Epitope-specific antibody fragments block aggregation of AGelD187N, an aberrant peptide in gelsolin amyloidosis.

Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We employed phage display technology to identify novel single-chain variable fragments (scFvs) that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size exclusion chromatography combined with multi-angle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.

Development of an in vitro aggregation assay for long synthetic polypeptide, amyloidogenic gelsolin fragment AGelD187N 173-242.

Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173-242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173-242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173-242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.

Novel delivery systems for improving the clinical use of peptides.

Peptides have long been recognized as a promising group of therapeutic substances to treat various diseases. Delivery systems for peptides have been under development since the discovery of insulin for the treatment of diabetes. The challenge of using peptides as drugs arises from their poor bioavailability resulting from the low permeability of biological membranes and their instability. Currently, subcutaneous injection is clinically the most common administration route for peptides. This route is cost-effective and suitable for self-administration, and the development of appropriate dosing equipment has made performing the repeated injections relatively easy; however, only few clinical subcutaneous peptide delivery systems provide sustained peptide release. As a result, frequent injections are needed, which may cause discomfort and additional risks resulting from a poor administration technique. Controlled peptide delivery systems, able to provide required therapeutic plasma concentrations over an extended period, are needed to increase peptide safety and patient compliancy. In this review, we summarize the current peptidergic drugs, future developments, and parenteral peptide delivery systems. Special emphasis is given to porous silicon, a novel material in peptide delivery. Biodegradable and biocompatible porous silicon possesses some unique properties, such as the ability to carry exceptional high peptide payloads and to modify peptide release extensively. We have successfully developed porous silicon as a carrier material for improved parenteral peptide delivery. Nanotechnology, with its different delivery systems, will enable better use of peptides in several therapeutic applications in the near future.

Sympathetic nervous system-targeted neuropeptide Y overexpression in mice enhances neointimal formation in response to vascular injury.

Sympathetic neurotransmitter neuropeptide Y (NPY) is associated with vascular remodelling, neointimal hyperplasia and atherosclerosis in experimental animal models and clinical studies. In order to study the role of sympathetic nerve-produced NPY in vascular diseases, transgenic mouse model overexpressing NPY in central and peripheral noradrenergic neurons under the dopamine-beta-hydroxylase (DBH) promoter was recently created (OE-NPY(DBH) mouse). This study aimed to examine the effect of NPY overexpression on arterial neointimal hyperplasia in an experimental model of vascular injury. Transgenic OE-NPY(DBH) mice and wildtype control mice of two different inbred strains (C57BL/6 and FVB/n) underwent a femoral artery surgery with a transluminar injury by a 0.38-mm guide wire insertion. Arteries were harvested 4 weeks from the surgery, and they were stained for basic morphology. Both strains of OE-NPY(DBH) mice, as compared with wildtype control mice, showed on average 50% greater formation of the neointima (P<0.01) and an increase in the medial area (P=0.05). The results suggest that moderately increased neuronal NPY causes the arteries to be more susceptible to femoral artery thickening after endothelial injury. The OE-NPY(DBH) mouse provides a novel tool to explore the role of NPY in the development of vascular disease related to metabolic disorders.

The intracellular mobility of NPY and a putative mitochondrial form of NPY in neuronal cells.

Preproneuropeptide Y is a precursor peptide to mature neuropeptide Y (NPY), which is a universally expressed peptide in the central and peripheral nervous system. NPY is normally routed to endoplasmic reticulum and secretory vesicles in cells, which secrete NPY. In our previous studies, we found a functional Leucine7 to Proline7 (L7P) polymorphism in the signal peptide sequence of preproNPY. This polymorphism affects the secretion of NPY and causes multiple physiological effects in humans. The sequence of NPY mRNA contains two in frame kozak sequences that allow translation initiation to shift, and translation of two proteins. In addition to mature NPY(1-36) also a putative truncated NPY(17-36) with mitochondrial targeting signal is produced. The purpose of this study was to investigate the protein mobility of the putative mitochondrial fragment and the effect of the L7P polymorphism on the cellular level using GFP tagged constructs. The mobility was studied with fluorescence recovery after photobleaching technique in a neuronal cell line. We found that the mobility of the secretory vesicles with NPY(1-36) in cells with L7P genotype was increased in comparison to vesicle mobility in cells with the more abundant L7L genotype. The mobility in the cells with the putative mitochondrial construct was found to be very low. According to the results of the present study, the mitochondrial truncated peptide stays in the mitochondrion. It can be hypothesized that this could be one of the factors affecting energy balance of the membranes of the mitochondrion.

Leucine 7-proline 7 polymorphism in the signal peptide of neuropeptide Y is not a risk factor for exudative age-related macular degeneration.

PURPOSE: Because of the regulatory role of neuropeptide Y (NPY) in angiogenesis, we set out to determine the presence of the leucine 7-proline 7 (Leu7Pro) polymorphism in exudative age-related macular degeneration (AMD) patients and to analyse its implications. METHODS: Genotype analysis of the Leu7Pro polymorphism in the signal peptide region of the human prepro-NPY was performed in blood samples from exudative AMD patients (n = 240) and control subjects (n = 79). RESULTS: In all, 11% of exudative AMD patients and 14% of control subjects exhibited the NPY signal peptide Leu7Pro polymorphism. There were no statistically significant differences in Leu7Pro polymorphism frequency between the exudative AMD and control cases, as analysed by Fisher's exact two-sided test. CONCLUSIONS: Leu7Pro polymorphism in the signal peptide region of the human prepro-NPY is not a risk factor for exudative AMD.

Mitochondrial targeting signal in human neuropeptide Y gene.

Neuropeptide Y (NPY) is universally expressed in many different neuronal and non-neuronal cells. Human NPY gene has two in-frame kozak sequences and thus, has potentially two translation initiation sites producing two NPY peptides with different molecular weights. In the present study, the intracellular location of NPY was studied in endothelial cells endogenously expressing NPY, and in neuronal (SK-N-BE) and non-neuronal (CHO-K1) cells transfected with NPY-GFP-constructs. By mutating kozak sequences we discovered that kozak-1 directs the NPY peptide to secretory vesicles, and kozak-2 is a prerequisite for mitochondrial targeting. If both kozak sequences are present, non-neuronal cells seem to benefit leaky scanning to initiate translation at both initiation sites, in contrast to neuronal cells, which prefer the kozak-1. This finding suggests that both the kozak sequences of NPY mRNA can be used in the translation depending on the cell type. The size and the function of the novel NPY fragment routed to mitochondria remains to be determined.

The Leu7Pro polymorphism of preproNPY is associated with decreased insulin secretion, delayed ghrelin suppression, and increased cardiovascular responsiveness to norepinephrine during oral glucose tolerance test.

CONTEXT: Neuropeptide Y (NPY) plays a role in angiogenesis, cardiovascular regulation, and hormone secretion. The leucine7 to proline7 (Leu7Pro) polymorphism of preproNPY is associated with vascular diseases and has an impact on hormone levels in healthy subjects. OBJECTIVE: The current study investigated the role of the Leu7Pro polymorphism in metabolic and cardiovascular autonomic regulation. DESIGN AND SUBJECTS: A 5-h oral glucose tolerance test was performed on 27 healthy volunteers representing two preproNPY genotypes (Leu7/Pro7 and Leu7/Leu7) matched for age, sex, body mass index and physical activity. MAIN OUTCOME MEASURES: Simultaneously we performed cardiovascular autonomic function tests and plasma measurements of sympathetic transmitters, glucose, insulin, and ghrelin. RESULTS: The subjects with Leu7/Pro7 genotype had decreased plasma NPY, norepinephrine (NE), and insulin concentrations and insulin to glucose ratios. The suppression of ghrelin concentrations after glucose ingestion was delayed in these subjects. They also had increased heart rate variability indices and baroreflex sensitivity. However, they displayed significant negative association of NE concentration with variability of low-frequency R-R-intervals and with baroreflex sensitivity. CONCLUSIONS: The Leu7Pro polymorphism of preproNPY is related to decreased level of basal sympathetic activity, decreased insulin secretion, and delayed ghrelin suppression during oral glucose tolerance test. The increased responsiveness of autonomic functions to NE associated with the polymorphism may be connected to increased cardiovascular vulnerability.

Obesity in transgenic female mice with constitutively elevated luteinizing hormone secretion.

Transgenic (TG) female mice, expressing a chimeric bovine luteinizing hormone (LH) beta-subunit/human chorionic gonadotropin beta-subunit COOH-terminal extension (bLHbeta-CTP) gene, produce high levels of circulating LH and serve as a model for functional ovarian hyperandrogenism and follicular cysts. We report here that obesity is a typical feature of these female mice. The mean body weight of the bLHbeta-CTP females was significantly higher than in controls at, and beyond 5 wk of age, and at 5 mo, it was 32% increased. At this age, the amount of white adipose tissue in the bLHbeta-CTP females was significantly increased, as reflected by the weight difference of the retroperitoneal fat pad. In addition, the expression of leptin mRNA in white adipose tissue of the TG females was elevated about twofold. Serum leptin and insulin levels, and food intake, were also increased significantly in the TG females. Brown adipose tissue (BAT) thermogenic activity, as measured by GDP binding to BAT mitochondria, was reduced (P < 0.05). Ovariectomy at the age of 3 wk totally prevented the development of obesity. In summary, the present results show that intact female bLHbeta-CTP mice are obese, have increased food consumption, and reduced BAT thermogenic activity. The weight gain can be explained partly by elevated androgens but is probably also contributed to the increased adrenal steroidogenesis. Hence, the bLHbeta-CTP mice provide a useful model for studying obesity related to elevated LH secretion, with consequent alterations in ovarian and adrenal function.

Deletion polymorphism in the alpha2B-adrenergic receptor gene is associated with flow-mediated dilatation of the brachial artery.

A deletion variant of the alpha(2B)-adrenergic receptor (alpha(2B)-AR) has been associated with an increased risk of acute cardiac events in middle-aged men. Our aim was to determine the possible associations between the alpha(2B)-AR gene deletion variant and indicators of subclinical atherosclerosis in the brachial and carotid arteries. A total of 148 middle-aged men participating in an epidemiological twin study on risk factors for subclinical coronary heart disease were genotyped using PCR. Flow-mediated dilatation (FMD) of the brachial artery, carotid artery compliance and carotid intima-media thickness were measured using high-resolution ultrasound. FMD was 6.2+/-5.0% in subjects with the I/I (insertion/insertion) genotype, 5.5+/-4.1% in the I/D (insertion/deletion) group and 4.1+/-3.8% in the D/D (deletion/deletion) group ( P =0.03 for trend). In multivariate regression analysis controlling for age, presence of hypertension, smoking, use of angiotensin-converting enzyme inhibitors and plasma levels of low-density lipoprotein cholesterol and lipoprotein (a), the association between the alpha(2B)-AR genotype and FMD remained significant ( P =0.04 for trend). The alpha(2B)-AR genotype was not associated with intima-media thickness or carotid artery compliance. These findings indicate that subjects homozygous for the deletion allele of alpha(2B)-AR appear to have an increased risk of impaired endothelial function, which may provide an explanation for the previously observed increased risk of myocardial infarction in male subjects with this genotype. It is not known whether the association of the alpha(2B)-AR polymorphism with endothelial function is direct, or is mediated via altered sympathetic activation.