scholarly journals Identification of amino acids at the junction of exons 3 and 7 that are used for the generation of glycosylation-related human CD45RO and CD45RO-like antigen specificities.

1994 ◽  
Vol 179 (3) ◽  
pp. 1035-1040 ◽  
Author(s):  
R Pulido ◽  
S F Schlossman ◽  
H Saito ◽  
M Streuli
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Protein ◽  
Tyrosine Phosphatase ◽  
Cell Lineage ◽  
Lymphocyte Activation ◽  
Structural Basis ◽  
Common Precursor ◽  
A Cell ◽  
T Cell Maturation

The CD45 transmembrane protein tyrosine phosphatase plays an essential role in lymphocyte activation. In humans, CD45 is composed of five isoforms that are generated by alternative splicing of three exons of a common precursor mRNA. Expression of the smallest molecular mass 180-kD CD45 isoform (CD45-O) results from splicing out of exons 4(A), 5(B), and 6(C), which encode peptide regions near the NH2 terminus, and is regulated during T cell maturation and activation. Two monoclonal antibodies (mAb), UCHL1 (anti-CD45RO) and A6 (anti-CD45RO-like), were studied that selectively bind to murine transfectant cells expressing the human CD45-O isoform. The anti-CD45RO-like A6 mAb, but not the anti-CD45RO UCHL1 mAb, also weakly reacted with transfectant cells expressing the human CD45 isoforms that contained exons 4 and 5(AB), or exon 5(B) encoded sequences. The structural basis of the antigen specificities of these two different human anti-CD45RO mAbs was investigated at the molecular level by using potential glycosylation-defective CD45-O isoform variants containing amino acid substitutions at the junction of exons 3 and 7. Replacement of the threonine residue at position 8 (last amino acid encoded in exon 3 and a putative O-linked carbohydrate anchorage site) by an alanine, completely abrogated the reactivity of the UCHL1 mAb, but did not affect that of the A6 mAb. Conversely, replacement of either the asparagine at position 174 or the serine at position 176 (the first two putative carbohydrate anchorage sites in exon 7) by alanine, abrogated the reactivity of the A6 mAb, but not that of the UCHL1 mAb. Both the UCHL1 and A6 epitopes were dependent on the presence of O-linked carbohydrates; and the UCHL1, but not the A6 epitope, was dependent on the presence of sialic acid. These results demonstrate a pivotal role for the amino acids encoded at the junction of exons 3 and 7 for the generation of glycosylation-related CD45RO epitopes that are expressed in a cell lineage- and activation-regulated fashion.

scholarly journals An Aspartate-Specific Solute-Binding Protein Regulates Protein Kinase G Activity To Control Glutamate Metabolism in Mycobacteria

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Nabanita Bhattacharyya ◽  
Irene Nailain Nkumama ◽  
Zaccheus Newland-Smith ◽  
Li-Ying Lin ◽  
Wen Yin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Protein Kinase ◽  
Transmembrane Protein ◽  
Nutrient Utilization ◽  
Protein Kinase G ◽  
Structural Basis ◽  
Ligand Specificity ◽  
Content Type

ABSTRACTSignaling by serine/threonine phosphorylation controls diverse processes in bacteria, and identification of the stimuli that activate protein kinases is an outstanding question in the field. Recently, we showed that nutrients stimulate phosphorylation of the protein kinase G substrate GarA inMycobacterium smegmatisandMycobacterium tuberculosisand that the action of GarA in regulating central metabolism depends upon whether it is phosphorylated. Here we present an investigation into the mechanism by which nutrients activate PknG. Two unknown genes were identified as co-conserved and co-expressed with PknG: their products were a putative lipoprotein, GlnH, and putative transmembrane protein, GlnX. Using a genetic approach, we showed that the membrane protein GlnX is functionally linked to PknG. Furthermore, we determined that the ligand specificity of GlnH matches the amino acids that stimulate GarA phosphorylation. We determined the structure of GlnH in complex with different amino acid ligands (aspartate, glutamate, and asparagine), revealing the structural basis of ligand specificity. We propose that the amino acid concentration in the periplasm is sensed by GlnH and that protein-protein interaction allows transmission of this information across the membrane via GlnX to activate PknG. This sensory system would allow regulation of nutrient utilization in response to changes in nutrient availability. The sensor, signaling, and effector proteins are conserved throughout theActinobacteria, including the important human pathogenMycobacterium tuberculosis, industrial amino acid producerCorynebacterium glutamicum, and antibiotic-producingStreptomycesspecies.IMPORTANCETuberculosis (TB) kills 5,000 people every day, and the prevalence of multidrug-resistant TB is increasing in every country. The processes by which the pathogenMycobacterium tuberculosissenses and responds to changes in its environment are attractive targets for drug development. Bacterial metabolism differs dramatically between growing and dormant cells, and these changes are known to be important in pathogenesis of TB. Here, we used genetic and biochemical approaches to identify proteins that allowM. tuberculosisto detect amino acids in its surroundings so that it can regulate its metabolism. We have also shown how individual amino acids are recognized. The findings have broader significance for other actinobacterial pathogens, such as nontuberculous mycobacteria, as well asActinobacteriaused to produce billions of dollars of amino acids and antibiotics every year.

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

scholarly journals Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

2020 ◽  
Vol 117 (20) ◽  
pp. 10806-10817 ◽  
Author(s):  
Michael P. Torrens-Spence ◽  
Ying-Chih Chiang ◽  
Tyler Smith ◽  
Maria A. Vicent ◽  
Yi Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Convergent Evolution ◽  
Structural Features ◽  
Divergent Evolution ◽  
Structural Basis ◽  
Acid Decarboxylase ◽  
Substrate Selectivity ◽  
Amino Acid Decarboxylase ◽  
Catalytic Mechanisms

Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

scholarly journals PTP1B Modulates the Association of β-Catenin with N-cadherin through Binding to an Adjacent and Partially Overlapping Target Site

2002 ◽  
Vol 277 (51) ◽  
pp. 49989-49997 ◽  
Author(s):  
Gang Xu ◽  
Carlos Arregui ◽  
Jack Lilien ◽  
Janne Balsamo
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Tyrosine Phosphatase ◽  
Target Site ◽  
Target Domain ◽  
Chick Retina ◽  
Cell Permeable Peptide ◽  
A Cell ◽  
Relationship Of

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to competein vitrobinding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function.

scholarly journals Incorporation of 3H from δ-(l-α-amino (4,5-3H)adipyl)-l-cysteinyl-d-(4,4-3H)valine into isopenicillin N

1979 ◽  
Vol 184 (2) ◽  
pp. 421-426 ◽  
Author(s):  
J O'Sullivan ◽  
R C Bleaney ◽  
J A Huddleston ◽  
E P Abraham
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Adipic Acid ◽  
Experimental Error ◽  
Acid Oxidase ◽  
Cephalosporium Acremonium ◽  
Amino Acid Oxidase ◽  
Free System ◽  
A Cell ◽  
Isopenicillin N

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.

scholarly journals A Novel Cytoplasmic Tail MXXXL Motif Mediates the Internalization of Prostate-specific Membrane Antigen

2003 ◽  
Vol 14 (12) ◽  
pp. 4835-4845 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Gopalakrishnapillai Anilkumar ◽  
Eri Oshima ◽  
James U. Bowie ◽  
He Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Protein ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Cytoplasmic Tail ◽  
Prostate Specific Membrane Antigen ◽  
Amino Acid Residues ◽  
Terminal Amino ◽  
Specific Membrane

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the α-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative μ2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

scholarly journals The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 5053
Author(s):  
Alina K. Bakunova ◽  
Alena Yu. Nikolaeva ◽  
Tatiana V. Rakitina ◽  
Tatiana Y. Isaikina ◽  
Maria G. Khrenova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Structural Analysis ◽  
Active Site ◽  
Active Sites ◽  
Structural Basis ◽  
Active Site Residues ◽  
Type Iv ◽  
Fold Type ◽  
Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

Phylogenetic and topological analyses of the bovine interferon-induced transmembrane protein (IFITM3)

2021 ◽  
Author(s):  
Yong-Chan Kim ◽  
Byung-Hoon Jeong
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Multiple Sequence Alignment ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Multiple Sequence ◽  
Interspecific Differences ◽  
Distinct Features

AbstractInterferon-induced transmembrane protein 3 (IFITM3) plays a pivotal role in antiviral capacity in several species. However, to date, investigations of the IFITM3 protein in cattle have been rare. According to recent studies, interspecific differences in the IFITM3 protein result in several unique features of the IFITM3 protein relative to primates and birds. Thus, in the present study, we investigated the bovine IFITM3 protein based on nucleotide and amino acid sequences to find its distinct features. We found that the bovine IFITM3 gene showed a significantly different length and homology relative to other species, including primates, rodents and birds. Phylogenetic analyses indicated that the bovine IFITM3 gene and IFITM3 protein showed closer evolutionary distance with primates than with rodents. However, cattle showed an independent clade among primates, rodents and birds. Multiple sequence alignment of the IFITM3 protein indicated that the bovine IFITM3 protein contains 36 bovine-specific amino acids. Notably, the bovine IFITM3 protein was predicted to prefer inside-to-outside topology of intramembrane domain 1 (IMD1) and inside-to-outside topology of transmembrane domain 2 by TMpred and three membrane embedding domains according to the SOSUI system.

scholarly journals Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

2007 ◽  
Vol 73 (17) ◽  
pp. 5598-5606 ◽  
Author(s):  
Jeong Min Seo ◽  
Geun Eog Ji ◽  
Sang Hee Cho ◽  
Myung Soo Park ◽  
Hyong Joo Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Bifidobacterium Longum ◽  
Deae Cellulose ◽  
Cell Extract ◽  
Coding Region ◽  
Tryptic Fragment ◽  
C Terminus ◽  
A Cell

ABSTRACT A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.

Peptidsynthese im zellfreien System unter dem Einfluß von Hefe-Ribonucleinsäure

1968 ◽  
Vol 23 (1) ◽  
pp. 50-58 ◽  
Author(s):  
W. Duntze ◽  
W. Atzpodien ◽  
B. Ulrich ◽  
H. Holzer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Gel Filtration ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Total Rna ◽  
Acid Incorporation ◽  
Yeast Ribosomes ◽  
A Cell

Total RNA from yeast stimulates the incorporation of 14C-amino acids into material insoluble in hot trichloroacetic acid in a cell-free E. coli system. In a sucrose gradient the stimulating RNA fraction sediments together with the 28 S fraction of ribosomal RNA. RNA from isolated yeast ribosomes preincubated with RNase was active in amino acid incorporation as well.Preincubation of ribosomal RNA at 70° resulted in an increased incorporation activity of the RNA. However, attempts to separate an active messenger fraction from total RNA as well as from 28 S RNA by heating were unsuccessful. The presented data indicate that ribosomal RNA itself is active in cell-free amino acid incorporation.By hydrolysis of the incorporation products it could be shown that the 14C-amino acids used in the cell free system were incorporated into peptides. The bulk of the radioactive peptides had a molecular weight below 2000 as estimated by Sephadex gel filtration.

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