scholarly journals The nuclear transport receptor TNPO1 binds macrophage immunometabolism regulator MACIR via a PY-NLS motif

2019 ◽  
Author(s):  
Gavin McGauran ◽  
Emma Dorris ◽  
Razvan Borza ◽  
Niamh Morgan ◽  
Denis C. Shields ◽  
...  
Keyword(s):  
Amino Acid ◽  
Autoimmune Disease ◽  
Structure Prediction ◽  
Nuclear Transport ◽  
Quantitative Mass Spectrometry ◽  
Disordered Protein ◽  
Acid Protein ◽  
Recognition Motifs ◽  
Transport Receptor ◽  
Amino Acid Protein

AbstractExpression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and the regulation of macrophage biology through unknown mechanisms. The 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. Here we identify specific interactions of MACIR using a fragment complementation-based affinity pull down of cellular proteins prepared with a membrane solubilization buffer. Quantitative mass spectrometry showed enrichment of nuclear and mitochondrial proteins and of 63 significant interacting proteins, binding to the nuclear transport receptor TNPO1 and trafficking proteins UNC119 homolog A and B were validated by immunoprecipitation. Analysis of mutations in two candidate recognition motifs in the MACIR amino acid sequence confirmed TNPO1 binds via a PY-NLS motif (aa98-117). Characterizing nuclear MACIR activity in macrophage and fibroblasts is a priority with respect to developing strategies for treatment of autoimmune disease.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

2006 ◽  
Vol 398 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Family Members ◽  
Fatty Acyl ◽  
Ceramide Synthase ◽  
Broad Substrate Specificity ◽  
Acid Protein ◽  
Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

scholarly journals The Vicious Circle of Hepatic Glucagon Resistance in Non-Alcoholic Fatty Liver Disease

2020 ◽  
Vol 9 (12) ◽  
pp. 4049
Author(s):  
Katrine D. Galsgaard
Keyword(s):  
Liver Disease ◽  
Amino Acid ◽  
Fatty Liver ◽  
Protein Metabolism ◽  
Fatty Liver Disease ◽  
Plasma Concentrations ◽  
Alcoholic Fatty Liver ◽  
Acid Protein ◽  
Alcoholic Fatty Liver Disease ◽  
Amino Acid Protein

A key criterion for the most common chronic liver disease—non-alcoholic fatty liver disease (NAFLD)—is an intrahepatic fat content above 5% in individuals who are not using steatogenic agents or having significant alcohol intake. Subjects with NAFLD have increased plasma concentrations of glucagon, and emerging evidence indicates that subjects with NAFLD may show hepatic glucagon resistance. For many years, glucagon has been thought of as the counterregulatory hormone to insulin with a primary function of increasing blood glucose concentrations and protecting against hypoglycemia. However, in recent years, glucagon has re-emerged as an important regulator of other metabolic processes including lipid and amino acid/protein metabolism. This review discusses the evidence that in NAFLD, hepatic glucagon resistance may result in a dysregulated lipid and amino acid/protein metabolism, leading to excess accumulation of fat, hyperglucagonemia, and increased oxidative stress contributing to the worsening/progression of NAFLD.

scholarly journals Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum

1999 ◽  
Vol 341 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Lu-Ping CHOW ◽  
Ning-Yuan SU ◽  
Chia-Jung YU ◽  
Bor-Luen CHIANG ◽  
Horng-Der SHEN
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Catalytic Triad ◽  
Penicillium Citrinum ◽  
E Coli ◽  
Acid Protein ◽  
Amino Acid Signal Peptide ◽  
Cloned Cdna ◽  
Allergic Disorders ◽  
Amino Acid Protein

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.

scholarly journals Genetic Organization and Molecular Analysis of the EcoVIII Restriction-Modification System of Escherichia coli E1585-68 and Its Comparison with Isospecific Homologs

2003 ◽  
Vol 69 (5) ◽  
pp. 2638-2650 ◽  
Author(s):  
Iwona Mruk ◽  
Tadeusz Kaczorowski
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Start Codon ◽  
Sequence Motif ◽  
Specific Sequence ◽  
Modification System ◽  
Acid Protein ◽  
Restriction Modification ◽  
Amino Acid Protein

ABSTRACT The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R · EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M r of 35,554. The convergently oriented EcoVIII methyltransferase (M · EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M r of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R · EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M · EcoVIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to N 6-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M · EcoVIII with M · HindIII and M · LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m6 N-adenine β-class methyltransferases. The deduced amino acid sequence of R · EcoVIII shows weak homology with its two isoschizomers, R · HindIII (26%) and R · LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · EcoVIII (D108X12DXK123), as well as in the primary structures of R · LlaCI and R · HindIII. Polyclonal antibodies raised against R · EcoVIII did not react with R · HindIII, while anti-M · EcoVIII antibodies cross-reacted with M · LlaCI but not with M · HindIII. R · EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M · EcoVIII enzyme. The biological implications of this finding are discussed.

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