NOVA: a software to analyze complexome profiling data.

SUMMARY: We introduce nova, a software for the analysis of complexome profiling data. nova supports the investigation of the composition of complexes, cluster analysis of the experimental data, visual inspection and comparison of experiments and many other features. AVAILABILITY AND IMPLEMENTATION: nova is licensed under the Artistic License 2.0. It is freely available at http://www.bioinformatik.uni-frankfurt.de. nova requires at least Java 7 and runs under Linux, Microsoft Windows and Mac OS. CONTACT: ina.koch@bioinformatik.uni-frankfurt.de.

Superoxide generation by complex III: from mechanistic rationales to functional consequences.

Apart from complex I (NADH:ubiquinone oxidoreductase) the mitochondrial cytochrome bc1 complex (complex III; ubiquinol:cytochrome c oxidoreductase) has been identified as the main producer of superoxide and derived reactive oxygen species (ROS) within the mitochondrial respiratory chain. Mitochondrial ROS are generally linked to oxidative stress, aging and other pathophysiological settings like in neurodegenerative diseases. However, ROS produced at the ubiquinol oxidation center (center P, Qo site) of complex III seem to have additional physiological functions as signaling molecules during cellular processes like the adaptation to hypoxia. The molecular mechanism of superoxide production that is mechanistically linked to the electron bifurcation during ubiquinol oxidation is still a matter of debate. Some insight comes from extensive kinetic studies with mutated complexes from yeast and bacterial cytochrome bc1 complexes. This review is intended to bridge the gap between those mechanistic studies and investigations on complex III ROS in cellular signal transduction and highlights factors that impact superoxide generation. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

A common mechanism links differently acting complex II inhibitors to cardioprotection: modulation of mitochondrial reactive oxygen species production.

In this study, we have analyzed the effect of different cardioprotective complex II inhibitors on the mitochondrial production of reactive oxygen species (ROS) because ROS seem to be essential for signaling during preconditioning to prevent ischemia/reperfusion injury. Despite different binding sites and concentrations required for half-maximal inhibition-ranging from nanomolar for the Q site inhibitor atpenin A5 to millimolar for the succinate analog malonate-all inhibitors modulated ROS production in the same ambivalent fashion: they promoted the generation of superoxide at the Q(o) site of complex III under conditions of "oxidant-induced reduction" but attenuated ROS generated at complex I due to reverse electron transfer. All inhibitors showed these ambivalent effects independent of the presence of K(+). These findings suggest a direct modulation of mitochondrial ROS generation during cardioprotection via complex II inhibition and question the recently proposed role of complex II as a regulatory component of the putative mitochondrial K(ATP) channel.

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

Generator-specific targets of mitochondrial reactive oxygen species.

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.

Quantification of protein complexes by blue native electrophoresis.

Blue native electrophoresis (BNE) is a long established method for the analysis of native protein complexes. Applications of BNE range from investigating subunit composition, stoichiometry, and assembly of single protein complexes to profiling of whole complexomes. BNE is an indispensible tool to diagnostically analyze cells and tissues from patients with mitochondrial disorders or model organisms. Since functional proteomic studies often require quantification of protein complexes, we describe here different quantification methods subsequent to protein complex separation by BNE.

Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.

Macromolecular complexes are essential players in numerous biological processes. They are often large, dynamic, and rather labile; approaches to study them are scarce. Covering masses up to ∼30 MDa, we separated the native complexome of rat heart mitochondria by blue-native and large-pore blue-native gel electrophoresis to analyze its constituents by mass spectrometry. Similarities in migration patterns allowed hierarchical clustering into interaction profiles representing a comprehensive analysis of soluble and membrane-bound complexes of an entire organelle. The power of this bottom-up approach was validated with well-characterized mitochondrial multiprotein complexes. TMEM126B was found to comigrate with known assembly factors of mitochondrial complex I, namely CIA30, Ecsit, and Acad9. We propose terming this complex mitochondrial complex I assembly (MCIA) complex. Furthermore, we demonstrate that TMEM126B is required for assembly of complex I. In summary, complexome profiling is a powerful and unbiased technique allowing the identification of previously overlooked components of large multiprotein complexes.