Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein

2001 ◽  
Vol 359 (3) ◽  
pp. 707-714 ◽  
Author(s):  
Dong CHENG ◽  
Rupalie L. MEEGALLA ◽  
Bokang HE ◽  
Debra A. CROMLEY ◽  
Jeffery T. BILLHEIMER ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Gel Filtration ◽  
Human Adipose Tissue ◽  
Cross Linking ◽  
Triacylglycerol Synthesis ◽  
Putative Active Site ◽  
Membrane Enzyme ◽  
C Terminus ◽  
Sds Page

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane enzyme that catalyses the last step of triacylglycerol synthesis from diacylglycerol and acyl-CoA. Here we provide experimental evidence that DGAT is a homotetramer. Although the predicted molecular mass of human DGAT protein is 55kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150kDa on gel-filtration chromatography. Cross-linking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108kDa) and the tetramer (214kDa) in SDS/PAGE. Finally, when two differently epitope-tagged forms of DGAT were co-transfected into mammalian cells, they could be co-immunoprecipitated. From a human adipose tissue cDNA library we cloned a cDNA encoding a novel splice variant of DGAT (designated DGATsv) that contained a 77nt insert of unspliced intron with an in-frame stop codon. This resulted in a truncated form of DGAT that terminated at Arg-387, deleting 101 residues from the C-terminus containing the putative active site. DGATsv was enzymically inactive when transfected in HEK-293E cells but was still able to form dimer and tetramer on cross-linking, indicating that the ability to form tetramers resides in the N-terminal region. When co-expressed in HEK-293E cells, DGATsv did not inhibit the activity of full-length DGAT, suggesting that the subunits of DGAT catalyse triacylglycerol synthesis independently.

scholarly journals MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

2019 ◽  
Author(s):  
Zhihao Wu ◽  
Ishaq Tantray ◽  
Junghyun Lim ◽  
Songjie Chen ◽  
Yu Li ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mitochondrial Dysfunction ◽  
Complex I ◽  
Mammalian Cells ◽  
Protein C ◽  
Stop Codon ◽  
Translation Termination ◽  
Disease Model ◽  
C Terminus ◽  
Translational Termination

SUMMARYMitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here we describe a phenomenon termed MISTERMINATE (mitochondrial stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs including complex-I 30kD subunit (C-I30) mRNA, occurring on mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C-terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally-extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson’s disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

scholarly journals Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Swathi Nageswara ◽  
Girijasankar Guntuku ◽  
Bhagya Lakshmi Yakkali
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
3D Structure ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Structural Elucidation ◽  
C Terminus ◽  
Sds Page ◽  
Sequence Similarities

Abstract Background Serratiopeptidase is an alkaline metalloendopeptidase, which acquired wide significance because of its therapeutic applications. The present study was undertaken for purification, characterization, and structural elucidation of serratiopeptidase produced from Streptomyces hydrogenans var. MGS13. Result The crude enzyme was purified by precipitating with ammonium sulfate, dialysis, and Sephadex gel filtration, resulting in 34% recovery with a 12% purification fold. The purified enzyme S.AMP13 was spotted as a single clear hydrolytic band on casein zymogram and whose molecular weight was found to be 32 kDa by SDS-PAGE. The inhibitor and stability studies revealed that this enzyme is metalloprotease, thermostable, and alkaline in nature. The maximum serratiopeptidase activity was observed at 37 °C and pH 9.0. The partial amino acid sequence of the purified enzyme S.AMP13 by LC-MS/MS analysis shows the closest sequence similarities with previously reported alkaline metalloendopeptidases. The amino acid sequence alignment of S.AMP13 shared a conserved C-terminus region with peptidase-M10 serralysin superfamily at amino acid positions 128–147, i.e., ANLSTRATDTVYGFNSTAGR revealed that this enzyme is a serralysin-like protease. The kinetic studies of the purified enzyme revealed a Km of 1 mg/mL for its substrate casein and Vmax of 319 U/mL/min. The 3D structure of the purified enzyme was modeled by using SWISS-MODEL, and the quality of the structure was authenticated by assessing the Ramachandran plot using PROCHECK server, which suggested that the enzyme was stable with good quality. Conclusion Inhibitor, stability, electrophoretic, and bioinformatic studies suggested that the purified enzyme obtained from S. hydrogenans var. MGS13 is a serralysin-like protease.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. 665-672 ◽  
Author(s):  
Samantha A M Young ◽  
Haruhiko Miyata ◽  
Yuhkoh Satouh ◽  
Masanaga Muto ◽  
Martin R Larsen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Male Fertility ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Cytoplasmic Tail ◽  
Binding Partner ◽  
C Terminus ◽  
Manipulation System

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm–egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.

scholarly journals Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions

1986 ◽  
Vol 234 (1) ◽  
pp. 43-48 ◽  
Author(s):  
E J Bergey ◽  
M J Levine ◽  
M S Reddy ◽  
S D Bradway ◽  
I Al-Hashimi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Gel Filtration ◽  
Cross Linking ◽  
Oral Streptococci ◽  
Bacterial Surface ◽  
Acetylneuraminic Acid ◽  
Streptococcus Sanguis ◽  
Surface Examination ◽  
Inhibition Studies

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.

scholarly journals Identification and characterization of Fep15, a new selenocysteine-containing member of the Sep15 protein family

2006 ◽  
Vol 394 (3) ◽  
pp. 575-579 ◽  
Author(s):  
Sergey V. Novoselov ◽  
Deame Hua ◽  
Alexey V. Lobanov ◽  
Vadim N. Gladyshev
Keyword(s):  
Mammalian Cells ◽  
Insertion Sequence ◽  
Phylogenetic Analyses ◽  
Dependent Manner ◽  
Putative Active Site ◽  
A Genome ◽  
Sequence Elements ◽  
Identification And Characterization ◽  
Insertion Sequence Elements

Sec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec. When transiently expressed in mammalian cells, Fep15 incorporated Sec in an SECIS- and SBP2 (SECIS-binding protein 2)-dependent manner and was targeted to the endoplasmic reticulum by its N-terminal signal peptide. Phylogenetic analyses of Sep15 family members suggest that Fep15 evolved by gene duplication.

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