Biomedical subjects

U Brandt

Publications and source records attributed to U Brandt.

At least 19 recordsLinked to original sources

PubMed2006

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme.

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.

Cell Membrane↗

PubMed2006

Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy.

After reduction with nicotinamide adenine dinucleotide (NADH), NADH:ubiquinone oxidoreductase (complex I) of the strictly aerobic yeast Yarrowia lipolytica shows clear signals from five different paramagnetic iron-sulfur (FeS) clusters (N1-N5) which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The ligand environment and the assignment of several FeS clusters to specific binding motifs found in several subunits of the complex are still under debate. In order to characterize the hyperfine interaction of the surrounding nuclei with FeS cluster N1, one- and two-dimensional electron spin echo envelope modulation experiments were performed at a temperature of 30 K. At this temperature only cluster N1 contributes to the overall signal in a pulsed EPR experiment. The hyperfine and quadrupole tensors of a nitrogen nucleus and the isotropic and dipolar hyperfine couplings of two sets of protons could be determined by numerical simulation of the one- and two-dimensional spectra. The values obtained are in perfect agreement with a ferredoxin-like binding structure by four cysteine amino acid residues and allow the assignment of the nitrogen couplings to a backbone nitrogen nucleus and the proton couplings to the beta-protons of the bound cysteine residues.

Electron Spin Resonance Spectroscopy↗

PubMed2006

Expression of the chemokine receptor CXCR1 in human glomerular diseases.

Leukocyte infiltration, a hallmark of renal diseases, is orchestrated in part by the actions of chemokines. The chemokine CXCL8/interleukin (IL)-8 is expressed during renal diseases and allograft rejection, whereas the corresponding receptor CXCR1 has not been described previously. Expression of CXCR1 was characterized in peripheral blood using multicolor fluorescence-activated cell sorter analysis (FACS). CXCR1 was localized in 81 formalin-fixed, paraffin-embedded renal specimens by immunohistochemistry using a monoclonal antibody against human CXCR1. Included were biopsies with crescentic glomerulonephritis (CGN, n = 22), immunoglobulin (Ig) A nephropathy (n = 15), membranoproliferative glomerulonephritis (MPGN, n = 17), lupus nephritis (n = 12), membranous nephropathy (n = 11), and non-involved parts of tumor nephrectomies (n = 4). Consecutive tissue sections of human tonsils, allograft explants, and renal biopsies were stained for CD15- and CD68-positive cells. Expression of CXCR1 and CXCL8/IL-8 mRNA was quantified by real-time reverse transcriptase-polymerse chain reaction of microdissected renal biopsies (n = 35) of the same disease entities. By FACS CXCR1 expression was found on polymorphonuclear CXCR1 expression by polymorphonuclear leukocytes (PMNs), natural killer cells, and a subpopulation of monocytes. By immunohistochemistry, CXCR1 expression was found on infiltrating inflammatory cells (predominantly PMNs), as well as on intrinsic renal cells (arterial smooth muscle cells, endothelial cells of peritubular capillaries). The distribution pattern of CXCR1 differed between disease entities. The highest numbers of glomerular CXCR1-positive cells were present in biopsies with MPGN, followed by lupus nephritis, and CGN. CXCR1 might be involved in the recruitment of PMNs to the glomerular tuft, which could be targeted by CXCR1-blocking agents.

Flow Cytometry↗

PubMed2005

Structure-function relationships in mitochondrial complex I of the strictly aerobic yeast Yarrowia lipolytica.

The obligate aerobic yeast Yarrowia lipolytica has been established as a powerful model system for the analysis of mitochondrial complex I. Using a combination of genomic and proteomic approaches, a total of 37 subunits was identified. Several of the accessory subunits are predicted to be STMD (single transmembrane domain) proteins. Site-directed mutagenesis of Y. lipolytica complex I has provided strong evidence that a significant part of the ubiquinone reducing catalytic core resides in the 49 kDa and PSST subunits and can be modelled using X-ray structures of distantly related enzymes, i.e. water-soluble [NiFe] hydrogenases from Desulfovibrio spp. Iron-sulphur cluster N2, which is related to the hydrogenase proximal cluster, is directly involved in quinone reduction. Mutagenesis of His226 and Arg141 of the 49 kDa subunit provided detailed insight into the structure-function relationships around cluster N2. Overall, our findings suggest that proton pumping by complex I employs long-range conformational interactions and ubiquinone intermediates play a critical role in this mechanism.

Aerobiosis↗

PubMed2001

Three molecules of ubiquinone bind specifically to mitochondrial cytochrome bc1 complex.

Bifurcated electron flow to high potential "Rieske" iron-sulfur cluster and low potential heme b(L) is crucial for respiratory energy conservation by the cytochrome bc(1) complex. The chemistry of ubiquinol oxidation has to ensure the thermodynamically unfavorable electron transfer to heme b(L). To resolve a central controversy about the number of ubiquinol molecules involved in this reaction, we used high resolution magic-angle-spinning nuclear magnetic resonance experiments to show that two out of three n-decyl-ubiquinones bind at the ubiquinol oxidation center of the complex. This substantiates a proposed mechanism in which a charge transfer between a ubiquinol/ubiquinone pair explains the bifurcation of electron flow.

Animals↗

PubMed2001

A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I.

We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit of [NiFe] hydrogenases. None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of iron-sulfur cluster N2, a prosthetic group not located in the 49-kDa subunit. In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster. We propose that the fold around the hydrogen reactive site of [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region. We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.

Amino Acid Sequence↗

PubMed2001

Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.

Chromatography, Affinity↗

PubMed2001

Exploring the catalytic core of complex I by Yarrowia lipolytica yeast genetics.

We have developed Yarrowia lipolytica as a model system to study mitochondrial complex I that combines the application of fast and convenient yeast genetics with efficient structural and functional analysis of its very stable complex I isolated by his-tag affinity purification with high yield. Guided by a structural model based on homologies between complex I and [NiFe] hydrogenases mutational analysis revealed that the 49 kDa subunit plays a central functional role in complex I. We propose that critical parts of the catalytic core of complex I have evolved from the hydrogen reactive site of [NiFe] hydrogenases and that iron-sulfur cluster N2 resides at the interface between the 49 kDa and PSST subunits. These findings are in full agreement with the "semiquinone switch" mechanism according to which coupling of electron and proton transfer in complex I is achieved by a single integrated pump comprising cluster N2, the binding site for substrate ubiquinone, and a tightly bound quinone or quinoid group.

Amino Acid Sequence↗

PubMed2001

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica.

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

Biological Transport↗

PubMed2000

Application of the obligate aerobic yeast Yarrowia lipolytica as a eucaryotic model to analyse Leigh syndrome mutations in the complex I core subunits PSST and TYKY.

We have used the obligate aerobic yeast Yarrowia lipolytica to reconstruct and analyse three missense mutations in the nuclear coded subunits homologous to bovine TYKY and PSST of mitochondrial complex I (proton translocating NADH:ubiquinone oxidoreductase) that have been shown to cause Leigh syndrome (MIM 25600), a severe progressive neurodegenerative disorder. While homozygosity for a V122M substitution in NDUFS7 (PSST) has been found in two siblings with neuropathologically proven Leigh syndrome (R. Triepels et al., Ann. Neurol. 45 (1999) 787), heterozygosity for a P79L and a R102H substitution in NDUFS8 (TYKY) has been found in another patient (J. Loeffen et al., Am. J. Hum. Genet. 63 (1998) 1598). Mitochondrial membranes from Y. lipolytica strains carrying any of the three point mutations exhibited similar complex I defects, with V(max) being reduced by about 50%. This suggests that complex I mutations that clinically present as Leigh syndrome may share common characteristics. In addition changes in the K(m) for n-decyl-ubiquinone and I(50) for hydrophobic complex I inhibitors were observed, which provides further evidence that not only the hydrophobic, mitochondrially coded subunits, but also some of the nuclear coded subunits of complex I are involved in its reaction with ubiquinone.

Amino Acid Sequence↗

PubMed2000

Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica.

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.

Amino Acid Sequence↗

PubMed2000

Binding of detergents and inhibitors to bovine complex I - a novel purification procedure for bovine complex I retaining full inhibitor sensitivity.

Mitochondrial complex I exhibits some peculiar and poorly understood features regarding the effects of detergents on activity and sensitivity to hydrophobic inhibitors that are not seen with other membrane complexes using ubiquinone as a substrate. Therefore, we investigated the interaction of complex I from bovine heart mitochondria with different types of detergents by monitoring activity, degree of inhibition and inhibitor binding in the presence of increasing concentrations of detergent. It is shown that apart from their nature as solubilizing and delipidating agents the polyoxyethylene-ether detergents Triton X-100, Brij-35 and Thesit act as specific inhibitors of complex I and compete with classical complex I inhibitors for a common binding domain. These findings were used to develop a novel large-scale chromatographic procedure for isolation of inhibitor-sensitive NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxylapatite, ion-exchange and gel-exclusion chromatography. By switching detergents from Triton X-100 to dodecylmaltoside after hydroxylapatite chromatography the procedure yields highly pure, monodisperse and fully inhibitor-sensitive enzyme.

Amines↗

PubMed2000

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.

Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.

Amino Acid Sequence↗

PubMed1999

Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Three classes of inhibitors share a common binding domain in mitochondrial complex I (NADH:ubiquinone oxidoreductase).

We have developed two independent methods to measure equilibrium binding of inhibitors to membrane-bound and partially purified NADH:ubiquinone oxidoreductase (complex I) to characterize the binding sites for the great variety of hydrophobic compounds acting on this large and complicated enzyme. Taking advantage of a partial quench of fluorescence upon binding of the fenazaquin-type inhibitor 2-decyl-4-quinazolinyl amine to complex I in bovine submitochondrial particles, we determined a Kd of 17 +/- 3 nM and one binding site per complex I. Equilibrium binding studies with [3H]dihydrorotenone and the aminopyrimidine [3H]AE F119209 (4(cis-4-[3H]isopropyl cyclohexylamino)-5-chloro-6-ethyl pyrimidine) using partially purified complex I from Musca domestica exhibited little unspecific binding and allowed reliable determination of dissociation constants. Competition experiments consistently demonstrated that all tested hydrophobic inhibitors of complex I share a common binding domain with partially overlapping sites. Although the rotenone site overlaps with both the piericidin A and the capsaicin site, the latter two sites do not overlap. This is in contrast to the interpretation of enzyme kinetics that have previously been used to define three classes of complex I inhibitors. The existence of only one large inhibitor binding pocket in the hydrophobic part of complex I is discussed in the light of possible mechanisms of proton translocation.

Animals↗

PubMed1999

Proton translocation in the respiratory chain involving ubiquinone--a hypothetical semiquinone switch mechanism for complex I.

The protonmotive Q-cycle is the generally accepted reaction scheme of the cytochrome bc1 complex of the respiratory chain. It employs the redox-dependent protonation and deprotonation of ubiquinone (coenzyme Q10) to translocate protons across the inner mitochondrial or bacterial plasma membrane. The requirements for the operation of a similar mechanism in proton translocating NADH:ubiquinone oxidoreductase (complex I) and the specific roles of the 'Rieske' iron-sulfur center in the cytochrome bc1 complex and iron-sulfur center N-2 in complex I are discussed. Recent results suggest that there is only one ubiquinone-reactive site in complex I which seems to exclude a classical Q-cycle type mechanism. Therefore, a "semiquinone switch" mechanism is proposed involving one tightly bound and one substrate quinone. It is based on the same principles as a Q-cycle, but is a localized rather than a ligand conduction type mechanism.

Animals↗

PubMed1999

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.

Animals↗

PubMed1999

Control of ubiquinol oxidation at center P (Qo) of the cytochrome bc1 complex.

The unique bifurcated oxidation of ubiquinol at center P (Qo) of the cytochrome bc1 complex is the reaction within the Q-cycle reaction scheme that is most critical for the link between electron transfer and vectorial proton translocation. While there is a general consensus about the overall reaction at center P, the nature of the intermediates and the way the reaction is controlled to ensure obligatory bifurcation is still controversial. By reducing the reaction to its essential steps, a kinetic net rate model is developed in which the activation barrier is associated with the deprotonation of ubiquinol, but the steady state rate is kinetically controlled by the occupancy of the ubiquinol anion and the semiquinone state. This concept is used to interpret experimental data and is discussed in terms of various mechanistic models that are under discussion. It is outlined how other aspects of the center P mechanism like the proposed "prosthetic" ubiquinone and the moving domain of the "Rieske" protein could be incorporated in the kinetic framework.

Animals↗