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...
Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5. Screening of this gene on genomic DNA revealed a mutation that interferes with correct splicing and results in the skipping of exon 2. Exon skipping was confirmed on the mRNA level. The mutation in this accessory subunit causes reduced activity and disturbed assembly of complex I. Furthermore, the mutation is associated with a mitochondrial depolarization. The expression and activity of complex I and the depolarization was (partially) rescued with a baculovirus system expressing the NDUFA2 gene.
Diffuse brain infiltration by glioma cells causes detrimental disease progression, however its multicellular coordination is poorly understood. We here show that glioma cells infiltrate brain collectively, as multicellular networks. Contacts between moving glioma cells were adaptive epithelial-like or filamentous junctions stabilized by N-cadherin, β-catenin and p120-catenin, which underwent kinetic turnover , transmitted intercellular calcium transients and mediated directional persistence. Downregulation of p120-catenin compromised cell-cell interaction and communication, disrupted collective networks, and both the cadherin and RhoA binding domains of p120-catenin were required for network formation and migration. Deregulating p120-catenin further prevented diffuse glioma cell infiltration of the mouse brain with marginalized Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Intracellular chemical reactions generally constitute reaction-diffusion systems located inside nanostructured compartments like the cytosol, nucleus, endoplasmic reticulum, Golgi, and mitochondrion. Understanding the properties of such systems requires quantitative information about solute diffusion. Here we present a novel approach that allows determination of the solvent-dependent solute diffusion constant (D solvent ) inside cell compartments with an experimentally quantifiable nanostructure. In essence, our method consists of the matching of synthetic fluorescence recovery after photobleaching (FRAP) curves, generated by a mathematical model with a realistic nanostructure, and experimental FRAP data. As a proof of principle, we assessed D solvent of a monomeric fluorescent protein (AcGFP1) and its tandem fusion (AcGFP1 2 ) in the mitochondrial matrix of HEK293 cells. Our results demonstrate that diffusion of both proteins is substantially slowed by barriers in the mitochondrial matrix (cristae), suggesting that cells can control the dynamics of biochemical reactions in this compartment by modifying its nanostructure. molecular dynamics | quantitative random-walk model | systems biology A major challenge facing biochemistry is to understand the dynamics of chemical reactions within inhomogeneous cell compartments like the cytosol, nucleus, endoplasmic reticulum (ER), Golgi, and mitochondrion (1). In general, intracompartment reactions involve the conversion of (im)mobile substrates by (im)mobile enzymes into (im)mobile products and therefore constitute reaction-diffusion systems. Obviously, gaining insight into the behavior of such systems requires quantitative information about solute diffusion. The latter depends on solvent and solute properties, the dimensions and shape of the compartment, and the internal structure of the compartment (2-6).A widely used strategy to investigate solute diffusion involves expressing a fluorescent tracer protein (FP) in the compartment of interest. Next, FP mobility is measured using FCS (fluorescence correlation spectroscopy) or FRAP (fluorescence recovery after photobleaching). This is then followed by curve fitting and/ or mathematical modeling of the experimental data to obtain the diffusion constant of the FP (7-16). However, these analysis methods generally do not include realistic (i.e., experimentally determined) information concerning the spatial dimensions and nanostructure of the compartment. Moreover, the temporal scale of most FRAP models does not quantitatively match with that of FRAP experiments. Therefore it was already recognized some time ago (8-17) that the above approaches will only yield an "apparent" (biased) value for the diffusion constant (D app ) of a given FP, which represents an underestimation of the "real" (i.e., purely solvent-dependent) diffusion constant (D solvent ).In this study we present a strategy to determine D solvent inside cell compartments with an experimentally accessible nanostructure. Our method consists of matching synthetic FRA...
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