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Hartmut Michel - One of the best experts on this subject based on the ideXlab platform.
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The 18 kDa Cytochrome c553 from Heliobacterium gestii: Gene Sequence and Characterization of the Mature Protein‡
Biochemistry, 1998Co-Authors: Ingrid Albert, Hans J. Grav, A.w. Rutherford, Josef Kellermann, Hartmut MichelAbstract:The 18 kDa cytochrome c553 is the dominant c-type cytochrome in cell membranes of Heliobacterium gestii. After solubilization, this cytochrome was purified in three steps as a complex with two other proteins of 32 and 42 kDa. The redox midpoint potential of the cytochrome c553 was determined to be +215 mV. The EPR spectra clearly show the presence of an ascorbate-reducible low-spin heme with gz = 3.048 and gy = 2.238. The gx = trough could not be detected. In addition, a Cu(II) signal with g = 2.058 was observed, indicating that one component of the cytochrome c553 complex contains a bound copper ion. The gene for the 18 kDa cytochrome c553, cyhA, consists of 429 bp coding for a protein of 142 amino acids. The association of the cytochrome with the cytoplasmic membrane is mediated by two fatty acid molecules, one palmitate and one stearate, that could be identified by mass spectrometry. Both fatty acids are most likely bound to the cysteine residue of the N-terminally processed protein via a glycerol moiety. The amino acid sequence deduced from the DNA sequence exhibits Partial Identity to the membrane-bound cytochrome c551 from Bacillus PS3 [Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N. (1993) Biochem. Biophys. Res. Commun. 1144, 213-219] and to the cytochrome c subunit (NorC) of the nitrous reductase from Pseudomonas stutzeri [Zumft, W. G., Braun, C., and Cuypers, H. (1994) Eur. J. Biochem. 219, 481-490].
Ingrid Albert - One of the best experts on this subject based on the ideXlab platform.
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The 18 kDa Cytochrome c553 from Heliobacterium gestii: Gene Sequence and Characterization of the Mature Protein‡
Biochemistry, 1998Co-Authors: Ingrid Albert, Hans J. Grav, A.w. Rutherford, Josef Kellermann, Hartmut MichelAbstract:The 18 kDa cytochrome c553 is the dominant c-type cytochrome in cell membranes of Heliobacterium gestii. After solubilization, this cytochrome was purified in three steps as a complex with two other proteins of 32 and 42 kDa. The redox midpoint potential of the cytochrome c553 was determined to be +215 mV. The EPR spectra clearly show the presence of an ascorbate-reducible low-spin heme with gz = 3.048 and gy = 2.238. The gx = trough could not be detected. In addition, a Cu(II) signal with g = 2.058 was observed, indicating that one component of the cytochrome c553 complex contains a bound copper ion. The gene for the 18 kDa cytochrome c553, cyhA, consists of 429 bp coding for a protein of 142 amino acids. The association of the cytochrome with the cytoplasmic membrane is mediated by two fatty acid molecules, one palmitate and one stearate, that could be identified by mass spectrometry. Both fatty acids are most likely bound to the cysteine residue of the N-terminally processed protein via a glycerol moiety. The amino acid sequence deduced from the DNA sequence exhibits Partial Identity to the membrane-bound cytochrome c551 from Bacillus PS3 [Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N. (1993) Biochem. Biophys. Res. Commun. 1144, 213-219] and to the cytochrome c subunit (NorC) of the nitrous reductase from Pseudomonas stutzeri [Zumft, W. G., Braun, C., and Cuypers, H. (1994) Eur. J. Biochem. 219, 481-490].
Nathalie Salome - One of the best experts on this subject based on the ideXlab platform.
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nonstructural proteins ns2 of minute virus of mice associate in vivo with 14 3 3 protein family members
Journal of Virology, 1996Co-Authors: Katrin Brockhaus, Serge Plaza, David J Pintel, Jean Rommelaere, Nathalie SalomeAbstract:The nonstructural NS2 proteins of the prototype strain of minute virus of mice (MVMp) were previously shown to be involved in parvoviral DNA amplification as well as in efficient virus production in a host cell-specific manner (L. K. Naeger, N. Salome, and D. J. Pintel, J. Virol. 67:1034-1043, 1993). NS2 polypeptides were also reported to participate in the cytotoxic activity of parvoviruses (C. Legrand, J. Rommelaere, and P. Caillet-Fauquet, Virology 195:149-155, 1993), for which transformed cells are preferential targets. To identify cellular partners of NS2 proteins, coimmunoprecipitation experiments were performed with various antibodies directed against the parvoviral products. Two cellular proteins with molecular masses of 30 and 32 kDa were found to associate in vivo with the NS2 polypeptides. From amino acid sequence homology, these NS2 partners were assigned to the 14-3-3 family of cellular proteins, showing at least Partial Identity with the epsilon and beta or zeta 14-3-3 isoforms. In agreement with this assignment, NS2-30/32-kDa protein immune complexes displayed an activating function for exoenzyme S in vitro, a hallmark of 14-3-3 polypeptides. Interactions with 14-3-3 proteins did not appear sufficient for NS2 functions, since they were not disrupted by NS2 C-terminal modifications that impaired virus replication. Binding of NS2 to 14-3-3 proteins was detected in various cells of mouse, rat, hamster, monkey, and human origin, irrespective of NS2 dispensability and host cell transformation or permissiveness. The ubiquitous 14-3-3 proteins were recently reported to associate with several other cellular or viral polypeptides involved in signal transduction and/or cell cycle regulation pathways (A. Aitken, Trends Biochem. Sci. 20:95-97, 1995). The NS2 products may connect with one of these pathways through their interaction with specific 14-3-3 polypeptides.
Michele P Calos - One of the best experts on this subject based on the ideXlab platform.
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nucleofection of phic31 integrase protein mediates sequence specific genomic integration in human cells
Journal of Molecular Biology, 2020Co-Authors: Tuhin Kumar Guha, Michele P CalosAbstract:The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having Partial Identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo-attP site. This work demonstrates for the first time the ability of 605- and 613-amino-acid versions of phiC31 integrase protein to mediate efficient, site-specific integration into the genome of human cells when co-nucleofected with full-sizedattB-containing donor plasmids or linear 2.5-kb PCR fragments. This protein-mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA.
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site specific genomic integration produces therapeutic factor ix levels in mice
Nature Biotechnology, 2002Co-Authors: Eric C Olivares, Roger P Hollis, Thomas W Chalberg, Leonard Meuse, Mark A Kay, Michele P CalosAbstract:We used the integrase from phage φC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was coinjected, hFIX serum levels increased more than tenfold to ∼4 µg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after Partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with Partial Identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration. Lasting gene therapy may be facilitated by the permanent introduction of exogenous DNA into the chromosomes of patients. Here we describe the use of bacteriophage φC31 integrase to achieve site-specific genomic integration of therapeutic genes into the genomes of adult mice. In nature, the integrase mediates unidirectional recombination of the phage genome into the bacterial chromosome through a site-specific reaction between the phage attP site and the host attB site1,2 .W e have shown that this integrase is functional in mammalian cells3, and that it can efficiently integrate plasmids containing an attB site into pseudo-attP sites in mammalian genomes 4 .T hese pseudo sites have enough Identity with wild-type attP to support integrase-mediated recombination, and persistent gene expression occurs from vectors integrated at such sites 4 .I n this study, we have applied these principles in vivo to integrate attB vectors bearing the gene for human blood clotting Factor IX (hFIX) into the chromosomes of mice at native pseudoattP sites, resulting in the generation of persistent normal levels of the protein. Results Integrase-mediated expression of hAAT in mouse liver. We constructed plasmids that contained a φC31 attB site and either a cDNA expression construct of the human α 1-antitrypsin gene (hAAT) 5
Jackie De Belleroche - One of the best experts on this subject based on the ideXlab platform.
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a 14 3 3 mrna is up regulated in amyotrophic lateral sclerosis spinal cord
Journal of Neurochemistry, 2008Co-Authors: Andrea Malaspina, Narendra Kaushik, Jackie De BellerocheAbstract:We have recently isolated a 2.2-kb cDNA clone (1C5) from a human spinal cord cDNA library with Partial Identity to the 14-3-3 protein mRNA encoding the theta protein (YWHAQ). 14-3-3 protein transcripts are highly expressed in large projection neurones of the hippocampus, cerebellum, and spinal cord and have been found to be significantly up-regulated in rat motor neurones following hypoglossal nerve axotomy. In this study we investigated whether the 1C5 transcript (YWHAQ) isolated from spinal cord was involved in amyotrophic lateral sclerosis (ALS). We found a significant up-regulation of 1C5 (YWHAQ) in lumbar spinal cord from patients with sporadic ALS compared with controls, with the highest levels of expression being found in individuals with predominant lower motor neurone involvement. A 6-bp tandem repeat in the 5'-untranslated region of the gene was found to be polymorphic, but no significant association with disease was found following genomic analysis of this region. The localisation of 1C5 (YWHAQ) to chromosome 2 was determined and coincides with that reported for clone HS1 (EMBL accession no. X57347). These results show the marked up-regulation of the 14-3-3 isoform (YWHAQ) in ALS spinal cord and indicate the involvement of a potential 14-3-3-mediated survival pathway in the pathogenesis of ALS.