Coding Sequence Insertions in Fungal Genomes are Intrinsically Disordered and can Impart Functionally-Important Properties on the Host Protein.

Insertion and deletion mutations (indels) are important mechanisms of generating protein diversity. Indels in coding sequences are under considerable selective pressure to maintain reading frames and to preserve protein function, but once generated, indels provide raw material for the acquisition of new protein properties and functions. We reported recently that coding sequence insertions in the NDU1 protein, a mitochondrial protein involved in the assembly of the NADH:ubiquinone oxidoreductase are imperative for respiration, biofilm formation and pathogenesis.

An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in Candida albicans.

A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.

Evolution, structure and membrane association of NDUFAF6, an assembly factor for NADH:ubiquinone oxidoreductase (Complex I).

The NADH:ubiquinone oxidoreductase (complex I) is the largest member of the mitochondrial respiratory chain. Its FMN cofactor accepts two electrons from NADH and transfers them to ubiquinone via a chain of iron-sulphur centers. A central core of 14 highly conserved subunits can couple electron transfer to proton translocation.

Glutathione metabolism links FOXRED1 to NADH:ubiquinone oxidoreductase (complex I) deficiency: A hypothesis.

FOXRED1 mutations result in complex I (NADH:ubiquinone oxidoreductase) deficiencies and Leigh syndrome (subacute necrotizing encephalomyelopathy). FOXRED1 is a mitochondrial flavoprotein related to N-methyl amino acid dehydrogenases. How is FOXRED1 required for the biogenesis of complex I? I present a hypothesis that suggests FOXRED1 catalytic activity as a sarcosine oxidase protects the developing fetus from oxidative stress during pregnancy.

A structural model for FOXRED1, an FAD-dependent oxidoreductase necessary for NADH: Ubiquinone oxidoreductase (complex I) assembly.

The biogenesis of mitochondrial respiratory chain components is complex. Mammalian complex I (NADH:ubiquinone oxidoreductase) contains 44 different subunits, an FMN and seven iron-sulfur centers. Its assembly involves at least twelve additional proteins, called assembly factors.

Evolution of FOXRED1, an FAD-dependent oxidoreductase necessary for NADH:ubiquinone oxidoreductase (Complex I) assembly.

Complex I (NADH:ubiquinone oxidoreductase) is the major entry point for electrons into the respiratory chains of bacteria and mitochondria. Mammalian complex I is composed of 45 subunits and harbors FMN and iron-sulfur cluster cofactors. A heterogeneous disease profile is associated with complex I deficiency.

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p paralogs results in catalytically active succinate dehydrogenase isoenzymes.

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors.

(1)H NMR-based metabolic profiling reveals inherent biological variation in yeast and nematode model systems.

The application of metabolomics to human and animal model systems is poised to provide great insight into our understanding of disease etiology and the metabolic changes that are associated with these conditions. However, metabolomic studies have also revealed that there is significant, inherent biological variation in human samples and even in samples from animal model systems where the animals are housed under carefully controlled conditions. This inherent biological variability is an important consideration for all metabolomics analyses.

Fluorescence method for determining the mechanism and speed of action of surface-active drugs on yeast cells.

New antifungal agents are needed to treat life-threatening fungal infections, particularly with the development of resistance. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a time-dependent drug exposure assay that allows us to rapidly investigate the mechanism of surface-active antifungal drug action.

Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through (1)H NMR-based metabolic footprinting.

Metabolomics is a powerful method of examining the intricate connections between mutations, metabolism, and disease. Metabolic footprinting examines the extracellular metabolome or exometabolome. We employed NMR-based metabolic footprinting and multivariate statistical analysis to examine a yeast model of mitochondrial dysfunction.

C. elegans longevity pathways converge to decrease mitochondrial membrane potential.

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals.

Mutations in the C. elegans succinate dehydrogenase iron-sulfur subunit promote superoxide generation and premature aging.

The mitochondrial succinate dehydrogenase (SDH) is an iron-sulfur flavoenzyme linking the Krebs cycle and the mitochondrial respiratory chain. Mutations in the human SDHB, SDHC and SDHD genes are responsible for the development of paraganglioma and pheochromocytoma, tumors of the head and neck or the adrenal medulla, respectively. In recent years, SDH has become recognized as a source of reactive oxygen species, which may contribute to tumorigenesis.

Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans.

Mitochondria are essential organelles with central roles in diverse cellular processes such as apoptosis, energy production via oxidative phosphorylation, ion homeostasis, and the synthesis of heme, lipid, amino acids, and iron-sulfur clusters. Defects in the mitochondrial respiratory chain lead to or are associated with a wide variety of diseases in humans. The nematode Caenorhabditis elegans provides a powerful genetic and developmental model system for reproducing deleterious mutations causing mitochondrial dysfunction and investigating their metabolic consequences and their mechanisms of pathology.

The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction.

The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S.

Mitochondrial genetics.

The mitochondrial genome is vital for Caenorhabditis elegans metabolism, physiology, and development. The C. elegans mitochondrial DNA is typical of animal mitochondrial genomes in its size and gene content.

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease.

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration.

Ubiquinone-binding site mutations in the Saccharomyces cerevisiae succinate dehydrogenase generate superoxide and lead to the accumulation of succinate.

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma.

DiS-C3(3) monitoring of in vivo mitochondrial membrane potential in C. elegans.

The mitochondrial respiratory chain plays a crucial role in cellular and organismal health. In addition to being the major source of energy for most cells, mitochondrial respiratory chain function regulates or modulates redox and metabolite homeostasis, apoptosis and the generation of reactive oxygen species. In order to measure the relative in vivo mitochondrial membrane potential of different strains of the nematode, Caenorhabditis elegans, we have developed a fluorescence assay using the cationic, lipophilic carbocyanine dye, diS-C(3)(3).

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase.

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q(P)) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species.

Riboflavin enhances the assembly of mitochondrial cytochrome c oxidase in C. elegans NADH-ubiquinone oxidoreductase mutants.

Mitochondrial respiratory chain dysfunction is responsible for a large variety of early and late-onset diseases. NADH-ubiquinone oxidoreductase (complex I) defects constitute the most commonly observed mitochondrial disorders. We have generated Caenorhabditis elegans strains with mutations in the 51 kDa active site subunit of complex I.

Introduction of an additional pathway for lactate oxidation in the treatment of lactic acidosis and mitochondrial dysfunction in Caenorhabditis elegans.

Mitochondrial dysfunction, with an estimated incidence of 1 in 5,000 births, is associated with a wide variety of multisystem degenerative diseases. Among the most prevalent forms of dysfunction are defects in the NADH:ubiquinone oxidoreductase (complex I). Caenorhabditis elegans strains with complex I mutations exhibit characteristic features of human mitochondrial disease including decreased rates of respiration and lactic acidosis.

Caenorhabditis elegans development requires mitochondrial function in the nervous system.

The mitochondrial respiratory chain (MRC) supplies the majority of the energy requirements of most eucaryotic cells. A null mutation in the Caenorhabditis elegans nuo-1 gene encoding a subunit of complex I (NADH-ubiquinone oxidoreductase) is lethal, leading to a developmental arrest at the third larval stage. To identify the tissues that regulate development in response to mitochondrial dysfunction, we restored nuo-1 expression with tissue-specific promoters.

Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase.

Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain. The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits. We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation.

The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies.

Succinate dehydrogenases and fumarate reductases are complex mitochondrial or bacterial respiratory chain proteins with remarkably similar structures and functions. Succinate dehydrogenase oxidizes succinate and reduces ubiquinone using a flavin adenine dinucleotide cofactor and iron-sulfur clusters to transport electrons. A model of the quaternary structure of the tetrameric Saccharomyces cerevisiae succinate dehydrogenase was constructed based on the crystal structures of the Escherichia coli succinate dehydrogenase, the E.

Mitochondrial complex I mutations in Caenorhabditis elegans produce cytochrome c oxidase deficiency, oxidative stress and vitamin-responsive lactic acidosis.

Mitochondrial dysfunction, with an estimated incidence of 1 in 10 000 live births, is among the most common genetically determined conditions. Missense mutations in the human NDUFV1 gene, which encodes the 51 kDa active site subunit of the NADH-ubiquinone oxidoreductase or complex I, can lead to severe neurological disorders. Owing to the rare and often sporadic nature of mitochondrial disorders, the mechanisms of pathogenesis of most mutations remain poorly understood.