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PubMed1981

Phospholipid requirements for the reconstitution of complex-III vesicles exhibiting controlled electron transport.

Phospholipid requirements for the reconstitution of Complex-III vesicles exhibiting respiratory control (electron-transport control) were studied. Vesicles prepared from pure phosphatidylethanolamine gave maximal control ratios. Phosphatidylcholine alone did not support respiratory control, although these vesicles were capable of maintaining stable K+-diffusion gradients. Apparently Complex III cannot insert into a bilayer of phosphatidylcholine. Formation of mixed phosphatidylcholine/phosphatidylethanolamine (6:1, w/w) vesicles was sufficient, however, to allow Complex-III insertion and to restore respiratory control. Mixtures of acidic phospholipids with either phosphatidylethanolamine or phosphatidylcholine did not improve respiratory control over that obtained with pure phosphatidylethanolamine. Phosphatidylethanolamine from bovine heart mitochondria, soya beans or Escherichia coli was equally effective in reconstituting respiratory control, suggesting that the specificity is referable to the head group and not to the fatty-acid moiety.

Animals↗
PubMed1985

Thermodynamic control of electron flux through mitochondrial cytochrome bc1 complex.

The redox states of exogenously added ubiquinone-2 and cytochrome c, and the protonmotive force (delta p) of rat liver mitochondria were measured as the respiration rate was titrated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone. The force ratio delta Eh/delta p across the bc1 complex was close to 1:1 in State 4, indicating an H+/e- stoichiometry of 1:1 for the cytochrome bc1 complex, excluding protons moved by pool ubiquinone. Assuming a constant stoichiometry the rate of electron transport increased linearly with the disequilibrium (delta Eh - delta p) across the complex.

Animals↗
PubMed2002

Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. In order to address the question of a putative role of mitochondrial dysfunction in the pathogenesis of ALS, we studied the mitochondrial DNA (mtDNA) and mitochondrial respiratory chain enzyme activities in spinal cords of ALS patients and in control subjects without neuropathologic abnormalities. Using a "double PCR and digestion" technique to estimate the levels of randomly distributed point mutations in two small regions of the mtDNA, we found significantly higher levels of mutant mtDNA in the spinal cord of ALS patients compared to controls. No large-scale rearrangements were found, but the amount of mtDNA, measured by Southern blot, was significantly lower in the ALS samples. This reduction correlated well with a decrease of citrate synthase (CS) activity, a mitochondrial marker, as were the activities of respiratory chain complexes I + III, II + III, and IV, suggesting a loss of mitochondria in ALS spinal cords.

Adult↗
PubMed2001

Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex.

Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett., 439, 197-202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem. 267, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.

Antigen-Antibody Complex↗
PubMed1999

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.

Copper↗
PubMed1996

Chromosomal localization of the proteasome Z subunit gene reveals an ancient chromosomal duplication involving the major histocompatibility complex.

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon gamma, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC.

Amino Acid Sequence↗
PubMed1996

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.

Amino Acid Sequence↗
PubMed1996

Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease.

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.

Cysteine Endopeptidases↗
PubMed1996

Inhibitor probes of the quinone binding sites of mammalian complex II and Escherichia coli fumarate reductase.

The structural and catalytic properties of beef heart succinate dehydrogenase (succinate-ubiquinone oxidoreductase, complex II) and Escherichia coli fumarate reductase are remarkably similar. One exception is that whereas electron exchange between the mammalian enzyme and its quinone pool is inhibited by thenoyltrifluoroacetone and carboxanilides, the enzyme from E. coli is not sensitive to these inhibitors. The lack of good inhibitors has seriously hampered the elucidation of the mechanism of quinone oxidation/reduction in the E. coli enzyme. We have previously reported (Tan, A. K., Ramsay, R. R., Singer, T. P., and Miyoshi, H. (1993) J. Biol. Chem. 268, 19328-19333) that 2-alkyl-4,6-dinitrophenols inhibit mammalian complexes I, II, and III, but with different potencies and kinetic characteristics. Based on these studies we have selected a series of 2-alkyl-4,6-dinitrophenols which proved to be very effective noncompetitive inhibitors of mammalian complex II, particularly when acting in the direction of quinone reduction, the physiological event. These compounds turned out to be even more potent inhibitors of E. coli fumarate reductase, particularly when acting in the direction of quinol oxidation, again, the physiological event. Kinetic analysis revealed that with both enzymes 2 inhibitor binding sites seem to be involved in the oxidation of succinate by quinone, but one seems to be functioning when fumarate is reduced by external quinol. Since the E. coli enzyme can be modified by site-directed mutagenesis, these studies were extended to four mutants of fumarate reductase, impaired by single amino acid substitutions at either of the putative quinone binding sites (QA or QB) of the enzyme. The results were analyzed in terms of the model of these dual sites of quinone binding in fumarate reductase, as well as the nature of the substituent in the 2-position of the dinitrophenol inhibitors.

Animals↗
PubMed2000

Glucose-regulated turnover of mRNA and the influence of poly(A) tail length on half-life.

Glucose repression in Saccharomyces cerevisiae can now be seen to operate at two levels: regulation of transcription of certain genes and control of the half-life of the corresponding mRNAs (Scheffler, I. E., de la Cruz, B. J., and Prieto, S. (1998) Int. J. Biochem Cell Biol. 30, 1175-1193). For example, the steady state levels of SDH2 mRNA and SUC2 mRNA are significantly determined by their differential rates of turnover. A current model for the mechanism of mRNA turnover includes three distinct steps: a rate-limiting deadenylation, removal of the 5'-7-methyl-G (decapping), and 5'-3' exonuclease digestion. We have investigated the same three reactions during glucose-induced degradation of these transcripts. Our results indicate that while decapping (by Dcp1p) and 5'-3' exonuclease digestion (by Xrn1p) are obligatory steps for the rapid degradation of these mRNAs, the dependence on deadenylation is more complicated. At steady state in glycerol these transcripts have very short poly(A) tails but are nevertheless very stable; the addition of glucose causes immediate decapping and degradation without further deadenylation; in contrast, newly made SUC2 mRNA (after a shift from glucose to glycerol) has significantly longer poly(A) tails, and such transcripts are not rapidly degraded upon addition of glucose. A constitutive deadenylation reaction that is independent of the carbon source eventually makes the stability of these transcripts very sensitive to glucose. These results are interpreted in terms of a working hypothesis proposing a competition between translational initiation and decapping influenced by the carbon source. The presence of a long poly(A) tail may also affect this competition in favor of translational initiation and mRNA stabilization.

Base Sequence↗
PubMed2002

Neurodegeneration in methylmalonic aciduria involves inhibition of complex II and the tricarboxylic acid cycle, and synergistically acting excitotoxicity.

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonate (MMA) and alternative metabolites. There is growing evidence for basal ganglia degeneration in these patients. The pathomechanisms involved are still unknown, a contribution of toxic organic acids, in particular MMA, has been suggested. Here we report that MMA induces neuronal damage in cultures of embryonic rat striatal cells at a concentration range encountered in affected patients. MMA-induced cell damage was reduced by ionotropic glutamate receptor antagonists, antioxidants, and succinate. These results suggest the involvement of secondary excitotoxic mechanisms in MMA-induced cell damage. MMA has been implicated in inhibition of respiratory chain complex II. However, MMA failed to inhibit complex II activity in submitochondrial particles from bovine heart. To unravel the mechanism underlying neuronal MMA toxicity, we investigated the formation of intracellular metabolites in MMA-loaded striatal neurons. There was a time-dependent intracellular increase in malonate, an inhibitor of complex II, and 2-methylcitrate, a compound with multiple inhibitory effects on the tricarboxylic acid cycle, suggesting their putative implication in MMA neurotoxicity. We propose that neuropathogenesis of methylmalonic aciduria may involve an inhibition of complex II and the tricarboxylic acid cycle by accumulating toxic organic acids, and synergistic secondary excitotoxic mechanisms.

Animals↗
PubMed2003

Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans.

Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.

Electron Transport Chain Complex Proteins↗
PubMed2004

Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes.

Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes. Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway.

Cells, Cultured↗
PubMed1982

Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells.

Previous work has shown that injury of neoplastic cells by cytotoxic macrophages (CM) in cell culture is accompanied by inhibition of mitochondrial respiration. We have investigated the nature of this inhibition by studying mitochondrial respiration in CM-injured leukemia L1210 cells permeabilized with digitonin. CM-induced injury affects the mitochondrial respiratory chain proper. Complex I (NADH-coenzyme Q reductase) and complex II (succinate-coenzyme Q reductase) are markedly inhibited. In addition a minor inhibition of cytochrome oxidase was found. Electron transport from alpha-glycerophosphate through the respiratory chain to oxygen is unaffected and permeabilized CM-injured L1210 cells oxidizing this substrate exhibit acceptor control. However, glycerophosphate shuttle activity was found not to occur within CM-injured or uninjured L1210 cells in culture hence, alpha-glycerophosphate is apparently unavailable for mitochondrial oxidation in the intact cell. It is concluded that the failure of respiration of intact neoplastic cells injured by CM is caused by the nearly complete inhibition of complexes I and II of the mitochondrial electron transport chain. The time courses of CM-induced electron transport inhibition and arrest of L1210 cell division are examined and the possible relationship between these phenomena is discussed.

Animals↗
PubMed2000

Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes.

Interferon (IFN)-gamma-induced cells express the proteasome subunits low molecular weight protein (LMP)2, LMP7, and MECL-1 (multicatalytic endopeptidase complex-like 1), leading to the formation of immunoproteasomes. Although these subunits are thought to optimize MHC class I antigen processing, the extent of their role and the mechanistic aspects involved remain unclear. Herein, we study the proteolytic generation of an human histocompatibility leukocyte antigen (HLA)-Aw68-restricted hepatitis B virus core antigen (HBcAg) cytotoxic T lymphocyte (CTL) epitope that is recognized by peripheral blood lymphocytes from patients with acute self-limited but not chronic hepatitis B virus (HBV). Immunological data suggest that IFN-gamma-induced rather than uninduced HeLa cells process and present the HBV CTL epitope upon infection with HBcAg-expressing vaccinia viruses. Analyses of 20S proteasome digests of synthetic polypeptides covering the antigenic HBcAg peptide demonstrate that only immunoproteasomes efficiently perform the cleavages needed for the liberation of this HBV CTL epitope. Although the concerted presence of the three immunosubunits appears essential, we find that both catalytically active LMP7 and inactive LMP7 T1A support CTL epitope generation. We conclude that LMP7 influences the structural features of 20S proteasomes, thereby enhancing the activity of the LMP2 and MECL-1 catalytic sites, which provide cleavage specificity. Thus, LMP7 incorporation is of greater functional importance for the generation of an HBV CTL epitope than cleavage specificity.

Amino Acid Sequence↗
PubMed1996

Phylogenetic approaches to the identification and characterization of protein families and superfamilies.

With the advent of megabase genome sequencing, the need for computational analyses increases exponentially. Sequencing errors must be corrected, encoded proteins must be identified, functions must be assigned to these proteins, and distant phylogenetic relationships must be recognized in order to maximize the yield of information obtainable from genome sequencing projects. Both the computer and the human brain have their limitations, but using them in combination, the biologist can vastly extend his or her analytic capabilities. Computer techniques can be used to estimate protein structure, function, biogenesis, and evolution. In this review, the application of available computer programs to several protein families, particularly transport, receptor, and transcriptional regulatory protein families, illustrate our current capabilities and limitations. Although some multidomain protein families are evolutionarily homogeneous, others have mosaic origins. Evidence concerning the nature and frequency of occurrence of domain shuffling, splicing, fusion, deletion, and duplication during evolution of specific protein families is evaluated. It is shown that specific families of enzymes, receptors, transport proteins, and transcriptional regulatory proteins share a common evolutionary origin, frequently diverging in function because of domain splicing and ligation. Some large families arose gradually over evolutionary time, whereas others developed suddenly, due to bursts of intragenic or intergenic (or both) duplication events occurring over relatively short periods of time. It is argued that energy coupling to transport was a late occurrence, superimposed on preexisting mechanisms of solute facilitation. It is also shown that several transport protein families have evolved independently of each other, employing different routes, at different times in evolutionary history, to give topologically similar transmembrane protein complexes.

Bacterial Proteins↗
PubMed1999

Interaction of the Doa4 deubiquitinating enzyme with the yeast 26S proteasome.

e Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin-proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

Amino Acid Sequence↗