scholarly journals Further characterization of the platinum-reactive component of the α2-macroglobulin-receptor recognition site

1986 ◽  
Vol 238 (1) ◽  
pp. 217-225 ◽  
Author(s):  
S V Pizzo ◽  
P A Roche ◽  
S R Feldman ◽  
S L Gonias
Keyword(s):  
Recognition Site ◽  
Surface Binding ◽  
H2o2 Treatment ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Diethyl Dithiocarbamate ◽  
Receptor Recognition ◽  
Methionine Residues

alpha 2-Macroglobulin (alpha 2M)-methylamine that had been allowed to react with cis-dichlorodiammineplatinum(II) (cis-DDP) bound with greatly reduced affinity to specific alpha 2M receptors, as determined by macrophage binding studies in vitro and plasma-clearance experiments in vivo. Subsequent reaction with diethyl dithiocarbamate completely restored receptor recognition function. The optimal effect was obtained when the diethyl dithiocarbamate concentration was twice the total platinum concentration. alpha 2M-methylamine that was allowed to react with H2O2 competed less effectively for specific cell-surface binding sites, as demonstrated by studies both in vivo and in vitro. The apparent dissociation constant was increased nearly 7-fold by a 15 min exposure to H2O2. alpha 2M-methylamine was affected significantly less by the H2O2 exposure after pretreatment with cis-DDP. Amino acid analysis indicated that H2O2 treatment of alpha 2M modified 19 of the 25 methionine residues per alpha 2M subunit. Pretreatment with cis-DDP protected two to four of these methionine residues. The only other residue altered by H2O2 treatment of alpha 2M was histidine. A net decrease of two histidine residues per subunit was observed, but cis-DDP pretreatment did not alter this result. In order to rule out the slight possibility that histidine modification might account for the observed H2O2-induced loss in receptor recognition, diethyl pyrocarbonate was employed as a histidine-modifying reagent. This treatment modified 53 histidine residues in both native and fast-form alpha 2M. Fast-form alpha 2M was still recognized by the alpha 2M receptor, as determined by studies both in vivo and in vitro; however, a fraction of the modified protein now cleared via the acyl-low-density-lipoprotein receptor as well. Reaction of diethyl pyrocarbonate-treated alpha 2M with hydroxylamine reversed derivatization of 43 of the 53 histidine residues. Moreover, this treatment also resulted in an alpha 2M fast-form preparation that was recognized only by the alpha 2M receptor. It is concluded that cis-DDP and H2O2 modify a critical methionine residue in the primary sequence of the alpha 2M-receptor recognition site.

Surface binding and uptake of polycationic ferritin by neonatal piglet intestinal epithelium

1980 ◽  
Vol 46 (1) ◽  
pp. 235-252
Author(s):  
C.C. Chase ◽  
E.A. Munn
Keyword(s):  
Specific Interaction ◽  
Age Groups ◽  
Similar Distribution ◽  
Surface Binding ◽  
Protein Uptake ◽  
In Vivo Experiments ◽  
Absorptive Cells ◽  
Apical Vesicles

Pieces of small intestine from newborn, I- and 6-day-old piglets were incubated in vitro and ligated segments were incubated in vivo with polycationic ferritin (PCF) to determine the distribution and possible role in protein uptake of anionic sites on membranes at the luminal surfaces of the epithelial cells. Most PCF binding to the absorptive cells occurred on the upper third or half of the villi and to some non-absorptive cells (tuft cells) throughout the length of the villi. Pieces of intestine which were fixed before incubation had PCF on microvilli and apical invaginations of absorptive cells, but none in the sub-apical tubules. When samples were incubated with PCF in vitro before fixation PCF was bound to the surfaces of microvilli of absorptive cells and to the membranes lining the apical invaginations, some sub-apical vesicles and some sub-apical tubules in all age-groups. In vivo experiments with longer incubation times resulted in a similar distribution, with increased amounts of PCF in the sub-apical tubules in samples from newborn and 1-day-old piglets only. In the newborn there were small vesicles containing PCF which had apparently moved further into the cells. At the same concentration (2 mg/ml) ferritin did not enter the sub-apical tubules; but it did enter and was taken up by the cells in the presence of 4% (w/v) serum albumin. It is concluded that the mechanism of protein uptake does not involve the microvillar membrane and that non-specific interaction with the invaginated plasma membrane, as a function of charge-density, leads to opening of the sub-apical tubular system to protein. Movement further into the cell depends on other factors.

Inhibition of leukocyte locomotion by hyaluronic acid

1981 ◽  
Vol 48 (1) ◽  
pp. 315-331
Author(s):  
J.V. Forrester ◽  
P.C. Wilkinson
Keyword(s):  
Molecular Weight ◽  
Visual Analysis ◽  
Cell Movement ◽  
Chemotactic Factor ◽  
Major Constituent ◽  
Dependent Manner ◽  
Surface Binding ◽  
Micropore Filter

The effect of hyaluronate on neutrophil motility in vitro was studied by the micropore filter technique and by direct visual analysis of the locomotion of neutrophils on glass. Both directed and random locomotion of neutrophils was inhibited by physiological concentrations (0.5-6.0 mg ml(−1)) of hyaluronate in a dose- and molecular weight-dependent manner. Inhibition of cell movement was more pronounced for high molecular weight chemoattractants such as casein than for small chemotactic peptides such as f-Met-Leu-Phe. Chemotactic factor gradient formation in filter chambers was profoundly retarded by hyaluronate, which may partly explain the inhibitory effects of hyaluronate on directed neutrophil locomotion. In addition, hyaluronate inhibited the binding of chemotactic factor to the neutrophil surface. This effect, together with a reduction in cell-to-substratum adhesion, may provide an additional explanation for hyaluronate-induced inhibition of random neutrophil locomotion. Inhibition of locomotion by hyaluronate was easily reversed by washing the cells free of hyaluronate; thus competition by hyaluronate for cell-surface binding sites is unlikely, and physical effects such as steric exclusion or molecular sieving by the large hyaluronate polymer provide the most probable explanations of its inhibitory effect on cell locomotion. Since hyaluronate is a major constituent of tissue matrices, these results draw attention to the importance of the extracellular environment in regulating inflammatory cell movement in vivo.

scholarly journals Heme A Synthase Enzyme Functions Dissected by Mutagenesis of Bacillus subtilis CtaA

2005 ◽  
Vol 187 (24) ◽  
pp. 8361-8369 ◽  
Author(s):  
Lars Hederstedt ◽  
Anna Lewin ◽  
Mimmi Throne-Holst
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Mechanism ◽  
Enzyme Reaction ◽  
Prosthetic Group ◽  
Histidine Residues ◽  
Functional Roles ◽  
Heme A Synthase ◽  
Respiratory Oxidases

ABSTRACT Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.

scholarly journals Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

2000 ◽  
Vol 182 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
Volkmar Braun
Keyword(s):  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Binding Assays ◽  
Surface Binding ◽  
Citrate Transport ◽  
C Terminus ◽  
N Terminus ◽  
Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

Oxidation-resistant variants of recombinant anti-leucoprotease are better inhibitors of human-neutrophil-elastase-induced emphysema in hamsters than natural recombinant antileucoprotease

1991 ◽  
Vol 81 (6) ◽  
pp. 777-784 ◽  
Author(s):  
A. Rudolphus ◽  
R. Heinzel-Wieland ◽  
V. A. M. M. Vincent ◽  
D. Saunders ◽  
G. J. Steffens ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Site Directed Mutagenesis ◽  
Human Neutrophil Elastase ◽  
Polymorphonuclear Leucocytes ◽  
Inhibitory Effects ◽  
Oxidative Inactivation ◽  
Methionine Residues

1. Antileucoprotease, being sensitive to oxidative inactivation, can be produced by recombinant techniques. Via site-directed mutagenesis, two mutants of recombinant antileucoprotease were produced in which one or more of the oxidation-sensitive methionine residues were replaced by leucine: in rALP242, methionine-73 was replaced by leucine, and in rALP231, leucine was substituted for four methionine residues. In vitro, native antileucoprotease and the recombinant antileucoprotease preparations have similar inhibitory characteristics towards human neutrophil elastase. We hypothesized that replacement of methionine residues in the antileucoprotease molecule would result in a reduced oxidation sensitivity of the mutants. 2. After incubation of recombinant antileucoprotease and its mutants with increasing dosages of cis-platinum(II)diammine dichloride, we observed that native antileucoprotease and recombinant antileucoprotease were inactivated by this reagent to the same extent. Compared with this, rALP242 was less inactivated, whereas the inhibitory capacity of rALP231 was not influenced by cis-platinum(II)diammine dichloride at all. 3. After incubation of recombinant antileucoprotease, rALP242 and rALP231 with triggered polymorphonuclear leucocytes, which are thought to produce an excess of oxidants, we measured residual inhibitory activities towards human neutrophil elastase of 10%, 55% and 87%, respectively. 4. In vivo, the inhibitory effects of intratracheally administered rALP242 and rALP231 towards human-neutrophil-elastase-induced emphysema were significantly greater than that of recombinant antileucoprotease. There were no significant differences between the mutants. With respect to secretory cell metaplasia and haemorrhage, rALP231 tended to be a better inhibitor than recombinant antileucoprotease and rALP242. 5. We conclude that the recombinant antileucoprotease mutants are less sensitive to oxidation and consequently inhibit human-neutrophil-elastase-induced emphysema to a greater extent than recombinant antileucoprotease.

scholarly journals In Vitro and In Vivo Protective Effects of Lentil (Lens culinaris) Extract against Oxidative Stress-Induced Hepatotoxicity

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 59
Author(s):  
Yeon-Seop Jung ◽  
So-Hee Lee ◽  
So Young Chun ◽  
Dae Hwan Kim ◽  
Byung Ik Jang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Serum Levels ◽  
Liver Cells ◽  
Dependent Manner ◽  
Protective Effects ◽  
H2o2 Treatment ◽  
Antioxidant Genes ◽  
Hepatic Diseases

Excessive oxidative stress plays a role in hepatotoxicity and the pathogenesis of hepatic diseases. In our previous study, the phenolic extract of beluga lentil (BLE) showed the most potent in vitro antioxidant activity among extracts of four common varieties of lentils; thus, we hypothesized that BLE might protect liver cells against oxidative stress-induced cytotoxicity. BLE was evaluated for its protective effects against oxidative stress-induced hepatotoxicity in AML12 mouse hepatocytes and BALB/c mice. H2O2 treatment caused a marked decrease in cell viability; however, pretreatment with BLE (25–100 μg/mL) for 24 h significantly preserved the viability of H2O2-treated cells up to about 50% at 100 μg/mL. As expected, BLE dramatically reduced intracellular reactive oxygen species (ROS) levels in a dose-dependent manner in H2O2-treated cells. Further mechanistic studies demonstrated that BLE reduced cellular ROS levels, partly by increasing expression of antioxidant genes. Furthermore, pretreatment with BLE (400 mg/kg) for 2 weeks significantly reduced serum levels of alanine transaminase and triglyceride by about 49% and 40%, respectively, and increased the expression and activity of glutathione peroxidase in CCl4-treated BALB/c mice. These results suggest that BLE protects liver cells against oxidative stress, partly by inducing cellular antioxidant system; thus, it represents a potential source of nutraceuticals with hepatoprotective effects.

Bevacizumab Immune Complexes Induce Thrombocytopenia and Thrombosis in Mice Transgenic for Human IgG Receptor FcYIIa

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 114-114
Author(s):  
Ali Amirkhosravi ◽  
Todd V Meyer ◽  
Liza Robles-Carillo ◽  
Florian Langer ◽  
Theresa Robson ◽  
...  
Keyword(s):  
Immune Complexes ◽  
Binding Domain ◽  
Vascular Endothelial ◽  
Heparin Binding ◽  
Surface Binding ◽  
Platelet Surface ◽  
Heparin Binding Domain ◽  
Surface Localization

Abstract Background: The anti-VEGF drug, bevacizumab (Bev), has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. Prevailing opinion on the molecular mechanism behind Bev-associated bleeding and thrombosis is that tissue factor driven coagulation, secondary to vascular endothelial cell dysfunction, may cause thrombosis due to VEGF suppression by Bev. Bev forms immune complexes (IC) with VEGF (vascular endothelial growth factor), a heparin-binding protein. In our previous in vitro studies we showed that, in the presence of heparin, Bev+VEGF immune complexes activate platelets via the IgG receptor FcγRIIa —a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia (HIT). Objectives: First, we investigated whether Bev-associated thrombosis might be replicated in mice. Because mouse platelets do not carry FcγRIIa, we used mice transgenic for this human IgG receptor (hFcR mice) in order to enable the signaling pathway identified above. Second, using human platelets in vitro, we studied the functional roles of heparin and platelet surface localization of IC in Bev-induced FcγRIIa activation. Methods: Bev+VEGF IC were preformed using VEGF165 or VEGF121 (similar to VEGF165 but lacking the heparin-binding domain). Platelet dense granule release and aggregation were measured by the serotonin release assay (SRA) and Chrono- Log aggregometers, respectively. Platelet surface localization was assessed by flow cytometry (50,000 events/test condition) and fluorescence microscopy using Alexa488- labeled Bev (Bev488). For in vivo studies, Bev+VEGF+Heparin IC (60–500 nM) or control reagents were injected intravenously into wild-type (WT) or hFcR mice. Platelet counts were measured 10–60 minutes following IC injection after obtaining blood (0.45 ml) by cardiac puncture. Immediately afterward, lungs were processed for hematoxylin and eosin staining and analyzed microscopically for evidence of thrombosis. Results: IC consisting of Bev+VEGF165+Heparin (0.2U/ml) caused thrombotic thrombocytopenia in hFcR but not WT mice, showing a requirement for FcγRIIa. Injection of Bev+VEGF121+Heparin (0.2U/ml) into hFcR mice did not cause thrombocytopenia, suggesting a requirement for the VEGF165 heparin binding domain. Bev+VEGF165 was without effect in the absence of heparin or in the presence of excess (200 U/ml) heparin demonstrating that a limited range of heparin concentrations enable Bev-induced thrombocytopenia and thrombosis. This mechanism is similar to that observed in HIT and our in vivo results were consistent with SRA and aggregation in vitro studies. By flow cytometry, maximal Fab-dependent Bev488 platelet surface binding occured only with VEGF165+0.2U/ml heparin. Saturating IV.3 (anti-FcγRIIa antibody) concentrations, present in all samples, excluded Bev-Fc binding to FcγRIIa. Furthermore, binding of Bev488+VEGF121+0.2 U/ml heparin was not detected, suggesting the VEGF heparin binding domain is required for heparin-enhanced surface binding. Conclusions: In the presence of heparin, Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of FcγRIIa receptor. This mechanism may be relevant to the thromboembolic complications observed in patients receiving Bev therapy.

scholarly journals A reassessment of copper(II) binding in the full-length prion protein

2006 ◽  
Vol 399 (3) ◽  
pp. 435-444 ◽  
Author(s):  
Mark A. Wells ◽  
Graham S. Jackson ◽  
Samantha Jones ◽  
Laszlo L. P. Hosszu ◽  
C. Jeremy Craven ◽  
...  
Keyword(s):  
Prion Protein ◽  
Metal Ion ◽  
Tandem Repeats ◽  
Binding Mode ◽  
Full Length ◽  
Alternative Mode ◽  
Histidine Residues ◽  
Terminal Domain

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.

scholarly journals The dengue virus non-structural protein 1 (NS1) use the scavenger receptor B1 as cell receptor in human hepatic and mosquito cells

2019 ◽  
Author(s):  
Ana C. Alcalá ◽  
José L. Maravillas ◽  
David Meza ◽  
Octavio T. Ramirez ◽  
Juan E. Ludert ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Disease ◽  
Scavenger Receptor ◽  
Direct Interaction ◽  
Cell Receptor ◽  
Serum Lipoprotein ◽  
Structural Protein ◽  
Surface Binding

AbstractDengue is the most common virus disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional protein that form part of replication complexes. In addition, NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several different mechanisms. Cell binding and internalization of soluble NS1 result in the disruption of tight junctions and in down regulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells, and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptor for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh-7 cells, was demonstrated by confocal microcopy. Internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies and previous incubation of the cells with HDL significantly reduces NS1 internalization. In addition, the transient expression of SRB1 in Vero cells, which lack the receptor, renders these cells susceptible to NS1 entry. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in vivo by proximity ligation assays an in vitro by surface plasmon resonance. Finally, data is presented indicating that the SRB1 also act as cell receptor for zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients.

scholarly journals An Activity-Based Methionine Bioconjugation Approach to Developing Proximity-Activated Imaging Reporters

2019 ◽  
Author(s):  
Jun Ohata ◽  
Lakshmi Krishnamoorthy ◽  
Monica Gonzalez ◽  
Tong Xiao ◽  
Diana Iovan ◽  
...  
Keyword(s):  
Protein Detection ◽  
Specific Protein ◽  
Protein Activity ◽  
Protein Targets ◽  
Sensing Platform ◽  
Starting Point ◽  
Methionine Residues ◽  
Endogenous Activity

Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small-molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

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