustig, A.; Goloubinoff, P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase Nat.Chem.Biol. 2010, 6, 914-920. (78) Iosefson, O.; Sharon, S.; Goloubinoff, P.; Azem, A. Reactivation of protein aggregates by mortalin and Tid1--the human mitochondrial Hsp70 chaperone system Cell Stress.Chaperones. 2012, 17, 57-66. (79) Brust, D.; Hamann, A.; Osiewacz, H. D. Deletion of PaAif2 and PaAmid2, two genes encoding mitochondrial AIF-like oxidoreductases of Podospora anserina, leads to increased stress tolerance and lifespan extension Curr.Genet. 2010, 56, 225-235. (80) Guruharsha, K. G.; Rual, J. F.; Zhai, B.; Mintseris, J.; Vaidya, P.; Vaidya, N.; Beekman, C.; Wong, C.; Rhee, D. Y.; Cenaj, O.; McKillip, E.; Shah, S.; Stapleton, M.; Wan, K. H.; Yu, C.; Parsa, B.; Carlson, J. W.; Chen, X.; Kapadia, B.; VijayRaghavan, K.; Gygi, S. P.; Celniker, S. E.; Obar, R. A.; ArtavanisTsakonas, S. A protein complex network of Drosophila melanogaster Cell 2011, 147, 690-703. (81) Wiley, S. E.; Murphy, A. N.; Ross, S. A.; van der Geer, P.; Dixon, J. E. MitoNEET is an ironcontaining outer mitochondrial membrane protein that regulates oxidative capacity Proc.Natl.Acad.Sci.U.S.A 2007, 104, 5318-5323. (82) Ferecatu, I.; Goncalves, S.; Golinelli-Cohen, M. P.; Clemancey, M.; Martelli, A.; Riquier, S.; Guittet, E.; Latour, J. M.; Puccio, H.; Drapier, J. C.; Lescop, E.; Bouton, C. The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1 J.Biol.Chem. 2014, 289, 28070-28086. (83) Shulga, N.; Pastorino, J. G. Mitoneet mediates TNFalpha-induced necroptosis promoted by exposure to fructose and ethanol J.Cell Sci. 2014, 127, 896-907. (84) Li, M.; Wilson, D. M., III Human apurinic/apyrimidinic endonuclease 1 Antioxid.Redox.Signal. 2014, 20, 678-707. (85) Vascotto, C.; Bisetto, E.; Li, M.; Zeef, L. A.; D'Ambrosio, C.; Domenis, R.; Comelli, M.; Delneri, D.; Scaloni, A.; Altieri, F.; Mavelli, I.; Quadrifoglio, F.; Kelley, M. R.; Tell, G. Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function Mol.Biol.Cell 2011, 22, 3887-3901. (86) Cho, K. J.; Kim, H. J.; Park, S. C.; Kim, H. W.; Kim, G. W. Decisive role of apurinic/apyrimidinic endonuclease/Ref-1 in initiation of cell death Mol.Cell Neurosci. 2010, 45, 267-276.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1: Distribution of the 70 human FeS-Ps in different cellular processes


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2: Distribution of human FeS-Ps in different cellular compartments, grouped by cluster type. Two additional Fe2S2-binding proteins are not shown in the figure, as their intracellular location is unknown (Table 1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3: homology models of the three-dimensional structures of human RFESD (A) and AIFM3 (B), shown using ribbon representation. The FeS cluster is shown as spheres and its amino acidic ligands as sticks.


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Figure 4: Distribution of prokaryotic homologs of human FeS-Ps grouped by phyla (except proteobacteria) and cellular process (respiratory chain, DNA maintenance, metabolism of amino acids and nucleic bases, ribosome function). For each process, the y axis reports the average fraction of its human FeS-Ps that have at least one homolog in the organisms of the phylum on the x axis. Error bars refer to intra-phylum variability. The vertical bar separates archaeal from eubacterial phyla.

1.0

Archaea

Respiratory chain

Eubacteria

0.8 0.6 0.4 0.2

Fraction of human ISPs with homologs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.0 1.0 0.8

DNA maintenance

0.6 0.4 0.2 0.0 1.0

Aminoacid and nucleotide metabolism

0.8 0.6 0.4 0.2 0.0 1.0 0.8

Ribosome function and tRNA modification

0.6 0.4 0.2 0.0

i i ota ota ota ota ota ria ria ae ob iae lex ria cus tes tes ria tes ria ria ria ria tes tes ae res rae ria res ae ae ae ae ae cte cteuific lor yd rof cte oc icu ricu cteyce cte cte cte cte ae iste tog cte spi acte cte rcharcharchrarcharchdobanoba Aq ChhlamChlonobainocFirmene sobatom oba oba oba obairochergermorriba itrolfob roba a a e T Fu nc rote rote rote rote Sp Syn Th efe N su Fib C en ry m o no ci cti e Cy D Cr EuThau K Na A A D Plaa Pta P n P a P od h e ilo m erm alp bepsgam h T / lta de



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5: FeS-P-containing virtual operons of human genes in the cellular context. Nine virtual operons were identified. Genes in yellow boxes encode FeS-Ps. Genes in boxes outlined in cyan are associated to virtual operons by interactomics data of the corresponding proteins. Note that some proteins encoded by these genes can have more than one cellular location; for example, APEX1 and most of its partners are also nuclear proteins.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6: Bacterial operons giving rise to the virtual operon #6 of Figure 5. Genes in colored boxes have human orthologs. The human protein name is reported in brackets. Genes in white boxes do not have a human ortholog and therefore do not appear in the virtual operon of Figure 5.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 30 of 40

Tables Table 1: List of human FeS-Ps. Subcellular location include nucleus, mitochondrion and cytoplasm; we neglected other possible locations. A single gene is associated to multiple UniProt codes when the sequences of the various protein isoforms differ significantly.

1 2 3 4 5

Protein name AOX1 DPH1 DPH2 DPYD PPAT, GPAT

Q06278, C9J244 Q9BZG8, I3L1H5 Q9BQC3, H0YCR5 Q12882 Q06203

Cofactor(s) per subunit 2 x Fe2S2 Fe4S4 Fe4S4 4 x Fe4S4 Fe4S4

Cytoplasm Cytoplasm, Nucleus Cytoplasm Cytoplasm Cytoplasm

Amino acid and nucleotide metabolism Amino acid and nucleotide metabolism Amino acid and nucleotide metabolism Amino acid and nucleotide metabolism Amino acid and nucleotide metabolism

6

XDH

P47989

2 x Fe2S2

Cytoplasm

Amino acid and nucleotide metabolism

O75027

Fe2S2

Mitochondrion

CIA machinery

LFR, VYR

Q6FI81, H3BTZ8, H3BV90

2 x Fe2S2

CIA machinery

-

Q9NZ45

Fe2S2

CIA machinery

-

Q9H6Q4, H3BSH2

CIA machinery

VFR

Cytoplasm

CIA machinery

-

Q9Y5Y2, B7Z6P0, H3BNF0, H3BNS4, H3BQR2, H3BRE1

2 x Fe4S4 Fe4S4, Fe4S4 shared with NUBP2 Fe4S4 shared with NUBP1

Nucleus, Cytoplasm Mitochondrion, Cytoplasm Cytoplasm

Nucleus, Cytoplasm

CIA machinery

-

Q9BX63, J3QSE8

Fe4S4

Nucleus, Cytoplasm

DNA maintenance

IYR, LFR, PYK

Q96FC9

Fe4S4

Nucleus

DNA maintenance

LYR, LFK

Q92771, A8MPP1

Fe4S4

Nucleus

DNA maintenance

LYR, LFK

16 DNA2

P51530, F8VR31

Fe4S4

DNA maintenance

PYR, LFK, LFR

17 ERCC2, XPD

P18074, A8MX75, K7EIT8

Fe4S4

DNA maintenance

-

7 8 9 10 11 12 13 14 15

ABCB7, ATM1 CIAPIN1 CISD1 , MitoNEET NARFL, IOP1 NUBP1, NBP35 NUBP2, CFD1 BRIP1, FANCJ DDX11 , CHLR1 DDX12P, CHLR2

UniProt Codes

P53384, I3L3A0, I3L518, I3L531

Localization

Nucleus, Mitochondrion Cytoplasm, Nucleus 30

ACS Paragon Plus Environment

Process

LYR Motif LFK, PYK VFK, VFR LFK, LFR, LYK,PYK, VFR

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


18 MUTYH

Q9UIF7

Fe4S4

Nucleus Mitochondrion, Nucleus

DNA maintenance

LFR, VFR

19 NTHL1

P78549

Fe4S4

DNA maintenance

-

20 POLA1

P09884, A0A087WU64, A6NMQ1 Fe4S4

Nucleus

DNA maintenance

21 POLD1

P28340, M0R2B7

Fe4S4

Nucleus

DNA maintenance

22 POLE

Q07864

Fe4S4

Nucleus, Cytoplasm

DNA maintenance

23 PRIM2

P49643

Fe4S4

Nucleoplasm

DNA maintenance

24 REV3L

O60673

Fe4S4

Nucleus

DNA maintenance

Fe4S4

Nucleus

DNA maintenance

PYK

Fe2S2 Fe2S2 Fe2S2/ Fe4S4 Fe2S2/ Fe4S4

Mitochondrion Mitochondrion Mitochondrion Mitochondrion Mitochondrion (Iso 1), Cytoplasm (Iso 2), Nucleus (Iso 2) Mitochondrion, Nucleus, Cytoplasm

ISC machinery ISC machinery ISC machinery ISC machinery

-

ISC machinery

-

ISC machinery

IYK, LFR

Q9NZ71, D6RA96, F6WH68, X6R5I7 26 FDX1L, FDX2 Q6P4F2 27 GLRX5 Q86SX6 28 ISCA1 Q9BUE6, Q5TBE9 29 ISCA2 Q86U28 25 RTEL1

LYR, IFR, LYK, VFK IFR, LFR, PYK, VFK, VFR, VYK IYK, LFR, PFR LYR, LYK, LFK, LFR, VFK, VYK

30 ISCU

Q9H1K1, B3KQ30, B4DNC9, F5H5N2

Fe2S2

31 NFU1

Q9UMS0, C9J8Q1, F8W9P7, H7C537

Fe4S4

Q8TB37, F8W0A2

Fe2S2/ Fe4S4

Mitochondrion

ISC machinery

-

Q9Y697

Fe2S2

ISC machinery

-

Q8N5K1, D6RCF4

Fe2S2

Other processes

-

P21399 Q99798, A2A274 P48200, H0YNL8 P80404, H3BNQ7, H3BRN4 P10109

Fe4S4 Fe4S4 Fe4S4 Fe2S2 per homodimer Fe2S2

Cytoplasm, Nucleus Mitochondrion, Cytoplasm Cytoplasm Mitochondrion Cytoplasm Mitochondrion Mitochondrion

Other processes Other processes Other processes Other processes Other processes

LYK, VFK, LFR PYR, VFR -

32 33 34 35 36 37 38 39

NUBPL, IND1 NFS1 CISD2, MINER1 ACO1, IRP1 ACO2 IREB2 ABAT FDX1

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

O43766 Q9NZB8 Q9HA92 Q8WXG1, C9J674

Fe2S2 shared with partner 2 x Fe4S4 2 x Fe4S4 Fe4S4 Fe4S4

45 GLRX2

Q9NS18

Fe2S2 per homodimer

46 CMAHP FECH, 47 HEMH 48 NDUFS1 49 NDUFS2 50 NDUFS7

Q9Y471

Fe2S2

Mitochondrion Cytoplasm, Nucleus Mitochondrion Mitochondrion Mitochondrion (Iso 1), Nucleus (Iso 2) Cytoplasm

P22830

Fe2S2

Mitochondrion

Other processes

PYR, IYR

2 x Fe4S4, Fe2S2 Fe4S4 Fe4S4

Mitochondrion Mitochondrion Mitochondrion

Respiratory chain Respiratory chain Respiratory chain

PYR VFR

2 x Fe4S4

Mitochondrion

Respiratory chain

LYR, LFR

Fe4S4 Fe2S2

Mitochondrion Mitochondrion

Respiratory chain Respiratory chain

-

Fe2S2, Fe3S4 , Fe4S4

Mitochondrion

Respiratory chain

LYR, IYR

55 UQCRFS1 56 ETFDH

P28331, B4DJ81, C9JPQ5 O75306 O75251 O00217, E9PKH6, E9PPW7, Q08E91 P49821, B4DE93, G3V0I5 P19404, E7EPT4 P21912, A0A087WWT1, A0A087WXX8 P47985, P0C7P4 Q16134

Fe2S2 Fe4S4

Respiratory chain Respiratory chain

LYK, PFR

57 ABCE1

P61221, D6R9I9, D6RGF4

2 x Fe4S4

58 CDK5RAP1 59 CDKAL1

Q96SZ6 Q5VV42

2 x Fe4S4 2 x Fe4S4

60 ELP3

Q9H9T3 B4DKA4

Fe4S4

Mitochondrion Mitochondrion Cytoplasm, Mitochondrion Cytoplasm Cytoplasm Cytoplasm (Iso 2), Nucleus (Iso 1 and 2)

Q6NUM6, Q9NV66, A0A087WUV9, A0A087WWV6, A0A087WZB2 A0A087X1V1

Fe4S4

Cytoplasm

Ribosome function and tRNA modification VYR, LFK, LFR

40 41 42 43 44

GLRX3, PICOT LIAS MOCS1 RSAD1 RSAD2

Page 32 of 40

51 NDUFS8 52 NDUFV1 53 NDUFV2 54 SDHB

61

TYW1, TYW1B

O76003

Cytoplasm

Other processes

-

Other processes Other processes Other processes Other processes

VFR

Other processes

LYK

Other processes

LYK

32


Ribosome function and tRNA modification VFK Ribosome function and tRNA modification IFR Ribosome function and tRNA modification Ribosome function and tRNA modification VYR

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

62

CISD3, MINER2


P0C7P0

2 x Fe2S2

Mitochondrion

Unknown

-

Mitochondrion

Unknown

-

Cytoplasm

Unknown

-

Mitochondrion

Unknown

-

66 GRXCR1

Fe2S2 shared with GLRX Fe2S2 shared with GLRX Fe2S2 shared with GLRX Fe2S2 (predicted)

Unknown

Unknown

-

67

2 x Fe4S4

Nucleus

Unknown

IFR

Fe2S2 (predicted) Fe2S2 (predicted) Fe2S2 (predicted)

Unknown Mitochondrion Unknown

Unknown Unknown Unknown

PFK PFR, VFK -

63 BOLA1

Q9Y3E2

64 BOLA2

Q9H3K6, H3BV85

65 BOLA3

Q53S33

68 69 70

A8MXD5 Q9UHQ1, A0A088AWN8, NARF E7EP87, E9PH27, J3KRH7, J3KS48, J3KT43, J3QRB0 RFESD Q8TAC1, D6RBY0 AIFM3 Q96NN9, C9JPU8, C9K029 UQCRFS1P1 P0C7P4

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 34 of 40

Table 2: Human FeS-Ps that have homologs in at least one third of the prokaryotes analyzed. The second column reports the percentage of prokaryotes organisms containing a homolog. The functional annotation has been taken from Uniprot. The EC number is given only for enzymes.

Protein Name DPYD

%

Process

Functional Annotation

Example structure

EC number

49

1.3.1.2

86

None

3.1.3.2

XDH

42

Amino acid and nucleotide metabolism

2CKJ (holo)

1.17.1.4

MUTYH

73

DNA maintenance

3N5N (holo)

3.2.2.-

NTHL1

94

DNA maintenance

None

4.2.99.18

ACO1

54

Iron regulation

2B3X (holo)

4.2.1.3

GLRX5

34

ISC machinery

2MMZ (apo)

None

ISCA1

53

ISC machinery

None

None

ISCA2

48

ISC machinery

Involved in pyrimidine base degradation. Catalyzes the reduction of uracil and thymine. Dephosphorylates receptor tyrosine-protein kinase erbB-4 and inhibits the ligand-induced proteolytic cleavage. Catalyzes the oxidation of hypoxanthine to xanthine in purine degradation. Catalyzes the oxidation of xanthine to uric acid. Contributes to the generation of reactive oxygen species. Involved in oxidative DNA damage repair. Removes inappropriately paired adenine bases from the DNA backbone. Bifunctional DNA N-glycosylase with associated apurinic/apyrimidinic (AP) lyase function that catalyzes the first step in base excision repair (BER), the primary repair pathway for the repair of oxidative DNA damage. Iron sensor. Binds a 4Fe-4S cluster and functions as aconitase when cellular iron levels are high. Functions as mRNA binding protein that regulates uptake, sequestration and utilization of iron when cellular iron levels are low. Monothiol glutaredoxin involved in the biogenesis of iron-sulfur clusters Involved in the maturation of mitochondrial 4Fe-4S proteins functioning late in the iron-sulfur cluster assembly pathway. Involved in the maturation of mitochondrial 4Fe-4S

1H7W (holo)

PPAT

Amino acid and nucleotide metabolism Amino acid and nucleotide metabolism

None

None

34


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


ISCU

38

ISC machinery

NFU1

40

ISC machinery

NUBPL

72

ISC machinery

ACO2

82

Other processes

LIAS

72

Other processes

MOCS1

81

Other processes

RSAD1 NDUFS1

37 66

Other processes Respiratory chain

NDUFS8

72

Respiratory chain

NDUFV1

64

Respiratory chain

NDUFV2

60

Respiratory chain

SDHB

67

Respiratory chain

CDK5RAP1

83

CDKAL1

62

Ribosome function and tRNA modification Ribosome function and tRNA modification

proteins functioning late in the iron-sulfur cluster assembly pathway. Scaffold protein in the assembly or repair of the FeS present in iron-sulfur proteins. Scaffold protein the assembly of FeS and their deliver to target proteins. Required for the assembly of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It delivers FeS to complex I subunits. Catalyzes the isomerization of citrate to isocitrate via cis-aconitate. Catalyzes the insertion of two sulfur atoms into the C-6 and C-8 positions of the octanoyl moiety bound to the lipoyl domains of lipoate-dependent enzymes, thereby converting the octanoylated domains into lipoylated derivatives. Catalyzes the conversion of 5'-GTP to cyclic pyranopterin monophosphate May be involved in porphyrin cofactor biosynthesis. Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Iron-sulfur subunit of succinate dehydrogenase that is involved in complex II of the mitochondrial electron transport chain Inhibits CDK5 activation by CDK5R1. Catalyzes the methylthiolation of N6threonylcarbamoyladenosine, leading to the 35


1WFZ (apo)

None

2LTM (apo), 2M5O (apo) None

None

None

4.2.1.3

None

2.8.1.8

None

4.1.99.18

None None

1.3.99.1.6.5.3, 1.6.99.3

None

1.6.5.3, 1.6.99.3

None

1.6.5.3, 1.6.99.3

None

1.6.5.3, 1.6.99.3

None

1.3.5.1

None

None

None

2.8.4.5

None


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

AIFM3

45

Unknown

formation of 2-methylthio-N6threonylcarbamoyladenosine at position 37 in tRNAs that read codons beginning with adenine Induces apoptosis through a caspase dependent pathway. Reduces mitochondrial membrane potential.

36


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None

1.-.-.-.

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Table 3: Proteins in the human ISC machinery # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Protein name ISCU NFS1 ISD11 FXN FDX1L, FDX2 FDXR HSPA9 HSCB GRPEL1, MGE1 GLRX5 ISCA1 ISCA2 IBA57 NFU1 NUBPL, IND1

FeS cluster bound Fe2S2 Fe2S2 None None Fe2S2 None None None None Fe2S2 Fe2S2/ Fe4S4 Fe2S2/ Fe4S4 None Fe4S4 Fe2S2/ Fe4S4

Protein role within the ISC machinery Scaffold protein Sulfur donor Stabilizer of NFS1 Iron donor? Electron transfer Electron transfer FeS cluster transfer FeS cluster transfer Nucleotide exchange FeS cluster transfer Assembly of Fe4S4 clusters Assembly of Fe4S4 clusters Assembly of Fe4S4 clusters Dedicated ISC targeting factor Dedicated ISC targeting factor

37



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Table 4: Proteins in virtual operons #1 and #6 of Figure 5 without a clearly established functional role in the context of the virtual operon. Operon 1

1

1

Protein name DNAJA3

AIFM3

APEX1

6

PFAS

6

PAICS

Full protein name

Subcellular localization

Function

DnaJ protein Tid-1

Modulates apoptotic signal. Affect cytochrome C release from the mitochondria and caspase 3 activation

Apoptosis-inducing factor 3

Induces apoptosis through a caspase dependent pathway. Reduces mitochondrial membrane potential

Mitochondrion

Multifunctional protein that plays a central role in the cellular response to oxidative stress

Nucleus, Cytoplasm, Mitochondrion

It is involved in the purine biosynthetic pathway

DNA-(apurinic or apyrimidinic site) lyase Phosphoribosylformyl glycinamidine synthase Multifunctional protein ADE2

Mitochondrion Cytoplasm

Associated to: GRPEL1, HSPA9 ISCU,

FDX2 ISCA2,

Taxonomy Common in most bacterial species

Common in bacteria which possess SUF machinery Occasional in α, β and γ proteobacteria Common in α proteobacteria

NTH

NTH-APEX fusion in some bacterial species

Cytoplasm

BOLA-like, GLRX-like

Common in α proteobacteria

It is involved in the purine biosynthetic pathway

Cytoplasm

BOLA-like, GLRX-like

Common in α proteobacteria

Isoform 1: Nucleus, Cytoplasm, Isoform 2: Mitochondrion

BOLA-like

Common in β and γ proteobacteria

6

PIN4

Parvulin-14

Isoform 1 is involved in ribosome biogenesis. Isoform 2 binds to doublestranded DNA

6

MSRB3

Methionine-Rsulfoxide reductase B3

Catalyzes the reduction of free and protein-bound methionine sulfoxide to methionine

Mitochondrion

BOLA-like


CDH

Cystathionine gammalyase

Catalyzes the last step in the transsulfuration pathway from methionine to cysteine

Nucleus, Cytoplasm

PPAT, FPGS, PUSL1, PCCB


6

38


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Supporting information Table S-1. List of all organisms analyzed



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for TOC only 54x32mm (300 x 300 DPI)


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Engineered fluorescent proteins illuminate the bacterial periplasm.

Algal proteins illuminate epilepsy.

Exploiting rRNA operon copy number to investigate bacterial reproductive strategies.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

Exploiting genomic data to identify proteins involved in abalone reproduction.

Profiling bacterial communities by MinION sequencing of ribosomal operons.

Methods to Illuminate the Role of Salmonella Effector Proteins during Infection: A Review.

A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons.

Bacterial delivery of TALEN proteins for human genome editing.

Bacterial toxins induce heat shock proteins in human neutrophils.

Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins.

Exploiting gut bacteriophages for human health.

Case studies continue to illuminate the cognitive neuroscience of memory.

The diversity of membrane transporters encoded in bacterial arsenic-resistance operons.

Bacterial iron-sulfur proteins.

Exploiting radiation damage to map proteins in nucleoprotein complexes: the internal structure of bacteriophage T7.

Exploiting tRNAs to Boost Virulence.

BadR and BadM Proteins Transcriptionally Regulate Two Operons Needed for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris.

The effects of proteins on bacterial attachment to polystyrene.

Analysis of bacterial biotin-proteins.

Borrowed proteins in bacterial bioluminescence.

Bacterial ice crystal controlling proteins.

Bacterial adherence to human gallbladder epithelium.

Neuroscience. Exploiting sleep to modify bad attitudes.