Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein

Severe acute respiratory syndrome coronavirus (SCoV) 7a protein is one of the viral accessory proteins. In expressing cells, 7a protein exhibits a variety of biological activities, including induction of apoptosis, activation of the mitogen-activated protein kinase signaling pathway, inhibition of host protein translation, and suppression of cell growth progression. Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay, in which intact SCoV particles were specifically immunoprecipitated by anti-S protein monoclonal antibody, demonstrated that 7a protein was associated with purified SCoV particles. Coexpression of 7a protein with SCoV S, M, N, and E proteins resulted in production of virus-like particles (VLPs) carrying 7a protein, while 7a protein was not released from cells expressing 7a protein alone. Although interaction between 7a protein and another SCoV accessory protein, 3a, has been reported, 3a protein was dispensable for assembly of 7a protein into VLPs. S protein was not required for the 7a protein incorporation into VLPs, and yet 7a protein interacted with S protein in coexpressing cells. These data established that, in addition to 3a protein, 7a protein was a SCoV accessory protein identified as a SCoV structural protein.     

UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange

The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4. Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear. We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E. coli. UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA. UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein. The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.     

Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein

Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.     

Biochemical properties of the Escherichia coli recA430 protein. Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA

The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.     

Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.     

Proteolysis of a 34 kDa Phosphoprotein Coincident with a Decrease in Protein Kinase Activity During the Erythrocytic Schizont Stage of the Malaria Parasite

ABSTRACT. Protein phosphorylation events may play important roles in the replication and differentiation of the malarial parasite. Investigations into the lability of a Plasmodium protein kinase revealed that a 34 kDa parasite phosphoprotein is rapidly converted into a 19 kDa fragment. Coincident with this conversion is a nearly total loss of a protein kinase activity, as determined from the phosphorylation of endogenous protein substrates. Both the conversion of the 34 kDa protein to the 19 kDa protein and the loss of protein kinase activity are inhibited by thio-protease inhibitors. The presence of low levels of the intact 34 kDa protein restores the protein kinase activity to almost maximum levels. However, it was not possible to demonstrate protein kinase activity associated with the 34 kDa protein, thus suggesting that the 34 kDa protein is probably an activator or regulator of the protein kinase activity and not a protein kinase. The conversion to the 19 kDa fragment also occurs in vivo and only during the schizont stage prior to the appearance of ring forms. During this same period the protein kinase activity decreases suggesting that the proteolytic processing of the 34 kDa protein may be a physiological regulator of the protein kinase.     

6-Hydroxydopamine upregulates iron regulatory protein 1 by activating certain protein kinase C isoforms in the dopaminergic MES23.5 cell line

Iron-induced oxidative stress is thought to play a crucial role in the pathogenesis of Parkinson's disease. Our previous studies demonstrated that decreased expression of ferroportin 1 contributes to 6-hydroxydopamine induced intracellular iron accumulation and that decreased ferroportin 1 expression is caused by increased expression of iron regulatory protein 1. Iron regulatory protein 1 is a central regulator of iron homeostasis and is a likely target of extracellular agents to program changes in cellular iron metabolism. Therefore, the mechanism of iron regulatory protein 1 upregulation induced by 6-hydroxydopamine has become a significant focus of research. Iron regulatory protein 1 is regulated by protein kinase C, although this regulation is tissue specific. Therefore, in the present study, we aimed to determine whether alteration of protein kinase C activity modified iron regulatory protein 1 expression in the dopaminergic MES23.5 cell line, Furthermore, we investigated whether 6-hydroxydopamine induced iron regulatory protein 1 upregulation is mediated by protein kinase C, thus achieving regulation of cellular iron levels. The results showed that iron regulatory protein 1 was upregulated by phorbol 12-myristate-13-acetate, the PKC activator in dopaminergic MES23.5 cells, and ferroportin 1 expression and iron efflux were decreased as a result of iron regulatory protein 1 upregulation. The protein kinase C inhibitor bisindolylmaleimide I hydrochloride abolished the effect of phorbol 12-myristate-13-acetate. Protein kinase C-δ and protein kinase C-ζ, but not protein kinase C-? were activated by 6-hydroxydopamine. The protein kinase C-δ inhibitor rottlerin inhibited protein kinase C-δ phosphorylation and abolished iron regulatory protein 1 upregulation induced by 6-hydroxydopamine. The protein kinase C-ζ pseudo-substrate inhibitor inhibited protein kinase C-ζ phosphorylation and abolished iron regulatory protein 1 upregulation induced by 6-hydroxydopamine. These data indicate that iron regulatory protein 1 is regulated by protein kinase C in dopaminergic MES23.5 cells and that protein kinase C activated by 6-hydroxydopamine regulates iron regulatory protein 1 expression, thus achieving regulation of cellular iron levels.     

Regulation of dnaB function in DNA replication in Escherichia coli by dnaC and lambda P gene products

The dnaB protein of Escherichia coli, a multifunctional DNA-dependent ribonucleotide triphosphatase and dATPase, cross-links to ATP on ultraviolet irradiation under conditions that support rNTPase and dATPase activities of dnaB protein. The covalent cross-linking to ATP is specifically inhibited by ribonucleotides and dATP. Tryptic peptide mapping demonstrates that ATP cross-links to only the 33-kDa tryptic fragment (Fragment II) of dnaB protein. The presence of single-stranded DNA alters the covalent labeling of dnaB protein by ATP, suggesting a possible role of DNA on the mode of nucleotide binding by dnaB protein. Present studies demonstrate that the dnaC gene product binds ribonucleotides independent of dnaB protein. On dnaB-dnaC protein complex formation, covalent incorporation of ATP to dnaB protein decreases approximately 70% with a concomitant increase of ATP incorporation to dnaC protein by approximately 3-fold. The mechanism of this phenomenon has been analyzed in detail by titrating dnaB protein with increasing amounts of dnaC protein. The binding of dnaC protein to dnaB protein appears to be a noncooperative process. The lambda P protein, which interacts with dnaB protein in the bacteriophage lambda DNA replication, does not bind ATP in the presence or absence of dnaB protein. However, lambda P protein enhances the covalent incorporation of ATP to dnaB protein approximately 4-fold, suggesting a direct physical interaction between lambda P and dnaB proteins with a probable change in the modes of nucleotide binding to dnaB protein. The lambda P protein likely forms a lambda P-dnaB-ATP dead-end ternary complex. The implications of these results in the E. coli and bacteriophage lambda chromosomal DNA replication are discussed.     

Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.     

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.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.     

Endothelial protein C receptor-dependent inhibition of migration of human lymphocytes by protein C involves epidermal growth factor receptor

The protein C pathway is an important regulator of the blood coagulation system. Protein C may also play a role in inflammatory and immunomodulatory processes. Whether protein C or activated protein C affects lymphocyte migration and possible mechanisms involved was tested. Lymphocyte migration was studied by micropore filter assays. Lymphocytes that were pretreated with protein C (Ceprotin) or activated protein C (Xigris) significantly reduced their migration toward IL-8, RANTES, MCP-1, and substance P, but not toward sphingosine-1-phosphate. The inhibitory effects of protein C or activated protein C were reversed by Abs against endothelial protein C receptor and epidermal growth factor receptor. Evidence for the synthesis of endothelial protein C receptor by lymphocytes is shown by demonstration of receptor mRNA expression and detection of endothelial protein C receptor immunoreactivity on the cells' surface. Data suggest that an endothelial protein C receptor is expressed by lymphocytes whose activation with protein C or activated protein C arrests directed migration. Exposure of lymphocytes to protein C or activated protein C stimulates phosphorylation of Tyr845 of epidermal growth factor receptor, which may be relevant for cytoprotective effects of the protein C pathway.     

In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal

We expressed firstly the Capsid protein gene defecting the nuclear localization signal (NLS) of Porcine circovirus type II (PCV2) in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and the recombinant NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell and capsid protein (Cap protein) of PCV2 expressed in PK15 cells, respectively. mAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrated that rGST-dCap protein expressed in E. coli was folded correctly or at least partly, and mAbs to rdCap protein possessed the binding epitopes of PCV2 particles whereas mAbs 4C4 and 3F6 to rdCap protein remained the neutralization epitope of PCV2 particle, showing a possibility of neutralizing mAb to rdCap protein as an immnuotherapeutic agent and a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.     

Effect of low-calorie diets on plasma retinol-binding protein concentrations in overweight women

The concentrations of total protein, albumin and retinol-binding protein, a major transport protein for vitamin A, are significantly decreased by protein-calorie malnutrition. Weight-loss diets, sometimes involving severe energy deficits over prolonged periods of time, are common in the United States. The effect, if any, of prolonged low calorie weight-loss diets with normal intakes of protein on albumin, total protein and retinol-binding protein concentrations (and potentially on vitamin A metabolism) had not been extensively studied. We measured total protein, albumin, apo + holo retinol-binding protein and holo-free- and holo-transthyretin-bound retinol-binding protein concentrations during the course of a nutritionally adequate weight-loss diet (50% calorie restriction). We found that this type of dieting did not affect total protein, albumin or apo + holo, holo-free or holo-transthretin-bound retinol-binding protein concentrations significantly. This suggests that protein intake is more critical than caloric intake for retinol-binding protein status.     

Cross-linking of the proteins in the outer membrane of Escherichia coli.

1. The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A. Both forms of this protein, proteins A1 and A2, produced similar cross-linking products. No cross-linking of protein A to the peptidoglycan was detected. 2. The proteins of the isolated outer membrane varied in their ease of cross-linking. The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions. The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked. No linkage of protein A to protein B was detected. 3. Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. 4. These results suggest that specific protein-protein interactions occur in the outer membrane.     

Cultured bovine aortic endothelial cells promote activated protein C-protein S-mediated inactivation of factor Va

Previous studies have demonstrated that protein S is required for optimal activated protein C-mediated inactivation of Factor Va on the surface of either the platelet or phospholipid vesicles. In this report we demonstrate assembly of the activated protein C-protein S complex on the surface of cultured bovine aortic endothelial cells. Endothelial cell surface acceleration of Factor Va inactivation by activated protein C required the presence of protein S. Kinetic studies indicated that the rate of Factor Va inactivation was half-maximal at a protein S concentration of 0.2 nM and an activated protein C concentration of 0.05 nM. Binding of 125I-activated protein C to endothelial cell monolayers was absolutely dependent on the presence of protein S. At saturating levels of protein S, activated protein C binding was saturable with Kd = 0.04 nM. In contrast, specific, time-dependent, and saturable binding of 125I-protein S to endothelium occurred in the absence of activated protein C. Addition of activated protein C increased the affinity of protein S from Kd = 11 nM to 0.2 nM, but did not change the number of molecules bound per cell at saturation (85,000 molecules/cell). These studies suggest that activated protein C increases the affinity of protein S for pre-existing sites on the endothelial cell surface. The close correlation between the parameters of protein S-activated protein C binding to endothelium and Factor Va inactivation supports the concept that it is bound protein S and activated protein C that are the active species. Formation of functional activated protein C-protein S complexes thus occurs effectively on the endothelial cell surface and represents a new addition to the list of vessel wall anticoagulant properties.