Biomedical subjects

Immo E Scheffler

Publications and source records attributed to Immo E Scheffler.

9 recordsLinked to original sources

PubMed2006

Cyanide detoxification by the cobalamin precursor cobinamide.

Cyanide is a highly toxic agent that inhibits mitochondrial cytochrome-c oxidase, thereby depleting cellular ATP. It contributes to smoke inhalation deaths in fires and could be used as a weapon of mass destruction. Cobalamin (vitamin B12) binds cyanide with a relatively high affinity and is used in Europe to treat smoke inhalation victims. Cobinamide, the penultimate compound in cobalamin biosynthesis, binds cyanide with about 10(10) greater affinity than cobalamin, and we found it was several-fold more effective than cobalamin in (i) reversing cyanide inhibition of oxidative phosphorylation in mammalian cells; (ii) rescuing mammalian cells and Drosophila melanogaster from cyanide toxicity; and (iii) reducing cyanide inhibition of Drosophila Malpighian tubule secretion. Cobinamide could be delivered by oral ingestion, inhalation, or injection to Drosophila, and it was as effective when administered up to 5 mins post-cyanide exposure as when given pre-exposure. We conclude that cobinamide is an effective cyanide detoxifying agent that has potential use as a cyanide antidote, both in smoke inhalation victims and in persons exposed to cyanide used as a weapon of mass destruction.

Animals↗

PubMed2004

Molecular genetics of complex I-deficient Chinese hamster cell lines.

The work from our laboratory on complex I-deficient Chinese hamster cell mutants is reviewed. Several complementation groups with a complete defect have been identified. Three of these are due to X-linked mutations, and the mutated genes for two have been identified. We describe null mutants in the genes for the subunits MWFE (gene: NDUFA1) and ESSS. They represent small integral membrane proteins localized in the Ialpha (Igamma) and Ibeta subcomplexes, respectively [J. Hirst, J. Carroll, I.M. Fearnley, R.J. Shannon, J.E. Walker. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604 (7-10-2003) 135-150.]. Both are absolutely essential for assembly and activity of complex I. Epitope-tagged versions of these proteins can be expressed from a poly-cistronic vector to complement the mutants, or to be co-expressed with the endogenous proteins in other hamster cell lines (mutant or wild type), or human cells. Structure-function analyses can be performed with proteins altered by site-directed mutagenesis. A cell line has been constructed in which the MWFE subunit is conditionally expressed, opening a window on the kinetics of assembly of complex I. Its targeting, import into mitochondria, and orientation in the inner membrane have also been investigated. The two proteins have recently been shown to be the targets for a cAMP-dependent kinase [R. Chen, I.M. Fearnley, S.Y. Peak_Chew, J.E. Walker. The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. xx (2004) xx-xx.]. The epitope-tagged proteins can be cross-linked with other complex I subunits.

Amino Acid Sequence↗

PubMed2004

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain.

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.

Adenosine Triphosphate↗

PubMed2004

Development and characterization of a conditional mitochondrial complex I assembly system.

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE.HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another approximately 24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1-6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as "accessory." It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.

Animals↗

PubMed2004

Import and orientation of the MWFE protein in mitochondrial NADH-ubiquinone oxidoreductase.

The MWFE subunit of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a small, essential membrane protein of 70 amino acids that is made in the cytosol, imported into mitochondria, and assembled without further proteolytic processing. The experiments identify the first approximately 30 amino acids as a minimal mitochondrial targeting sequence, and establish its orientation in the inner membrane and in complex I. This sequence has a highly conserved glutamate at position 4, which is not typical of a mitochondrial targeting signal. However, it is not essential for MWFE function. Within this sequence there is also a 'stop-transfer' signal. The membrane anchor cannot be replaced by that from another subunit within complex I.

Journal Article↗

PubMed2004

The role of the ESSS protein in the assembly of a functional and stable mammalian mitochondrial complex I (NADH-ubiquinone oxidoreductase).

The ESSS protein is a recently identified subunit of mammalian mitochondrial complex I. It is a relatively small integral membrane protein (122 amino acids) found in the beta-subcomplex. Genomic sequence database searches reveal its localization to the X-chromosome in humans and mouse. The ESSS cDNA from Chinese hamster cells was cloned and shown to complement one complementation group of our previously described mutants with a proposed X-linkage. Sequence analyses of the ESSS cDNA in these mutants revealed chain termination mutations. In two of these mutants the protein is truncated at the C-terminus of the targeting sequence; the mutants are null mutants for the ESSS subunit. There is no detectable complex I assembly and activity in the absence of the ESSS subunit as revealed by blue native polyacrylamide gel electrophoresis (BN/PAGE) analysis and polarography. Complex I activity can be restored with ESSS subunits tagged with either hemagglutinin (HA) or hexahistidine (His6) epitopes at the C-terminus. Although, the accumulation of ESSS-HA is not dependent upon the presence of mtDNA-encoded subunits (ND1-6,4 L), it is incorporated into complex I only in presence of compatible complex I subunits from the same species.

Amino Acid Sequence↗

PubMed2003

The tumor suppressor cybL, a component of the respiratory chain, mediates apoptosis induction.

A genetic screen was established to clone apoptosis-inducing genes in a high-throughput format. It led to the isolation of several proapoptotic genes whose proteins are localized to mitochondria. One of the isolated genes is cytochrome bL (cybL also known as SDHC, CII-3, or QPs-1), a component of the respiratory chain complex II. It was further investigated because both cybL and another component of complex II, cybS, have recently been identified as tumor suppressor proteins, some of which act by controlling apoptosis. Our studies reveal that cell death induction by cybL expression is concomitant with a transient inhibition of complex II and the generation of reactive oxygen species. Importantly, cells that are constitutively deficient in cybL are resistant to a variety of proapoptotic cytostatic drugs and to the effects of the Fas receptor. Our results therefore identify complex II as a sensor for apoptosis induction and could explain the unexpected observation that complex II is inactivated in tumors.

Animals↗

PubMed2002

Species-specific and mutant MWFE proteins. Their effect on the assembly of a functional mammalian mitochondrial complex I.

The MWFE protein (70 amino acids) is highly conserved in evolution, but the human protein (80% identical to hamster) does not complement a null mutation in Chinese hamster cells. We have identified a small protein segment where significant differences exist between rodents and primates, illustrating very specifically the need for compatibility of the nuclear and mitochondrial genomes in the assembly of complex I. The segment between amino acids 39 and 46 appears to be critical for species-specific compatibility. Amino acid substitutions in this region were tested that caused a reduction of activity of the hamster protein or converted the inactive human protein into a partially active one. Such mutations could be useful in making mice with partial complex I activity as models for mitochondrial diseases. Their potential as dominant negative mutants was explored. More deleterious mutations in the NDUFA1 gene were also characterized. A conservative substitution, R50K, or a short C-terminal deletion makes the protein completely inactive. In the absence of MWFE, no high molecular weight complex was detectable by Blue Native-gel electrophoresis. The MWFE protein itself is unstable in the absence of assembled mitochondrially encoded integral membrane proteins of complex I.

Alleles↗

PubMed2002

The role of the 5' untranslated region (UTR) in glucose-dependent mRNA decay.

When S. cerevisiae are grown with glucose, SDH2 mRNA encoding the iron protein of the succinate dehydrogenase complex is unstable and present at low level. In yeast grown without glucose, SDH2 mRNA is stable and its level rises. Addition of glucose to a glucose-limited culture causes the SDH2 mRNA level to fall rapidly with a half-life of approximately 5-7 min. Previously the 5'UTR of the mRNA of SDH2 was shown to be necessary and sufficient to destabilize it in glucose (Lombardo et al., 1992). We now show that the SDH1 and SUC2 5'UTRs are capable of conferring glucose-sensitive mRNA instability. We also examine how changes in the SDH2 5'UTR affect glucose-triggered degradation. Finally, we show that changes in mRNA stability are correlated with changes in translational efficiency for these transcripts.

5' Untranslated Regions↗