Within living cells, mitochondria are considered relevant sources of reactive oxygen species (ROS) and are exposed to reactive nitrogen species (RNS). During the last decade, accumulating evidence suggests that mitochondrial (dys)function, ROS/RNS levels, and aberrations in mitochondrial morphology are interconnected, albeit in a cell- and context-dependent manner. Here it is hypothesized that ROS and RNS are involved in the short-term regulation of mitochondrial morphology and function via non-transcriptional pathways. We review the evidence for such a mechanism and propose that it allows homeostatic control of mitochondrial function and morphology by redox signaling.

Background: Antibodies directed to proteins containing the non-standard amino acid citrulline, are extremely specific for rheumatoid arthritis (RA). Peptidylcitrulline can be generated by post-translational conversion of arginine residues. This process, citrullination, is catalysed by a group of calcium dependent peptidylarginine deiminase (PAD) enzymes. Objective: To investigate the expression and activity of four isotypes of PAD in peripheral blood and synovial fluid cells of patients with RA. Results: The data presented here show that citrullination of proteins by PAD enzymes is a process regulated at three levels: transcription-in peripheral blood PAD2 and PAD4 mRNAs are expressed predominantly in monocytes; PAD4 mRNA is not detectable in macrophages, translation-translation of PAD2 mRNA is subject to differentiation stage-specific regulation by its 39 UTR, and activation-the PAD proteins are only activated when sufficient Ca 2+ is available. Such high Ca 2+ concentrations are normally not present in living cells. In macrophages, which are abundant in the inflamed RA synovium, vimentin is specifically citrullinated after Ca 2+ influx. Conclusion: PAD2 and PAD4 are the most likely candidate PAD isotypes for the citrullination of synovial proteins in RA. Our results indicate that citrullinated vimentin is a candidate autoantigen in RA.

Virtually every mammalian cell contains mitochondria. These double-membrane organelles continuously change shape and position and contain the complete metabolic machinery for the oxidative conversion of pyruvate, fatty acids, and amino acids into ATP. Mitochondria are crucially involved in cellular Ca 2+ and redox homeostasis and apoptosis induction. Maintenance of mitochondrial function and integrity requires an inside-negative potential difference across the mitochondrial inner membrane. This potential is sustained by the electron-transport chain (ETC). NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest protein complex of the ETC, couples the oxidation of NADH to the reduction of ubiquinone. During this process, electrons can escape from CI and react with ambient oxygen to produce superoxide and derived reactive oxygen species (ROS). Depending on the balance between their production and removal by antioxidant systems, ROS may function as signaling molecules or induce damage to a variety of biomolecules or both. The latter ultimately leads to a loss of mitochondrial and cellular function and integrity. In this review, we discuss (a) the role of CI in mitochondrial functioning; (b) the composition, structure, and biogenesis of CI; (c) regulation of CI function; (d) the role of CI in ROS generation; and (e) adaptive responses to CI deficiency.

Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the[Keywords: Mitochondria; oxidative phosphorylation; complex I; NADH:ubiquinone oxidoreductase; Ecsit; NDUFAF1] Supplemental material is available at http://www.genesdev.org.

Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.Mitochondrial ATP is produced by the oxidative phosphorylation (OXPHOS) 3 system. This system consists of five complexes, composed of at least 75 nuclear DNA-encoded and 13 mitochondrial DNA (mtDNA)-encoded proteins, and is a prominent example of coordinated assembly. The first four OXPHOS complexes (CI-CIV) constitute the respiratory chain, which transfers electrons from substrates NADH (at CI) and FADH 2 (at CII) to the final electron acceptor molecular oxygen (CIV). Energy released by this electron transport is used to drive proton translocation across the mitochondrial inner membrane at CI, CIII, and CIV. The resulting proton gradient is used to drive the conversion of ADP and inorganic phosphate into ATP by complex V (1). CI (NADH:ubiquinone oxidoreductase complex; EC 1.6.5.3) constitutes the largest and least understood of the OXPHOS complexes (2, 3). Electron microscopy revealed that CI has an L-shaped structure that consists of a hydrophobic arm embedded in the lipid bilayer of the mitochondrial inner membrane and a hydrophylic peripheral arm exposed to the mitochondrial matrix (4). Using chaotropic salts and the detergent N,N-dimethyldodecylamine N-oxide, CI can be fractionated into several fragments (5, 6) that together encompass 45 distinct subunits in bovine CI (7,8). The recent appearance of the first crystal structure of the hydrophilic domain of CI in Thermus thermophilus is an example of the increasing insight that is gained in this area of research (9).In contrast, the many steps involved in the assembly of these 45 subunits still remain puzzling. Studies in the fungus Neurospora crassa demonstrated that the membrane and peripheral...