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Bioinformatics of Mitochondria, …a top-down lecture…

Bioinformatics of Mitochondria, …a top-down lecture…. Martijn Huynen. Parkinson. Leigh syndrome. Diabetes. Myopathies. Friedreich’s ataxia. Alzheimer. Leber’s syndrome. Central role of mitochondria in metabolism. Calcium signaling. Coenzyme synthesis. Citric acid cycle. Urea cycle.

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Bioinformatics of Mitochondria, …a top-down lecture…

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  1. Bioinformatics of Mitochondria,…a top-down lecture… Martijn Huynen

  2. Parkinson Leigh syndrome Diabetes Myopathies Friedreich’s ataxia Alzheimer Leber’s syndrome Central role of mitochondria in metabolism Calcium signaling Coenzyme synthesis Citric acid cycle Urea cycle Heme synthesis FeS clusters Apoptosis Electrical signaling ATP production Fatty acids oxidation Heat generation

  3. Endosymbiotic origin of mitochondria 16S Ribosomal RNA

  4. Original rationales for the endosymbiosis 1: ATP ? (MCF family is strictly eukaryotic) 2: Oxygen sink ? (Andersson & Kurland) 3: H2 ? (Martin & Muller) O2 ATP H2

  5. Free-living, alpha-proteobacterial ancestor Gene transfer Gene loss Gain (Andersson & Kurland) and retargeting of proteins Rickettsia Mitochondria

  6. Identifying eukaryotic proteins with an alpha-proteobacterial origin based on their phylogeny A B Eukaryotic + alpha-proteobacterial proteinsg in the same branch Alpha-proteobacterial proteins with the rest of the bacteria and archaea

  7. GENOMES SELECTION OF HOMOLOGS, (Smith&Waterman) LIST ALIGNMENTS AND TREE (Clustalx, Kimura+Dayhoff) PHYLOME Detecting eukaryotic genes of alpha-proteobacterial ancestry GENOME 6 alpha-proteobacteria (22 500 genes) 6 alpha-proteobacteria 9 eukaryotes 56 Bacteria+Archaea TREE SCANNING

  8. species Genome size Selected % Groups Rickettsia prowazekii 835 196 23,5 173 Rickettsia conorii 1374 235 17,1 192 Caulobacter crescentus 3718 668 17,9 480 Brucella melitensis 3188 578 18,1 403 Rhizobium loti 7260 969 13,3 516 Rhizobium meliloti 6150 821 13,3 Alpha-proteobacterial genes monophyletic with eukaryotic genes 446 630 Non redundant orthologous groups:

  9. Estimating false positives and false negatives,630 orthologous groups appears a lower bound: • False positives: Of the unrelated Deinococcus radiodurans the algorithm selects 1.3 % • False negatives: Of the 66 proteins encoded in the mitochondrial genome of Reclinomonas americana the procedure selects 49%

  10. Saturation in the estimated size of the proto-mitochondrial proteome with an increasing number of sequenced alpha-proteobacterial genomes

  11. Increasing the number of genomes leads to more accurate results: less false negatives, less false positives

  12. Proto-mitochondrial metabolism • Catabolism of fatty acids, glycerol and amino acids-Some pathways are not mitochondrial in present day mitochondria non-mitoch.. mitochondrial not in yeast/human

  13. The majority of the proto-mitochondrial proteome is not mitochondrial (anymore) 566 Yeast mitochondrial proteome: Proto-mitochondrial proteins in S.cerevisiae Eric Schon, Methods Cell Biol 2001 (manually curated) 35 303 59 293 10 Huh et al., Nature 2003 (green fluorescent genomics) 527 Proto-mitochondrial proteins in H.sapiens 755 Human mitochondrial proteome: Eric Schon, Methods Cell Biol 2001 113 508

  14. ~65% of the alpha-proteobacteria derived set is not mitochondrial. ~16% of the mitochondrial yeast proteins are of alpha-proteobacterial origin. From endosymbiont to organell, not only loss and gain of proteins but also “retargeting”: proteins loss re-targeting Ancestor Modern mitochondria gain t Gabaldon and Huynen, Science 2003

  15. Original rationales for the endosymbiosis • Aerobic (…no hydrogenosomal eukaryotes have been published yet….) • Catabolizing lipids, glycerol and amino-acids, incomplete TCA • Benefit for host wider than either H2, ATP or O2 consumption: iron-sulfur clusters and a variety of metabolic pathways

  16. From endosymbiont to organell: a turnover of protein in functional classes and an increase in specialization (middle columns: yeast, right columns: human) COG functional classification F: Nucleotide metabolism G: Carbohydrate metabolism M: Cell envelope biogenesis C: Energy conversion O: Protein turnover, chaperones J: Translation, Ribosomal structure Very low throughout: D: Cell division

  17. Gene loss in the evolution of mitochondria • Mitochondrial FtsZ in a chromophyte alga.Beech PL, Nheu T, Schultz T, Herbert S, Lithgow T, Gilson PR, McFadden GI Science 2000 • A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the alpha-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.

  18. Kiefel BRGilson PRBeech PL.Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ. Protist. 2004 Mar;155(1):105-15.Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

  19. Zooming in on one mitochondrial complex, NADH:ubiquinone oxidoreductase (Complex I), and using gene loss for function prediction • -Complex I deficiency is a severe hereditary disease (patients < 5 year) without therapy • -For 60% of the patients no mutation is found in known CI genes

  20. Tracing the evolution of Complex I from 14 subunits in the Bacteria to 46 subunits in the Mammals by comparative genome analysis Fungi: 37 Mammals: 46 Bacteria: 14 subunits Plants: 30 Algae: 30

  21. Issues in homology detection: that we do not detect sequence similarity does not mean that proteins are not homologous. Latest developments in homology detection: profile vs. profile searches The fungal ComplexI subunit NUVM is homologous to the Bovine subunit NB5M (B15), This homology can only be detected by profile vs. profile searches

  22. Beyond Blastology, Cogoly: Phylogenies for orthology prediction The Complex I assembly protein CI30 has been duplicated in the Fungi. This can explain the presence of a CIA30-homolog in Complex I-less S.pombe

  23. Mining the proto-mitochondrion for new Complex I proteins } Metazoa } Fungi } Alpha-proteobacteria A methyltransferase derived from the alpha-proteobacterial ancestor of the mitochondria has a phylogenetic distribution identical to Complex I proteins, suggesting involvement of this protein in Complex I Gabaldon and Huynen, Bioinformatics 2005

  24. Function prediction of Complex I proteins NUEM Eukaryotes CIA30 Cyanobacteria CIA30 is inserted in NueM in Cyanobacteria, suggesting an interaction between CIA30 and NueM in the eukaryotes as well.

  25. Plants,algae Mammals Insects Nematode Fungi Fish Distribution of Complex I subunits among model species Experimentally verified Homolog present in genome, predicted gene Homolog present in genome, not predicted Absent from genome

  26. Reconstructing Complex I evolution by mapping the variation onto a phylogenetic tree. After an initial “surge” in complexity (from 14 to 35 subunits in early eukaryotic evolution) new subunits have been gradually added and incidentally lost. Complex I loss is not always “complete”, S.cerevisiae and S.pombe have retained 1 and 3 proteins respectively Six of the eukaryotic Complex I proteins have been “recruited” from the alpha-proteobacteria

  27. Tinkering in the eukaryotic evolution of Complex I: new subunits have been added “all over” the complex Gabaldon et al. (2005) J. Mol. Biol. See also Science (2005) 308, 167

  28. Deconstructing protein complexes by tracing their evolution: The phylogenetic distribution of Complex I subunits suggests the presence of submodules and the functions of the individual proteins In eukaryotes evolution appears less “sub-modular” than in prokaryotes T. Friedrich’s model Huynen et al., FEBS lett. 2005

  29. How about the origin of the peroxisome? Like mitochondria an organell involved in oxidative metabolism, but without a genome. Multiplication by fission has suggested an endosymbiotic origin.

  30. Scenarios for the origin of the peroxisome and mitochondria A) Independent endosymbiotic origins B) A single origin followed by fission C) Retargeting of mitochondrial proteins Time

  31. The yeast and rat peroxisomal proteomes contain a large fraction of proteins of alpha-proteobacterial origin (18-19%), besides a large fraction of proteins of eukaryotic origin (37-38%) Alpha-proteobact unresolved Yeast (61 proteins) Rat (50 proteins)

  32. Most (90%) of the peroxisomal proteins of alpha-proteobacterial origin have paralogs in the mitochondria

  33. The retargeting of mitochondrial proteins to the peroxisome has continued in recent evolution } Peroxisomal Mitoch. Signal peptide cleavage site Within the Cit1/2 protein family all proteins have a mitochondrial location (exp. data and/or predictions), expect Cit2p which is peroxisomal, and has lost the cleavage site (YS)

  34. Tracing the evolution of the peroxisome: a continuous retargeting of proteins from various origins. (yellow = eukaryotic, green = alphaprot, red = actinomyc., blue = cyanobact.) The “ancestral peroxisome” was likely involved in b-oxidation, harboring Catalase to detoxify hydrogen peroxide.

  35. Scenarios for the origin of the peroxisome and mitochondria A) Independent endosymbiotic origins B) A single origin followed by fission C) Retargeting of mitochondrial proteins Time

  36. Studying evolution of organellar proteomes is not only interesting in itself, it also provides us with clues about the functions of proteins

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