Serpin-enzyme Complex Research Articles

Is the Serpin-Enzyme Complex Receptor an Orphan G-Protein Coupled Receptor?

Alpha-1 antitrypsin (α1-AT) is a member of serine proteinase inhibitors, collectively known as serpins, that inactivate their target enzymes by forming a stabilized serpin-enzyme complex. α1-AT molecules interact with elastase, forming biologically active complexes. It was suggested that the formation of α1-AT-elastase complex would expose a domain from α1-AT molecule that is recognized by a specific cell receptor. Thus, a synthetic peptide, called 105Y, was made based on the sequence of the exposed domain. The peptide binds specifically to human hepatoma HepG2 cells, human monocytes, human neutrophils, and rat pheochromocytoma cells (PC12). This binding is blocked by α1-AT-elastase complex and other serpin-enzyme complexes, suggesting an existence of a specific cell surface receptor, termed SEC receptor, that recognizes serpin enzyme complexes. However, the identity of SEC receptor is still unknown. Thus, we decided to use the Deductive Reasoning Strategy approach to investigate if the SEC receptor belongs to G-protein-coupled receptor (GPCR) family. We measured responsiveness of HepG2 cells as well as human neuroblastoma SHSY5Y cells to 105Y peptide by quantifying their expression of an early response gene c-Fos using real time quantitative polymerase chain reaction (qPCR) and measured their expression of orphan GPCRs using polymerase chain reaction (PCR) followed by gel electrophoresis. Our results indicate that the cells were responding to 105Y peptide and express certain orphan GPCRs. Our goal is to evaluate the necessity of these oGPCRs in 105Y peptide signaling in order to determine the best candidate(s) for the 105Y receptor. These results would shed light on the identity of the SEC receptor for better understanding of the molecular biology and physiology of the serpin-enzyme complexes and their receptors. National Institute of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-Inflammatory Therapy for Acute Coronary Syndromes: Is It Time for a Shift in the Treatment Paradigm?

Anti-Inflammatory Therapy for Acute Coronary Syndromes: Is It Time for a Shift in the Treatment Paradigm?

Aβ31-35 Decreases Neprilysin-Mediated Alzheimer's Amyloid-β Peptide Degradation.

Alzheimer's disease is associated with the deposition of extracellular senile plaques, made primarily of amyloid-β (Aβ), particularly peptides Aβ1-42 and Aβ1-40. Neprilysin, or neutral endopeptidase (NEP), catalyzes proteolysis of the amyloid peptides (Aβ) and is recognized as one of the major regulators of the levels of these peptides in the brain, preventing Aβ accumulation and plaque formation. Here, we used a combination of techniques to elucidate the mechanism of Aβ binding and cleavage by NEP. Our findings indicate that the Aβ31-X cleavage products remain bound to the neprilysin active site, reducing proteolytic activity. Interestingly, it was already shown that this Aβ31-35 sequence is also critical for recognition of Aβ peptides by other targets, such as the serpin-enzyme complex receptor in neuronal cells.

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

Ligand-functionalized nanoliposomes for targeted delivery of galantamine

The purpose of this study was to design ligand-functionalized nanoliposomes that are proficient in providing effective intracellular delivery of an alkaloid drug (galantamine) into PC12 neuronal cells in response to managing Alzheimer's disease (AD). Ligand-functionalized nanoliposomes were produced and validated for their physicochemical properties, in silico molecular mechanics energy relationships, ex vivo cytotoxicity, peptide coupling efficiency (PCE), drug entrapment efficiency (DEE), drug release, fluorometry and confocal microscopy. Particle sizes of the nanoliposomes ranged from 127 nm to 165 nm (PdI=0.39-0.03), zeta potential values of -18 mV to -36 mV, PCE from 40% to 78% while DEE ranged from 42% to 79%. The surface morphology of the nanoliposomes was stable, spherically and uniform in shape. Thermal behavior and Fourier transform infrared (FTIR) analyses confirmed that galantamine and the peptide-ligand were incorporated into the inner core and surface of the nanoliposomes, respectively. The optimized formulation showed sustained drug release (30% of drug released within 48 h). Fluorometry and confocal microscopy revealed that the ligand-functionalized nanoliposomes facilitated galantamine uptake into PC12 neuronal cells via the Serpin Enzyme Complex Receptor in a mediated manner. CytoTox-Glo™ cytotoxicity assay established the low cytotoxicity on PC12 neuronal cells when exposed to native nanoliposomes and the ligand-functionalized nanoliposomes. Response surface analysis demonstrated there was a high degree of correlation between the experimental and fitted values. Furthermore, ex vivo studies showed that the high galantamine accumulation into PC12 neuronal cells was influenced by the post-engineering of peptides on the surface of the galantamine-loaded nanoliposomes. MMER analysis aptly corroborated the experimental findings.

Fabrication of 105Y, Alpha 1‐Anti Trypsin Peptide Conjugated Hybrid Nanoparticles for Biological Applications

AbstractIn this report, we describe the characterizations and applications of hybrid nanoparticles. These nanoparticles have been synthesized by combination of organometallic, polymerization process and functionalized with a specific peptide for targeting expressed serpin‐enzyme complex (SEC) receptor of human hepatoma HepG2 cells. By using peptide conjugated hybrid nanoparticles, the specific receptor targeting, collections of cells were successfully achieved. The cell collection results indicated that, the maximum up to 95.32% of HepG2 cell were collected. The 5‐dimethylthiazol‐2‐yl‐2,5‐diphenyltetrazolium bromide (MTT) assay of HepG2 cells incubated with these nanoparticles indicated that, the peptide conjugated hybrid nanoparticles did not possess significant cytotoxicity. The rotating magnetic field induced cell death studies indicated that, the HepG2 cell showed up to 70% of cell death was induced by hybrid nanoparticles under magnetic field. Concluding, these studies demonstrate that the hybrid nanoparticles have the capability of effective separation, imaging, targeting and killing of the human hepatoma cells.

Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells

Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. To anchor three serpins -α(1) -proteinase inhibitor (α(1) PI) (M358R), antithrombin (AT), and heparin cofactor II (HCII) - in the plasma membranes of transfected mammalian cells and assess their ability to inhibit thrombin. Serpin cDNAs were altered to include N-terminal, non-cleavable plasma membrane-targeting sequences from the human transferrin receptor (TR) (TR-serpin) or the human asialoglycoprotein receptor (AR) (AR-serpin), and used to transfect COS-1 or HEK 293 cells. Cells were analyzed for serpin expression by immunoblotting of subcellular fractions, by immunofluorescence microscopy, or by flow cytometry, with or without exposure to exogenous thrombin; AR-serpins and TR-serpins were also compared with their soluble recombinant counterparts. Both TR-α(1) PI (M358R) and AR-α(1) PI (M358R) were enriched in the integral membrane fraction of transfected COS-1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α(1) PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR-α(1) PI (M358R). Surface-displayed AR-AT also formed serpin-enzyme complexes with thrombin, but to a lesser extent than AR-α(1) PI (M358R); AR-HCII inhibitory function was not detected. Immunofluorescence detection and flow cytometric quantification of bound thrombin also supported the status of AR-α(1) PI (M358R) and AR-AT as thrombin inhibitors. Two of three thrombin-inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.

Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor

Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD.

C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.

Neuroserpin Binds Aβ and Is a Neuroprotective Component of Amyloid Plaques in Alzheimer Disease

Alzheimer disease is characterized by extracellular plaques composed of Abeta peptides. We show here that these plaques also contain the serine protease inhibitor neuroserpin and that neuroserpin forms a 1:1 binary complex with the N-terminal or middle parts of the Abeta(1-42) peptide. This complex inactivates neuroserpin as an inhibitor of tissue plasminogen activator and blocks the loop-sheet polymerization process that is characteristic of members of the serpin superfamily. In contrast neuroserpin accelerates the aggregation of Abeta(1-42) with the resulting species having an appearance that is distinct from the mature amyloid fibril. Neuroserpin reduces the cytotoxicity of Abeta(1-42) when assessed using standard cell assays, and the interaction has been confirmed in vivo in novel Drosophila models of disease. Taken together, these data show that neuroserpin interacts with Abeta(1-42) to form off-pathway non-toxic oligomers and so protects neurons in Alzheimer disease.

Investigating serpin–enzyme complex formation and stability via single and multiple residue reactive centre loop substitutions in heparin cofactor II

Introduction Following thrombin cleavage of the reactive centre (P1–P1′; L444–S445) of the serpin heparin cofactor II (HCII), HCII traps thrombin (IIa) in a stable inhibitory complex. To compare HCII to other serpins we substituted: the P13–P5′ residues of HCII with those of α 1-proteinase inhibitor (α 1-PI), α 1-PI (M358R), or antithrombin (AT); the P4–P1, P3–P1, and P2–P1 residues of HCII with those of AT; and made L444A/H/K/M or R point mutations. We also combined L444R with changes in the glycosaminoglycan binding domain collectively termed MutD. Materials and methods Variants were made by site-directed mutagenesis, expressed in bacteria, purified and characterized electrophoretically and kinetically. Results and conclusions Of the P13–P5′ mutants, only the α 1-PI-loop variant retained anti-IIa activity, but less than the corresponding L444M. Heparin-catalyzed rate constants for IIa inhibition were reduced vs. wild-type (WT) by at most three-fold for all P1 mutants save L444A (reduced 20-fold). L444R and L444K inhibited IIa > 50- and > 6-fold more rapidly than WT in heparin-free reactions, but stoichiometries of inhibition were increased for all variants. HCII–IIa complexes of all P1 variants were stable in the absence of heparin, but those of the L444K and L444R variants released active IIa over time with heparin. Limited proteolysis of these two groups of HCII–IIa complexes produced different fragmentation patterns consistent with conformational differences. The combination of either substituted AT residues at P2, P3, and P4, or the MutD mutations with L444R resulted in complex instability with or without heparin. This is the first description of HCII–IIa complexes of transient stability forming in the absence of heparin, and may explain the extent to which the reactive centre loop of HCII differs from that of AT.

201. C105Y, a Novel Cell Penetrating Peptide Enhances Gene Transfer of Sec-R Targeted Molecular Conjugates

We have used poly-L-lysine (polyK) condensed DNA complexes to deliver DNA to cells both in vitro and in vivo. In order to target and increase cellular uptake of these complexes, we covalently attached a small peptide ligand, C105Y (CSIPPEVKFNKPFVYLI), to polyK before DNA condensation. This peptide was initially identified as an amino acid sequence within the carboxy-terminal tail of a1-antitrypsin (a1-AT) that could mediate binding to the serpin enzyme complex receptor (sec-R). This putative receptor was characterized, mainly by biochemical means as a cell surface binding site on human hepatoma HepG2 cells and blood monocytes. The synthetic peptide C105Y based on amino acids sequence 346-374 of a1-AT, competed with the natural ligand for binding to the sec-R and provided gene transfer into cells when it was conjugated to polyK - DNA complexes. These particles, which average <50 nm in diameter by EM, provide gene transfer of the CFTR gene in vivo to airway epithelial cells from CFTR deficient mice.

1104. Imaging of sec-R Directed and PEG-Stabilized Gene Transfer Nanoparticles in CF Mice

Compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of human CFTR DNA sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the noses of CF knock out mice. To examine the distribution of these compacted DNA particles in vivo, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 |[mu]|g of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 |[mu]|Ci each), while 3 control mice received administration of free I-125 (specific activity 50 |[mu]|Ci each). We used a small animal gamma scanner to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control. Both test and control groups begin to exhibit thyroid uptake by 2 hrs, while significantly higher activities were observed in the nasal and lower airways for animals that received the targeted complexes vs. controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p<0.05) higher activities for the targeted delivery in nose, lung and liver vs. controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. We also examined expression conferred by second generation PEG-stabilized compacted nanoparticles containing the Firefly luciferase gene using bioluminescent imaging. Expression was detected in lung 2 and 4 days following intra-tracheal (IT) but not IN administration. Lung homogenates from both methods of administration exhibited luciferase activity although levels in the IT group were significantly higher. These data suggest that a threshold level of expression, achieved only with IT administration, is necessary for successful bioluminescent detection . Development of these real-time imaging techniques has allowed us to better assess the localization and site of activity of our gene transfer complex formulations.

Defining strategies to extend duration of gene expression from targeted compacted DNA vectors.

Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1 -/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally (n=10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting.

358. Route of Administration of sec-R Targeted Compacted DNA Influences Both the Site of Expression and Host Immune Response to the Vector

DNA can be condensed, using poly-L-lysine, under high salt conditions, into discrete unimolecular (with respect to DNA) particles, 12–30 nm in diameter. We have shown that modification of these complexes with a peptide ligand (C105Y) that binds the serpin enzyme complex receptor (sec-R) targets them to lung, liver and spleen after intravenous administration and to airway epithelial cells after intratracheal administration in mice. The receptor is present on the apical surface of airway epithelia in mice and humans. We tested the effect of different routes of administration, luminal or intravenous (IV), on host antibody response to vector and delivery of a secreted protein, Factor IX, into the blood. C57BL/6 mice (n = 10) received a transtracheal application of a 50 μl bolus of a 0.5 M NaCl solution containing 5 μg of sec-R targeted compacted pCMV FIX plasmid (codes for secretable human Factor IX driven by the CMV promoter). Control groups included animals that received 5 μg non-targeted compacted pCMV FIX in 0.5 M NaCl (n = 5), 0.5 M NaCl alone (n = 5), and 5 μg sec-R targeted pCMV FIX injected intravenously via the tail vein (n = 10). Blood was sampled on days 2, 10, and 28. Human Factor IX was detected only in the plasma of animals that received sec-R targeted complexes. The luminal application group achieving expression levels of 65–90 ng/ml plasma while the IV group achieved 59–145 ng/ml plasma. Expression persisted for 10 days but diminished by 28 days, which may be due in part to development of anti-Factor IX-antibodies. When the complexes were administered IV (n = 36), 19 of 36 immuno-competent mice dosed with sec-R targeted human Factor IX, following initial dosing with sec-R targeted lac Z complexes 3 weeks earlier, developed antibodies against the vector by 28 days, but the antibodies did not affect the efficacy of re-administration. Animals treated with saline alone or non-targeted complexes did not produce antibodies to targeted complexes. When luminal administration was used, no antibody response is observed even in animals that receive targeted complexes. In conclusion, compacted DNA, targeted to the sec-R, is effective in delivering genes in vivo following luminal or intravenous administration. The route of administration influences both the site of expression as well as the production of antibodies against the complex. Furthermore, luminal administration achieves levels of secreted protein expression and delivery to the blood comparable to what is achieved by the intravenous route.

829. Preferential Transfection of Tissue Macrophages Following Intravenous Administration of sec-R Targeted hCFTR DNA Enhances Survival of CF Mice Inoculated with Pseudomonas aeruginosa Agar Beads

Previously, we have shown that modification of Poly K compacted DNA complexes with a peptide ligand (C105Y) that binds the serpin enzyme complex receptor (sec-R) targets them to lung, liver and spleen after intravenous (IV) administration and to airway epithelial cells after intranasal (IN) administration in CF mice. Predominantly, tissue macrophages were transfected following IV dosing. Since both epithelial cells and macrophages express CFTR, we took advantage of these predilection to test whether CFTR delivery to macrophages or to epithelial cells protected against the consequences of Pseudomonas aeruginosa infection. We tested the effect of different routes of administration of sec-R targeted hCFTR, IN versus IV, on alleviating the consequence of the inoculation of CF mice with Pseudomonas aeruginosa laden agar beads. This method of infection induces inflammation in CF mice that models advanced disease in CF patients. Three groups of S489X/FABP-hCFTR mice (n=14 per group) housed in sterile micro-isolator cages were studied. Group 1 received a 50 μl bolus of a 1.0 M NaCl solution containing 10 μg of sec-R targeted compacted hCFTR plasmid (codes for human CFTR driven by the elongation factor 1 promoter) via the tail vein injection and 50 μl 1.0 M NaCl alone IN. Group 2 received 50 μl sec-R targeted hCFTR IN and 50 μl 1.0 M NaCl alone IV. Control group animals received 50 μl 1.0 M NaCl IV and IN. One day following dosing the mice were inoculated with 5 × 104 cfu P. aeruginosa embedded in agar beads, and their weights recoded. We monitored the mice for 10 days following infection. Ten of fourteen mice that received IV administration of sec-R targeted hCFTR survived the course of the experiment, while 6/14 survived for the IN dosed group and 3/14 survived for the control group (p=0.03, ANOVA). Weight loss that occurs following infection was reversed one day earlier on day 4 for groups 1 and 2 versus day 5 for control animals. These results were reproduced in 2 other experiments. In a fourth experiment, analysis of bronchoalveolar lavage (BAL) from animals similarly dosed and infected, but sacrificed on day 3 following infection revealed no significant differences in cell counts or production of TNF-α, IL-1β, IL-6, or mip-2/KC (mouse IL-8 homologs). Based on previous observations, these data suggest that delivery of hCFTR to tissue macrophages rather than airway epithelial cells allows for better protection against and/or recovery from infection in CF mice. Subtle decreases in cytokine production may be sufficient for this, although other event involved in infection and inflammation not assayed for by us may have been impacted more significantly by hCFTR gene transfer to tissue macrophages.

832. Radioscintigraphy of sec-R Directed CFTR Gene Transfer in CF Mice Following Intranasal Administration

Previously, we have shown that compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of a human CFTR vector sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the CF knock out mouse nose. To examine the distribution of these compacted DNA particles in vivo following administration, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 μg of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 μCi each), while 3 control mice received administration of free I-125 (specific activity 50 μCi each). We used a small animal gamma scanner produced at Thomas Jefferson National Lab to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control at the assayed time points. Both test and control groups begin to exhibit thyroid uptake by 2 hrs. By 2 hrs, scintigraphic and autoradiographic data indicated high activities in the nasal and lower airways in animals that received the targeted complexes compared to controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p < 0.05) higher activities for the targeted delivery in nose, lung and liver compared with controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways (nasal region) and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. Intranasal administration may also result in aspiration or ingestion of the targeted complexes. Alternatively, these complexes or their components may have access to and enter the circulation and accumulate in the liver.

Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease.

The extracellular accumulation of amyloid-beta (Abeta) in neuritic plaques is one of the characteristic hallmarks of Alzheimer's disease (AD), a progressive dementing neurodegenerative disorder of the elderly. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteins and proteoglycans. The binding of the various forms of Abeta (soluble or fibrillar) to plasma membranes has been studied with regard to the direct toxicity of Abeta to neurons, and the activation of a local inflammation phase involving microglia. The binding of Abeta to membrane lipids facilitates Abeta fibrillation, which in turn disturbs the structure and function of the membranes, such as membrane fluidity or the formation of ion channels. A subset of membrane proteins binds Abeta. The serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind the monomeric form of Abeta. The alpha7nicotinic acetylcholine receptor (alpha7nAChR), integrins, RAGE (receptor for advanced glycosylation end-products) and FPRL1 (formyl peptide receptor-like 1) are able to bind the monomeric and fibrillar forms of Abeta. In addition, APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), the P75 neurotrophin receptor (P75NTR), the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha6beta1-integrin and CD47 have been reported to bind the fibrillar form of Abeta. Heparan sulfate proteoglycans have also been described as cell-surface binding sites for Abeta. The various effects of Abeta binding to these membrane molecules are discussed.

Binding sites of amyloid beta-peptide in cell plasma membrane and implications for Alzheimer's disease.

The binding of amyloid beta peptides (Abeta) to plasma membranes appears to be a promising point of intervention in the events leading to the development of Alzheimer's disease (AD). This binding has been studied as regards the direct toxicity of Abeta on neurons, and the activation of a local inflammation phase involving microglia. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteoglycans and proteins. This review focuses on the membrane proteins that can mediate the interaction between Abeta and the plasma membranes in AD. On neurons, these are APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), integrins, the alpha7nicotinic acetylcholine receptor (alpha7nAChR), the P75 neurotrophin receptor (P75NTR) and the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV). On glial cells, FPRL1 (formyl peptide receptor-like 1), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha(6)beta(1)-integrin and CD47, and heparan sulfate proteoglycans have been reported to bind Abeta. It should be noted that integrins, RAGE (receptor for advanced glycosylation end-products), the Serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind Abeta and are present on neurons and on glial cells. After a presentation of the structure and the function of each of these proteins, the method used to prove their binding to Abeta is described, and the implication of this binding in AD is discussed. Finally, it is underlined that multireceptor complexes containing integrins may be involved in this interaction.

Squamous Cell Carcinoma Antigen 1-mediated Binding of Hepatitis B Virus to Hepatocytes Does Not Involve the Hepatic Serpin Clearance System

The cellular receptor for hepatitis B virus (HBV) has not yet been identified. A recent candidate is a homologue of squamous cell carcinoma antigen 1 (SCCA1), a serpin. This study confirms that transfection of SCCA1 into mammalian cells (both hepatocyte-derived and of non-hepatocyte origin) results in increased HBV binding. Furthermore, virus bound to transfected cells is protected significantly from degradation by trypsin (75% compared with 30% in untransfected cells). The possibility that HBV enters cells via the hepatic clearance system for serpin-enzyme complexes was investigated by analysis of the reactive site loop of SCCA1. Functional and deletion mutants of SCCA1 were constructed by site-directed mutagenesis and compared with the wild type construct. In no case was virus binding reduced by functional alterations or deletions within the reactive site loop. A possible role for the low density lipoprotein receptor-related protein (LRP) in binding virus was investigated. SCCA1 transfection of Huh7 cells was shown to result in up-regulation of LRP expression, reaching levels observed in total liver. However, the use of receptor-associated protein (RAP), a competitive ligand for LRP, suggests than LRP up-regulation is not responsible for enhanced virus binding to SCCA1-transfected cells.