Inhibition of the Bacterial Cell Wall Synthesis in vitro by Enduracidin,a New Polypeptide Antibiotic

Adenosine 5′-phosphosulfate (APS) kinase from a thermophilic bacterium, Bacillus st ear other mophilus, was purified to apparent homogeneity. The apparent molecular weight was 50 kDa, consisting of two 26-kDa subunits. The enzyme was very thermostable and lacked cysteine and methionine residues. Enzyme activity was more stimulated with Mn^{2 +} , Zn^{2 +}, or Co^{2 +} than with Mg^{2 +} and the K_m for ATP and APS were 220 µM and 42 µM, respectively.     

Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid–ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.     

Investigation of liver binding of pentachlorophenol based upon measurement of protein adducts

Abstract

Covalent binding of reactive metabolites of pentachloropheno (PCP) was investigated both in vitro andin vivo in the livers of male Sprague-Dawley rats via measurement of protein adducts. Cysteinyl adducts of quinones andsemiquinones in liver cytosolic (Cp) andnuclear (Np) proteins were assayed after catalytic cleavage by Raney nickel. Results from in vitro experiments confirmed that PCP metabolism produced tetrachlorobenzoquinones andthe comsponding tetrachlorobentosemiquinones which subsequently bound to sulphydryl groups in liver proteins. In vivo, the production of cysteinyl adducts increased with the administered dosage (0–40 mg PCP per kg body weight) andpresented evidence of saturable metabolism. Results suggest two metabolic pathways for PCP, including a high-affinity low-capacity pathway anda low-affinity high-capacity pathway. Time-course experiments in vivo andin vitro suggested that quinone adducts partlcipated in multiple substitution reactions with protein and/or non-protein thiols, andpointed to possible formation of protein-protein cross-links in vivo. The elimination rate constants of quinone adducts in vitro were about 0.35 h^?1 in liver Cp. The elimination of quinone adducts in vivo appeared to follow biphasic kinetics with rate constants for the terminal phase being 0.014 and0.008 h^?1 in liver Cp andNp, respectively.     

Overexpression and Purification of an Immunologically Reactive His–BIV Capsid Fusion Protein

The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)₆p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-β- -galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein inEscherichia coli,allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)₆p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.     

Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Cyclic nucleotide selectivity of protein kinase G isozymes

The intrinsic activity of the C‐terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)‐dependent protein kinases (PKG) is inhibited by interactions with the N‐terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R‐domain disrupts the inhibitory R–C interaction, leading to the release and activation of the C‐domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C‐domain are more cGMP selective and therefore critical for cGMP‐dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme‐specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB‐mediated dimeric contacts that indicate cGMP‐driven reorganization of domain–domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.     

Glycoproteomics Landscape of Asymptomatic and Symptomatic Human Alzheimer’s Disease Brain

Molecular changes in the brain of individuals afflicted with Alzheimer's disease (AD) are an intense area of study. Little is known about the role of protein abundance and posttranslational modifications in AD progression and treatment, in particular large-scale intact N-linked glycoproteomics analysis. To elucidate the N-glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography, and LC-MS–based glycoproteomics. We analyzed brain tissue from 10 persons with no cognitive impairment or AD, 10 with asymptomatic AD, and 10 with symptomatic AD, detecting over 300 glycoproteins and 1900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic or complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain N-glycoproteome, occurring at <9% among the glycoforms. We observed AD-associated differences in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among samples from our three groups. Further analysis revealed glycosylation differences in subcellular compartments across disease stage, including glycoproteins in the lysosome frequently modified with paucimannosidic glycans. These results illustrate the N-glycoproteomics landscape across the spectrum of AD clinical and pathologic severity and will facilitate a deeper understanding of progression and treatment development.