Molecular basis for passive immunotherapy of Alzheimer's disease

Amyloid aggregates of the amyloid-{beta} (A{beta}) peptide are implicated in the pathology of Alzheimer's disease. Anti-A{beta} monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-A{beta} mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize A{beta} monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the A{beta}(1–8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to A{beta}(1–8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target A{beta} with improved specificity and higher affinity

Isolation and structure elucidation of a neuropeptide from three species of Namib Desert tenebrionid beetles

The corpora cardiaca of three Namib Desert beetles, Onymacris plana, O. rugatipennis and Physadesmia globosa (family Tenebrionidae), contain hyperlipaemic and/or hypertrehalosaemic factors as shown by heterologous bioassays in migratory locusts and American cockroaches. The compounds have been isolated by reversed-phase high performance liquid chromatography from the three species. The retention times of the compounds for all three species were identical. Amino acid composition data, in combination with determination of the primary structure by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue, reveals an identical octapeptide for the three investigated species: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-TrpNH2. This peptide, code-named Tem-HrTH, is a member of the large adipokinetic hormone/red pigment-concentrating hormone family and was previously found in two other tenebrionid beetles which are only very distantly related to the Namib beetles. Thus, it appears that during evolution of tenebrionid beetles only one AKH/RPCH-family peptide was recruited

Gonadotropin-Releasing Hormone and Adipokinetic Hormone Signaling Systems Share a Common Evolutionary Origin

Gonadotropin-releasing hormone (GnRH) is a critical and central hormone that regulates vertebrate reproduction. The high conservation of GnRH signaling within the chordates (deuterostomians) raises the important question as to whether its appearance might date back prior to the divergence of protostomian and deuterostomian lineages, about 700 million years ago. This leads to several important questions regarding the evolution of the GnRH family. Has GnRH been retained in most protostomian lineages? And was regulation of reproduction already a function of ancestral GnRH? The first question can undoubtedly be answered affirmatively since several GnRH-like sequences have been found in wide variety of protostomian and deuterostomian phyla. However, based on their different primary functions in different phyla – which implies a less unanimous answer on the second question – consistency in the nomenclature of this peptide family has been lost. A comparative and phylogenetic approach shows that the ecdysozoan adipokinetic hormones (AKHs), lophotrochozoan GnRHs and chordate GnRHs are structurally related and suggests that they all originate from a common ancestor. This review supports the view that the AKH–GnRH signaling system probably arose very early in metazoan evolution, prior to the divergence of protostomians and deuterostomians

Mass spectrometric identification of pEGFYSQRYamide: A crustacean peptide hormone possessing a vertebrate neuropeptide Y (NPY)-like carboxy-terminus

In invertebrates, peptides possessing the carboxy (C)-terminal motif -RXRFamide have been proposed as the homologs of vertebrate neuropeptide Y (NPY). Using matrix assisted laser desorption/ionization mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation and chemical and enzymatic reactions, we have identified the peptide pEGFYSQRYamide from the neuroendocrine pericardial organ (PO) of the crab Pugettia producta. This peptide is likely the same as that previously reported, but misidentified, as PAFYSQRYamide in several earlier reports (e.g. [Li, L., Kelley, W.P., Billimoria, C.P., Christie, A.E., Pulver, S.R., Sweedler, J.V., Marder, E. 2003. Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis. J. Neurochem. 87, 642-656; Fu, Q., Kutz, K.K., Schmidt, J.J., Hsu, Y.W., Messinger, D.I., Cain, S.D., de la Iglesia, H.O., Christie, A.E., Li, L. 2005. Hormone complement of the Cancer productus sinus gland and pericardial organ: an anatomical and mass spectrometric investigation. J. Comp. Neurol. 493, 607-626.]). The -QRYamide motif contained in pEGFYSQRYamide is identical to that present in many vertebrate members of the NPY superfamily. Mass spectrometric analysis conducted on the POs of several other decapods showed that pEGFYSQRYamide is present in three other brachyurans (Cancer borealis, Cancer irroratus and Cancer productus) as well as in one species from another decapod infraorder (Lithodes maja, an anomuran). Thus, our findings show that at least some invertebrates possess NPY-like peptides in addition to those exhibiting an -RXRFamide C-terminus, and raise the question as to whether the invertebrate -QRYamides are functionally and/or evolutionarily related to the NPY superfamily. © 2007 Elsevier Inc. All rights reserved

The Effect of N-Terminal Cyclization on the Function of the HIV Entry Inhibitor 5P12-RANTES.

Despite effective treatment for those living with Human Immunodeficiency Virus (HIV), there are still two million new infections each year. Protein-based HIV entry inhibitors, being highly effective and specific, could be used to protect people from initial infection. One of the most promising of these for clinical use is 5P12-RANTES, a variant of the chemokine RANTES/CCL5. The N-terminal amino acid of 5P12-RANTES is glutamine (Gln; called Q0), a residue that is prone to spontaneous cyclization when at the N-terminus of a protein. It is not known how this cyclization affects the potency of the inhibitor or whether cyclization is necessary for the function of the protein, although the N-terminal region of RANTES has been shown to be critical for receptor interactions, with even small changes having a large effect. We have studied the kinetics of cyclization of 5P12-RANTES as well as N-terminal variations of the protein that either produce an identical cyclized terminus (Glu0) or that cannot similarly cyclize (Asn0, Phe0, Ile0, and Leu0). We find that the half life for N-terminal cyclization of Gln is roughly 20 h at pH 7.3 at 37 °C. However, our results show that cyclization is not necessary for the potency of this protein and that several replacement terminal amino acids produce nearly-equally potent HIV inhibitors while remaining CC chemokine receptor 5 (CCR5) antagonists. This work has ramifications for the production of active 5P12-RANTES for use in the clinic, while also opening the possibility of developing other inhibitors by varying the N-terminus of the protein

Identification of Candidate Biomarkers for Quality Assessment of Hatchery-reared Mussel Larvae Via GC/MS-based Metabolomics

To ensure environmental and economic sustainability of future aquaculture growth, large-scale hatchery production of mollusc larvae is required. However, variation in larval quality currently limits potential maximum yields. Identification of biomarkers which reflect the immediate physiological condition of larvae during hatchery production could help monitor and determine causes of variation. Metabolomics is well-suited to this task due to its capacity for providing an instantaneous snapshot of the physiology of an organism through analysis of its metabolite profile. As a test, we applied GC/MS-based metabolomics for this purpose. Using a variety of univariate and multivariate feature selection methods, we identified four metabolite–metabolite ratios involving levels of succinate, glycine, alanine, pyroglutamate and myristic acid as candidate biomarkers for assessing mussel larval quality. These metabolites are known to have roles in energy metabolism, osmotic regulation, immune function and cell–cell communication. We anticipate that further investigation of these metabolites and their associated biochemical pathways will yield a more complete understanding of the factors responsible for larval production variability

Structure and function of sea urchin neuropeptides

PhDThe subject of this thesis is the identification and functional characterization of sea urchin neuropeptides. Neuropeptides are important mediators of neural signalling in all known animals with a nervous system, including bilaterians, ctenophorans, and cnidarians. Sea urchin neuropeptides are of particular interest for three significant reasons; echinoderms have a radically different secondarily-derived pentaradial body structure, sea urchins have served as model organisms for research into embryonic development, and thirdly because the genome of a sea urchin, the purple sea urchin Strongylocentrotus purpuratus (Stimpson, 1857) has been sequenced (Sodergren et al., 2006). Only one family of neuropeptides, the SALMFamides, has previously been characterized in all classes of the phylum Echinodermata. The thesis reports the identification of putative neuropeptide GPCRs and at least seven novel sea urchin neuropeptide genes using genomic and Expressed Sequence Tag (EST) analysis. The novel sea urchin neuropeptides identified include putative homologues of vasotocin, the sea cucumber neuropeptide NGIWYamide, thyrotropin-releasing hormone, gonadotropin-releasing hormone, and calcitonin. A further three peptide precursor genes encoding peptides lacking strong homology to any known peptides were also identified and the peptides have been named GKamides and Pedal Peptide-like Neuropeptides. Two of the peptide precursor genes, those encoding peptides homologous to vasotocin and NGIWYamide, also each encode neurophysin domains, which have previously only been identified in association with vasopressin/oxytocin-like peptides. Biochemical and pharmacological techniques were employed to investigate the occurrence and functions of the putative neuropeptides identified. These included mass spectroscopy and in vitro bioassays, the former to detect the putative novel neuropeptides identified in this study and the latter to investigate bioactivity of the peptides in sea urchins. The thesis provides evidence of the neural expression and bioactivity of novel sea urchin neuropeptides and contributes to our understanding of the role of neuropeptides in echinoderm physiology and behaviour.University of London Central Research Fun

Insect neuropeptides regulating substrate mobilisation

Insect flight muscles perform their work completely aerobically, and working flight muscles are known to be the most metabolically active tissue in nature with respect to oxygen uptake. Various substrates can be oxidised and utilised as fuels for flight. Insects such as Diptera and Hymenoptera power their flight muscles by the breakdown of carbohydrates, whereas lipids are the predominant fuel for the contracting flight muscles of Lepidoptera and Orthoptera during long-distance flight. The amino acid proline can also be used as a substrate for flight, especially in tsetse flies and beetles (Colorado potato beetle, blister beetles, certain dung beetles). Neuropeptides from the corpus cardiacum are well-known to be responsible for carbohydrate and lipid mobilisation from the fat body. In this short overview, we show that peptides belonging to the large adipokinetic hormone/red pigment-concentrating hormone family are also thought to be the chemical messengers for initiating proline homeostasis. The peptides isolated and sequenced so far from glands of beetles from the genera Pachnoda, Scarabaeus and Onitis all have a tyrosine residue (at position 2 or 4) and seem to be related to each other

A peptidomic investigation into enteroendocrine cells and islets of humans and mice

Enteroendocrine cells (EECs) of the gastrointestinal (GI) tract and islets of Langerhans (islets) of the pancreas are endocrine cells capable of sensing nutrient intake or the nutrient content of the blood and responding by secreting peptide hormones to control various physiological functions. This thesis focuses on characterising the peptidome of EECs and islets by utilising liquid-chromatography coupled to mass spectrometry (LC-MS). Due to their ability to control food intake and glucose tolerance, hormones from EECs and islets are of great therapeutic interest for the treatment of type 2 diabetes (T2D) and obesity. I hypothesised that there may be undiscovered peptide hormones derived from EECs or islets that could also be of therapeutic interest for the treatment of T2D. In order to identify potentially novel hormone candidates, peptidomic analyses of mouse EECs, mouse islets and human GI tissue were carried out utilising LC-MS. 13 interesting peptide candidates identified in the peptidomic analyses were picked out to be synthesised for in vivo and/or in vitro characterisation. All 13 peptides were screened for activity at Gs or Gi signalling pathways in various cell lines although unfortunately no peptide seemed to exhibit activity through these signalling mechanisms in the cell lines tested. Despite this, studies were carried out to assess the ability of the 2 top peptide candidates to control food intake and glucose tolerance in vivo. 1 of these peptides, a peptide derived from progastrin, seemed to mildly improve glucose tolerance in lean mice, an effect that was seen in across several studies. I hypothesised that this progastrin derived peptide may be altering peptide secretions from islets to modulate glucose tolerance but no effect of this novel peptide was seen on insulin, somatostatin-14 or glucagon secretion from islets. The progastrin derived peptide was then administered to diet-induced obese mice to assess its therapeutic potential but unfortunately no effect was seen on glucose tolerance. More work needs to be performed to verifying my findings and investigating a possible mechanism of action before this novel progastrin derived peptide may be considered a gut hormone. In regard to the peptidome of islets, there are some gaps in the literature that our initial peptidomic analysis did not answer. Questions such as do islets produce GLP1 (glucagon-like peptide 1) receptor agonists and how is the islet peptidome altered in response to metabolic stress. I therefore carried out an investigation to address these questions. Targeted LC-MS methodology was used to search for intra-islet GLP1 receptor agonists. No peptides derived from glucose-dependent insulinotropic polypeptide (GIP) were found in mouse islets but active GLP-1 was found in both human and mouse islets. Comparisons were made between the abundance of active GLP-1 and glucagon to assess which of these peptides might be responsible for the intra-islet “incretin” effect. My peptidomic analysis of islets from T2D humans and obese mice may not have revealed new major findings but it has provided some clarity over how metabolic stress alters the peptidome of islets in T2D an obesity. In summary; a comprehensive analysis of the peptidome of EECs and islets was carried out, a number of novel peptide hormone candidates were synthesised and one of which exhibited a mild improvement in the glucose tolerance of lean mice, and clarity was provided on how metabolic stress alters the islet peptidome in mice and humans. I hope that the data produced in this thesis can contribute to a better understanding of the peptidome of the EECs and islets and may contribute to advances in therapeutic options for patients suffering from obesity or T2D