Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.     

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.     

Prion protein polymerisation triggered by manganese-generated prion protein seeds

Prion diseases are neurodegenerative diseases that can be transmitted between individuals. The exact cause of these diseases remains unknown. However, one of the key events associates with the disease is the aggregation of a cellular protein, the prion protein. The mechanism of this is still unclear. However, it is likely that the aggregation is trigged by a seeding mechanism in which an oligomer of the prion protein is able to catalyse polymerisation of further prion protein into larger aggregates. We have developed a model of this process using an oligomeric species generated from recombinant protein by exposure to manganese. On fractionation of the seeding species, we estimated that the smallest size the oligomer would be is an octomer. We analysed the catalytic mechanism of the seeding oligomer and its interaction with substrate. Different domains of the protein are necessary for the seeding ability of the prion protein as opposed to those required for it to form a substrate for the polymerisation reaction. Prion seeds formed from different sheep alleles are able to reproduce the characteristics of scrapie in terms of resistance to disease. However, we were also able to generate prion seed from chicken PrP a species where no prion disease is known. Our findings provide an insight into the aggregation process of the prion protein and its potential relation to disease progress.     

Regulation of activated protein C by thrombin-modified protein S

Protein S, a vitamin K-dependent plasma protein having Gla-residues, increases the rate of inactivation of Factor Va by activated protein C by enhancing the binding of activated protein C to phospholipid [Walker, J.F. (1981) J. Biol. Chem. 256, 11128-11131]. The present study aimed at elucidating the effect of thrombin-modified protein S on Factor Va inactivation by activated protein C. Nondigested protein S consisted 81% of intact form and 19% of modified form, and thrombin-digested protein S had 96% modified form. Protein S, both nondigested and digested, did not show any effects on the amidolytic activity of activated protein C towards synthetic peptide substrate. Nondigested protein S stimulated the Factor Va inactivation by activated protein C, whereas the digested protein appeared to suppress the inactivation. Protein-phospholipid binding experiments showed that although nondigested protein S enhanced the binding of activated protein C to phospholipid stoichiometrically, digested protein S appeared to not only suppress the complex formation, but also dissociate the complex. This evidence suggested that protein S modified by thrombin regulates the action of activated protein C towards Factor Va on phospholipid.     

Activation of cGMP-dependent protein kinase by protein kinase C

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.     

Altering biomineralization by protein design

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.     

Serum protein degradation by hypochlorite

The structural integrity of serum proteins: albumin, immunoglobulin G, transferrin, ceruloplasmin and superoxide dismutase, and the functional activity of the latter two enzymes after their interaction with hypochlorite were studied. It was shown that the interaction between the proteins and hypochlorite resulted in protein injury and degradation of their native structure. In the case of ceruloplasmin and transferrin, a practically complete protein "dissipation" occurred, the albumin and superoxide dismutase structures being injured in a lesser degree. The inactivation of ceruloplasmin was slower than that of superoxide dismutase. The protein degradation by hypochlorite seems to be the main factor restricting the ability of the proteins to act as antiinflammatory drugs.     

Membrane reshaping by protein condensates

Proteins can organize into dynamic, functionally important assemblies on fluid membrane surfaces. Phase separation has emerged as an important mechanism for forming such protein assemblies on the membrane during cell signaling, endocytosis, and cytoskeleton regulation. Protein-protein phase separation thus adds novel fluid mosaics to the classical Singer and Nicolson model. Protein condensates formed in this process can modulate membrane morphologies. This is evident from recent reports of protein condensate-driven membrane reshaping in processes such as endocytosis, autophagosome formation, and protein storage vacuole morphogenesis in plants. Lateral phase separation (on the membrane surface) of peripheral curvature coupling proteins can modulate such membrane morphological transitions. Additionally, three-dimensional protein phase separation can result in droplets that through adhesion can affect membrane shape changes. How do these condensate-driven curvature generation mechanisms contrast with the classically recognized scaffolding and amphipathic helix insertion activities of specific membrane remodeling proteins? A salient feature of these condensate-driven membrane activities is that they depend upon both macroscopic features (such as interfacial energies of the condensate, membrane, and cytosol) as well as microscopic, molecular-level interactions (such as protein-lipid binding). This review highlights the current understanding of the mechanisms underlying curvature generation by protein condensates in various biological pathways.     

Phosphorylation of a chromaffin granule-binding protein by protein kinase C

Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Cellular regulation by protein phosphorylation

A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca²⁺ and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.     

Signalling by cGMP-dependent protein kinases

The second messenger cGMP is a major intracellular mediator of the vaso-active agents nitric oxide and natriuretic peptides. The principal targets of cGMP are (i) phosphodiesterases, resulting in interference with the CAMP-signalling pathway, (ii) cGMPgated cation channels, and (iii) cGMP-dependent protein kinases (cGKs). Only two mammalian isotypes of cGK have been described so far: type I cGK, consisting of an and a isoform, presumably splice variants of a single gene, and identified as the most prominent cGK isotype in the cardio-vascular system; and type II cGK, expressed mainly in the intestine, the kidney and the brain. High levels of cGK I are found in vascular smooth muscle cells, endothelial cells and platelets. In these cells, cGK I is thought to counteract the increase in contraction provoked by Ca-mobilizing agonists, to reduce endothelial permeability and to inhibit platelet aggregation, respectively. Relatively low levels of cGK I are found in cardiomyocytes. In this cell type, cGK is implicated in the negative inotropic effect of cGMP, presumably through modulation of Ca channels and by diminishing the Ca-sensitivity of contractile proteins.     

Identification of protein bands in polyacrylamide gel by protein printing

Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.     

Enhancing recombinant protein quality and yield by protein stability profiling

The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.