scholarly journals Copper binding by a unique family of metalloproteins is dependent on kynurenine formation

2021 ◽  
Vol 118 (23) ◽  
pp. e2100680118
Author(s):  
Anastasia C. Manesis ◽  
Richard J. Jodts ◽  
Brian M. Hoffman ◽  
Amy C. Rosenzweig
Keyword(s):  
Biochemical Characterization ◽  
Protein A ◽  
Copper Ion ◽  
Copper Homeostasis ◽  
Metabolic Enzyme ◽  
Copper Binding ◽  
Methane Oxidizing Bacteria ◽  
And Function

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned ∼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Biochemical characterization of Yin Yang 1 – RNA complexes

2008 ◽  
Vol 86 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Zachery R. Belak ◽  
Andrew Ficzycz ◽  
Nick Ovsenek
Keyword(s):  
Rna Binding ◽  
Biochemical Characterization ◽  
Ribonucleoprotein Complexes ◽  
Yin Yang 1 ◽  
Significant Difference ◽  
Yin Yang ◽  
Rna Interaction ◽  
Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

scholarly journals Morphological and Biochemical Characterization of Macrophages Activated by Carrageenan and Lipopolysaccharide In Vivo

2004 ◽  
Vol 29 (2) ◽  
pp. 27-34 ◽  
Author(s):  
Valéria Pereira Nacife ◽  
Maria de Nazaré Correia Soeiro ◽  
Rachel Novaes Gomes ◽  
Heloísa D’Avila ◽  
Hugo Caire Castro-Faria Neto ◽  
...  
Keyword(s):  
Biochemical Characterization

scholarly journals Regulation of murine copper homeostasis by members of the COMMD protein family

2020 ◽  
Vol 14 (1) ◽  
pp. dmm045963
Author(s):  
Amika Singla ◽  
Qing Chen ◽  
Kohei Suzuki ◽  
Jie Song ◽  
Alina Fedoseienko ◽  
...  
Keyword(s):  
Copper Homeostasis ◽  
Copper Accumulation ◽  
Copper Transporter ◽  
Copper Excretion ◽  
Copper Absorption ◽  
Hepatic Copper ◽  
High Copper ◽  
Molecular Machinery ◽  
And Function

ABSTRACTCopper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominantly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be recycled actively between the endosomal network and the plasma membrane by a molecular machinery known as the COMMD/CCDC22/CCDC93 or CCC complex. In fact, mutations in COMMD1 can lead to impaired biliary copper excretion and liver pathology in dogs and in mice with liver-specific Commd1 deficiency, recapitulating aspects of this phenotype. Nonetheless, the role of the CCC complex in intestinal copper absorption in vivo has not been studied, and the potential redundancy of various COMMD family members has not been tested. In this study, we examined copper homeostasis in enterocyte-specific and hepatocyte-specific COMMD gene-deficient mice. We found that, in contrast to effects in cell lines in culture, COMMD protein deficiency induced minimal changes in ATP7A in enterocytes and did not lead to altered copper levels under low- or high-copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiological consequence. By contrast, deficiency of any of three COMMD genes (Commd1, Commd6 or Commd9) resulted in hepatic copper accumulation under high-copper diets. We found that each of these deficiencies caused destabilization of the entire CCC complex and suggest that this might explain their shared phenotype. Overall, we conclude that the CCC complex plays an important role in ATP7B endosomal recycling and function.

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo

2001 ◽  
Vol 38 (6) ◽  
pp. 1421-1429 ◽  
Author(s):  
Alessandra Boletta ◽  
Feng Qian ◽  
Luiz F. Onuchic ◽  
Alessandra Bragonzi ◽  
Marina Cortese ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bona Fide

scholarly journals N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joerg Behnke ◽  
Alejandro M. Cohen ◽  
Julie LaRoche
Keyword(s):  
Cell Biology ◽  
Biochemical Characterization ◽  
Solid Phase ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
Iron Starvation ◽  
Glycosylation Sites ◽  
Glycosylation Pathway ◽  
And Function

AbstractN-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

scholarly journals RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

2009 ◽  
Vol 90 (7) ◽  
pp. 1748-1756 ◽  
Author(s):  
M. Gopinath ◽  
M. S. Shaila
Keyword(s):  
Biochemical Characterization ◽  
Covalent Bond ◽  
Rinderpest Virus ◽  
Viral Mrnas ◽  
L Protein ◽  
Rna Triphosphatase ◽  
Rnp Complex

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of γ-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

scholarly journals Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei

2008 ◽  
Vol 190 (12) ◽  
pp. 4173-4180 ◽  
Author(s):  
Qiuhe Lu ◽  
Jing Han ◽  
Ligang Zhou ◽  
Jian Zhou ◽  
Hua Xiang
Keyword(s):  
Biochemical Characterization ◽  
Complete Loss ◽  
Pha Synthase ◽  
Significant Activity ◽  
Class Iii ◽  
Haloferax Mediterranei ◽  
Thermal Asymmetric Interlaced Pcr

ABSTRACT The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC Hme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE Hme and phaC Hme genes were constitutively expressed, and both the PhaEHme and PhaCHme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC Hme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC Hme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaCHme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaEHme/PhaCHme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaCHme and PhaEHme, accounted for the PHBV synthesis in H. mediterranei.

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