Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Mitochondrially generated ROS increase with age and are a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS, which may impair normal complex activity. This study of heart mitochondria from young, middle-aged, and old mice reveals that there is an age-related decline in complex I and V activity that correlates with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of the aged mouse heart.     

Cardiac mitochondria in heart failure: normal cardiolipin profile and increased threonine phosphorylation of complex IV

Mitochondrial dysfunction is a major contributor in heart failure (HF). We investigated whether the decrease in respirasome organization reported by us previously in cardiac mitochondria in HF is due to changes in the phospholipids of the mitochondrial inner membrane or modifications of the subunits of the electron transport chain (ETC) complexes. The contents of the main phospholipid species, including cardiolipin, as well as the molecular species of cardiolipin were unchanged in cardiac mitochondria in HF. Oxidized cardiolipin molecular species were not observed. In heart mitochondria isolated from HF, complex IV not incorporated into respirasomes exhibits increased threonine phosphorylation. Since HF is associated with increased adrenergic drive to cardiomyocytes, this increased protein phosphorylation might be explained by the involvement of cAMP-activated protein kinase. Does the preservation of cAMP-induced phosphorylation changes of mitochondrial proteins or the addition of exogenous cAMP have similar effects on oxidative phosphorylation? The usage of phosphatase inhibitors revealed a specific decrease in complex I-supported respiration with glutamate. In saponin-permeabilized cardiac fibers, pre-incubation with cAMP decreases oxidative phosphorylation due to a defect localized at complex IV of the ETC inter alia. We propose that phosphorylation of specific complex IV subunits decreases oxidative phosphorylation either by limiting the incorporation of complex IV in supercomplexes or by decreasing supercomplex stability.     

Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.     

Comparative genomics and structural biology of the molecular innovations of eukaryotes

Eukaryotes encode numerous proteins that either have no detectable homologs in prokaryotes or have only distant homologs. These molecular innovations of eukaryotes may be classified into three categories: proteins and domains inherited from prokaryotic precursors without drastic changes in biochemical function, but often recruited for novel roles in eukaryotes; new superfamilies or distinct biochemical functions emerging within pre-existing protein folds; and domains with genuinely new folds, apparently 'invented' at the outset of eukaryotic evolution. Most new folds emerging in eukaryotes are either alpha-helical or stabilized by metal chelation. Comparative genomics analyses point to an early phase of rapid evolution, and dramatic changes between the origin of the eukaryotic cell and the advent of the last common ancestor of extant eukaryotes. Extensive duplication of numerous genes, with subsequent functional diversification, is a distinctive feature of this turbulent era. Evolutionary analysis of ancient eukaryotic proteins is generally compatible with a two-symbiont scenario for eukaryotic origin, involving an alpha-proteobacterium (the ancestor of the mitochondria) and an archaeon, as well as key contributions from their selfish elements.     

Accelerated evolution of the electron transport chain in anthropoid primates

Mitochondria are both the power plant of the cell and a central integrator of signals that govern the lifespan, replication and death of the cell. Perhaps as a consequence, genes that encode components of the mitochondrial electron transport chain (ETC) are generally conserved. Therefore, it is surprising that many of these genes in anthropoid primates (New World monkeys, Old World monkeys and apes, including humans) have been major targets of darwinian positive selection. Sequence comparisons have provided evidence that marked increases of non-synonymous substitution rates occurred in anthropoid ETC genes that encode subunits of Complex III and IV, and the electron carrier molecule cytochrome c (CYC). Two important questions are: (i) how has evolution altered ETC function? and; (ii) how might functional changes in the ETC be linked to evolution of an expanded neocortical brain?     

Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I)

The increasing availability of sequenced genomes enables the reconstruction of the evolutionary history of large protein complexes. Here, we trace the evolution of NADH:ubiquinone oxidoreductase (Complex I), which has increased in size, by so-called supernumary subunits, from 14 subunits in the bacteria to 30 in the plants and algae, 37 in the fungi and 46 in the mammals. Using a combination of pair-wise and profile-based sequence comparisons at the levels of proteins and the DNA of the sequenced eukaryotic genomes, combined with phylogenetic analyses to establish orthology relationships, we were able to (1) trace the origin of six of the supernumerary subunits to the alpha-proteobacterial ancestor of the mitochondria, (2) detect previously unidentified homology relations between subunits from fungi and mammals, (3) detect previously unidentified subunits in the genomes of several species and (4) document several cases of gene duplications among supernumerary subunits in the eukaryotes. One of these, a duplication of N7BM (B17.2), is particularly interesting as it has been lost from genomes that have also lost Complex I proteins, making it a candidate for a Complex I interacting protein. A parsimonious reconstruction of eukaryotic Complex I evolution shows an initial increase in size that predates the separation of plants, fungi and metazoa, followed by a gradual adding and incidental losses of subunits in the various evolutionary lineages. This evolutionary scenario is in contrast to that for Complex I in the prokaryotes, for which the combination of several separate, and previously independently functioning modules into a single complex has been proposed.     

Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes

Chaperonins are oligomeric protein-folding complexes which are divided into two distantly related structural classes. Group I chaperonins (called GroEL/cpn60/hsp60) are found in bacteria and eukaryotic organelles, while group II chaperonins are present in archaea and the cytoplasm of eukaryotes (called CCT/TriC). While archaea possess one to three chaperonin subunit-encoding genes, eight distinct CCT gene families (paralogs) have been characterized in eukaryotes. We are interested in determining when during eukaryotic evolution the multiple gene duplications producing the CCT subunits occurred. We describe the sequence and phylogenetic analysis of five CCT genes from TRICHOMONAS: vaginalis and seven from GIARDIA: lamblia, representatives of amitochondriate protist lineages thought to have diverged early from other eukaryotes. Our data show that the gene duplications producing the eight CCT paralogs took place prior to the organismal divergence of TRICHOMONAS: and GIARDIA: from other eukaryotes. Thus, these divergent protists likely possess completely hetero-oligomeric CCT complexes like those in yeast and mammalian cells. No close phylogenetic relationship between the archaeal chaperonins and specific CCT subunits was observed, suggesting that none of the CCT gene duplications predate the divergence of archaea and eukaryotes. The duplications producing the CCTdelta and CCTepsilon subunits, as well as CCTalpha, CCTbeta, and CCTeta, are the most recent in the CCT gene family. Our analyses show significant differences in the rates of evolution of archaeal chaperonins compared with the eukaryotic CCTs, as well as among the different CCT subunits themselves. We discuss these results in light of current views on the origin, evolution, and function of CCT complexes.     

Mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase and succinate dehydrogenase complexes contain plant specific subunits

Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial samples and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their individual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9–10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis. Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.     

Initiation of Electron Transport Chain Activity in the Embryonic Heart Coincides with the Activation of Mitochondrial Complex 1 and the Formation of Supercomplexes

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.     

Cytonuclear coevolution: the genomics of cooperation

Without mitochondria we would be in big trouble, and there would be a global biological energy crisis if it were not for chloroplasts. Fortunately, genomic evolution over the past two billion years has ensured that the functions of these key organelles are with us to stay. Whole-genome analyses have not only proven that mitochondria and chloroplasts are descended from formerly free-living bacteria, but have also shown that it is difficult to define eukaryotes without reference to the fusion and coevolution of host and endosymbiont genomes. Here, we review how the macro- and microevolutionary insights that follow from the genomics of cytonuclear interactions are uniting molecular evolution, structural proteomics, population genetics and problems in aging and disease. Our goals are to clarify the coevolutionary events that have governed nuclear and organelle evolution, and to encourage further critical analyses of these interactions as problems in the study of co-adapted gene complexes.     

Age-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes

Age-associated mitochondrial dysfunction is a major source of reactive oxygen species (ROS) and oxidative modification to proteins. Mitochondrial electron transport chain (ETC) complexes I and III are the sites of ROS production and we hypothesize that proteins of the ETC complexes are primary targets of ROS-mediated modification which impairs their structure and function. The pectoralis, primarily an aerobic red muscle, and quadriceps, primarily an anaerobic white muscle, have different rates of respiration and oxygen-carrying capacity, and hence, different rates of ROS production. This raises the question of whether these muscles exhibit different levels of oxidative protein modification. Our studies reveal that the pectoralis shows a dramatic age-related decline in almost all complex activities that correlates with increased oxidative modification. Similar complex proteins were modified in the quadriceps, at a significantly lower level with less change in enzyme and ETC coupling function. We postulate that mitochondrial ROS causes damage to specific ETC subunits which increases with age and leads to further mitochondrial dysfunction. We conclude that physiological characteristics of the pectoralis vs quadriceps may play a role in age-associated rate of mitochondrial dysfunction and in the decline in tissue function.     

Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes

The coherence of mitochondrial biogenesis relies on spatiotemporally coordinated associations of 800-1000 proteins mostly encoded in the nuclear genome. We report the development of new quantitative analyses to assess the role of local protein translation in the construction of molecular complexes. We used real-time PCR to determine the cellular location of 112 mRNAs involved in seven mitochondrial complexes. Five typical cases were examined by an improved FISH protocol. The proteins produced in the vicinity of mitochondria (MLR proteins) were, almost exclusively, of prokaryotic origin and are key elements of the core construction of the molecular complexes; the accessory proteins were translated on free cytoplasmic polysomes. These two classes of proteins correspond, at least as far as intermembrane space (IMS) proteins are concerned, to two different import pathways. Import of MLR proteins involves both TOM and TIM23 complexes whereas non-MLR proteins only interact with the TOM complex. Site-specific translation loci, both outside and inside mitochondria, may coordinate the construction of molecular complexes composed of both nuclearly and mitochondrially encoded subunits.     

Biogenesis of beta-barrel membrane proteins of mitochondria

beta-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of beta-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of beta-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both beta-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane beta-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial beta-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.     

Mitochondrial morphology and dynamics in <Emphasis Type="Italic">Triticum aestivum</Emphasis> roots in response to rotenone and antimycin A

Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.     

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.     

Evolution of DNA Replication Protein Complexes in Eukaryotes and Archaea

Background

The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best.

Methodology/Principal Findings

While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex—all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea.

Conclusion/Significance

This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.     

Effects of aging on activities of mitochondrial electron transport chain complexes and oxidative damage in rat heart

Mitochondrial dysfunction and accumulation of oxidative damage have been implicated to be the major factors of aging. However, data on age-related changes in activities of mitochondrial electron transport chain (ETC) complexes remain controversial and molecular mechanisms responsible for ETC dysfunction are still largely unknown. In this study, we examined the effect of aging on activities of ETC complexes and oxidative damage to proteins and lipids in cardiac mitochondria from adult (6-month-old), old (15-month-old) and senescent (26-month-old) rats. ETC complexes I-IV displayed different extent of inhibition with age. The most significant decline occurred in complex IV activity, whereas complex II activity was unchanged in old rats and was only slightly reduced in senescent rats. Compared to adult, old and senescent rat hearts had significantly higher levels of malondialdehyde, 4-hydroxynonenal (HNE) and dityrosine, while thiol group content was reduced. Despite marked increase in HNE content with age (25 and 76 % for 15- and 26-month-old rats, respectively) Western blot analysis revealed only few HNE-protein adducts. The present study suggests that non-uniform decline in activities of ETC complexes is due, at least in part, to mitochondrial oxidative damage; however, lipid peroxidation products appear to have a limited impact on enzyme functions.     

Age-related decline in mitochondrial bioenergetics: Does supercomplex destabilization determine lower oxidative capacity and higher superoxide production?

Mitochondrial decay plays a central role in the aging process. Although certainly multifactorial in nature, defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Primarily, mitochondrial dysfunction affects the aging animal by limiting bioenergetic reserve capacity and/or increasing oxidative stress via enhanced electron leakage from the ETC. Even though the important aging characteristics of mitochondrial decay are known, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not completely understood. This review focuses on the potential role(s) that age-associated destabilization of the macromolecular organization of the ETC (i.e. supercomplexes) may be important for development of the mitochondrial aging phenotype, particularly in post-mitotic tissues.