Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β-1,4-exoceilobiohydrolase analogous to Trichoderma reesei CBH II

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.     

Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.     

FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport

Autophagy is the main eukaryotic degradation pathway for long-lived proteins, protein aggregates, and cytosolic organelles. Although the protein machinery involved in the biogenesis of autophagic vesicles is well described, very little is known about the mechanism of cytosolic transport of autophagosomes. In this study, we have identified an adaptor protein complex, formed by the two autophagic membrane-associated proteins LC3 and Rab7 and the novel FYVE and coiled-coil (CC) domain–containing protein FYCO1, that promotes microtubule (MT) plus end–directed transport of autophagic vesicles. We have characterized the LC3-, Rab7-, and phosphatidylinositol-3-phosphate–binding domains in FYCO1 and mapped part of the CC region essential for MT plus end–directed transport. We also propose a mechanism for selective autophagosomal membrane recruitment of FYCO1.     

MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

Background

Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.

Methods

To improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.

Results

The software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.

Conclusions

Software currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

Plasticity of proton pathway structure and water coordination in cytochrome c oxidase

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp(132)), near the protein surface, and glutamate Glu(286), which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu(286) affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser(197) (S197A or S197D), located approximately 7 A from Glu(286). Although Ser(197) is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of approximately 35% of that of the wild-type CytcO, and the O(2) reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.     

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA⁺ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE⁺ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.     

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope‐coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N‐termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS‐PAGE, in‐gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI‐TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of ¹²C and ¹³C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.     

The holocentric species Luzula elegans shows interplay between centromere and large‐scale genome organization

In higher plants, the large‐scale structure of monocentric chromosomes consists of distinguishable eu‐ and heterochromatic regions, the proportions and organization of which depend on a species' genome size. To determine whether the same interplay is maintained for holocentric chromosomes, we investigated the distribution of repetitive sequences and epigenetic marks in the woodrush Luzula elegans (3.81 Gbp/1C). Sixty‐one per cent of the L. elegans genome is characterized by highly repetitive DNA, with over 30 distinct sequence families encoding an exceptionally high diversity of satellite repeats. Over 33% of the genome is composed of the Angela clade of Ty1/copia LTR retrotransposons, which are uniformly dispersed along the chromosomes, while the satellite repeats occur as bands whose distribution appears to be biased towards the chromosome termini. No satellite showed an almost chromosome‐wide distribution pattern as expected for a holocentric chromosome and no typical centromere‐associated LTR retrotransposons were found either. No distinguishable large‐scale patterns of eu‐ and heterochromatin‐typical epigenetic marks or early/late DNA replicating domains were found along mitotic chromosomes, although super‐high‐resolution light microscopy revealed distinguishable interspersed units of various chromatin types. Our data suggest a correlation between the centromere and overall genome organization in species with holocentric chromosomes.     

Toward the identification of liver toxicity markers: a proteome study in human cell culture and rats

The effects of toxic and nontoxic compound treatments were investigated by high resolution custom developed 2-11 pH gradient NEPHGE (non equilibrium pH gradient electrophoresis) two-dimensional electrophoresis. Two models were compared: (i) in vivo rat and (ii) the human cell line HepG2, to test their suitability in a proteomics based approach to identify a toxicity marker. 163 and 321 proteins were identified from the rat liver and the HepG2 proteome. These represent various isoforms of 113 and 194 different NCBI annotated gene sequences, respectively. Nine compounds were selected to induce proteome variations associated with liver toxicity and metabolism. The rat liver proteome database consists of 78 gels, the HepG2 database of 52 gels. Variant proteins were assessed regarding their usefulness as a toxicity marker by evaluating their treatment specificity against multiple control treatments. Thirteen potential toxicity marker proteins were found in rat liver and eight in HepG2. Catalase and carbamoylphosphate synthetase-1 isoforms were found to be significantly changed after treatment by 4/4 and 3/4 toxic compounds in rat liver, respectively. Aldo-keto-reductase family 1, member C1 was implicated for 3/4 liver cell toxic compounds in HepG2. Our approach was able to differentiate the quality of potential toxicity markers and provided useful information for an ongoing characterization of more compounds in a wider number of toxicity classes.