Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni

Bacterial C-type haem-copper oxidases in the cbb3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCuAC (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c–1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.

Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.

Proteomic identification of the UDP-GlcNAc: PI α1–6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1–6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1–6 GlcNAc-transferase complex.

ACTIVITY AND ISOZYME COMPOSITION OF PEROXIDASE IN SCOTS PINE (PINUS SYLVESTRIS L.) NEEDLES EFFECTED BY TECHNOGENIC EMISSIONS FROM VARIOUS ENTERPRISES AND VEHICLES

Background. The technogenic pollution leads to excessive production of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to biomolecules. Plants have a complex antioxidant defense system that protects cells from the ROS and maintain homeostasis. The most important link this system is enzymes, in particular, peroxidase. It was of interest to determine the expression of the protective properties of one of the sensitive species of coniferous plants under the influence of technogenic emissions from various enterprises and vehicles. Purpose. Investigation the activity and isoenzyme composition of peroxidase in the needles of Pinus sylvestris L. under the influence of technogenic emissions of different compositions in the Baikal region. Materials and methods. The pine needles were collected on sample plots located near an aluminum plant, thermal power plant, chemical plant, coal mining enterprise, and the highway. The activity of soluble guaiacol-dependent peroxidases was defined by spectrophotometry in a reaction mixture with citrate-phosphate buffer, hydrogen peroxide, and guaiacol. Native polyacrylamide gel electrophoresis was used for determination of peroxidase isoforms. Results. It was shown that an increase in the total guaiacol-dependent peroxidase activity ranged from 6 to 22 times in the pine needles in polluted areas. Maximum enzyme activity was found in needle samples collected near the aluminum smelter, whose emissions are characterized by large amounts of fluorides and polycyclic aromatic hydrocarbons. The high variability of peroxidase isoform composition in Scots pine needles under industrial pollution was revealed. It was expressed in the emergence of new isoforms in the zone of fast-moving (Rf from 61 to 100) and medium-moving (Rf from 31 to 60) items. The maximum number of isoforms (nine) was found in pine needles near the aluminum smelter with only two ones detected in the background area. Conclusion. Peroxidase activity and the number of its newly formed isoforms can adequately reflect the degree of technogenic pollution and trees decline. The indicators can also be used in monitoring of coniferous forests condition.

Study of Blood Cells and Serum Proteins of Three Different Varieties of Fowls

Background: In the present study attempts have been made to study the blood cells and serum proteins comparatively among three different varieties of fowls, namely Turkey, RIR and Kadaknath. Total RBC and WBC count and serum protein analysis were performed. Methods: The total RBC and WBC were counted with the help of Neubauer’s Haemocytometer. The serum proteins were separated and analysed by native polyacrylamide gel electrophoresis (PAGE). Result: The results showed that, among all the fowl varieties tested, the RBC counts were ranged from 3.01 to 4.46 millions/mm3 and the WBC counts were ranged from 18.26 to 21.78 thousands/mm3. The serum protein analysis revealed that some proteins are present in specific species and specific sex. Hence, these proteins may be counted as markers to determine the species and sex of the species.

Simple isolation for two forms of malic enzyme from crustacean abdomen muscle

Two forms of NADP-dependent malic enzyme (ME, EC 1.1.1.40) were purified from the abdomen muscle of the crayfish Orconectes limosus (Rafinesque, 1817) and the shrimp Crangon crangon L., 1758 by affinity chromatography on 2′,5′-ADP-Sepharose 4B, with good qualitative recovery in a single step, using a substrate activation method with a malate–manganese chloride pair. The enzymes were identified by native polyacrylamide gel electrophoresis stained for protein and enzyme activity. The faster migrating mitochondrial enzyme from crayfish is inhibited by sulfhydryl reagent and loses its activity. Ellman’s Reagent, 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), can be used for the differentiation and measurement of cytoplasmic and mitochondrial malic enzyme in decapod crustacean tissue.

Abstract 517: Substrate Dependence of Mitochondrial Supercomplex Abundance in Murine Heart

Mitochondrial supercomplexes are prominent in mammalian tissues that have high energy demand. Nevertheless, the mechanisms that regulate supercomplex formation and abundance remain unclear. In this study, we examined how myocardial fuel preference regulated by constitutive changes in phosphofructokinase (PFK) activity in vivo or by differential substrate provision to isolated mitochondria affect mitochondrial supercomplexes. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain Complexes I, III, and IV as well as accessory proteins. Mitochondria from hearts with low PFK activity (Glyco Lo hearts) had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type (WT) or Glyco Hi hearts. Incubation of WT mitochondria with fatty acyl carnitine promoted higher supercomplex formation than did incubation with pyruvate, suggesting that substrate utilization is sufficient to regulate mitochondrial supercomplex abundance. These data are consistent with the hypothesis that mitochondrial supercomplex abundance is regulated in a substrate-dependent manner and suggest that metabolic scenarios favoring fat oxidation may promote supercomplex abundance.