Aggregation and neurotoxicity of alpha-synuclein and related peptides

Fibrillar deposits of alpha-synuclein occur in several neurodegenerative diseases. Two mutant forms of alpha-synuclein have been associated with early-onset Parkinson's disease, and a fragment has been identified as the non-amyloid-beta peptide component of Alzheimer's disease amyloid (NAC). Upon aging, solutions of alpha-synuclein and NAC change conformation to beta-sheet, detectable by CD spectroscopy, and form oligomers that deposit as amyloid-like fibrils, detectable by electron microscopy. These aged peptides are also neurotoxic. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified. NAC(8-18) is the smallest fragment that aggregates, as indicated by the concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. Fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Hence residues 8-16 of NAC comprise the region crucial for toxicity. Toxicity induced by alpha-synuclein, NAC and NAC(1-18) oligomers occurs via an apoptotic mechanism, possibly initiated by oxidative damage, since these peptides liberate hydroxyl radicals in the presence of iron. Molecules with anti-aggregational and/or antioxidant properties may therefore be potential therapeutic agents.

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

The complex array of bradykinin-related peptides (BRPs) in the peptidome of pickerel frog (Rana palustris) skin secretion is the product of transcriptional economy.

Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Flatworm nerve-muscle: structural and functional analysis

Platyhelminthes occupy a unique position in nerve-muscle evolution, being the most primitive of metazoan phyla. Essentially, their nervous system consists of an archaic brain and associated pairs of longitudinal nerve cords cross-linked as an orthogon by transverse commissures. Confocal imaging reveals that these central nervous system elements are in continuity with an array of peripheral nerve plexuses which innervate a well-differentiated grid work of somatic muscle as well as a complexity of myofibres associated with organs of attachment, feeding, and reproduction. Electrophysiological studies of flatworm muscles have exposed a diversity of voltage-activated ion channels that influence muscle contractile events. Neuronal cell types are mainly multi- and bi-polar and highly secretory in nature, producing a heterogeneity of vesicular inclusions whose contents have been identified cytochemically to include all three major types of cholinergic, aminergic, and peptidergic messenger molecules. A landmark discovery in flatworm neurobiology was the biochemical isolation and amino acid sequencing of two groups of native neuropeptides: neuropeptide F and FMRFamide-related peptides (FaRPs). Both families of neuropeptide are abundant and broadly distributed in platyhelminths, occurring in neuronal vesicles in representatives of all major flatworm taxa. Dual localization studies have revealed that peptidergic and cholinergic substances occupy neuronal sets separate from those of serotoninergic components. The physiological actions of neuronal messengers in flatworms are beginning to be established, and where examined, FaRPs and 5-HT are myoexcitatory, while cholinomimetic substances are generally inhibitory. There is immunocytochemical evidence that FaRPs and 5-HT have a regulatory role in the mechanism of egg assembly. Use of muscle strips and (or) muscle fibres from free-living and parasitic flatworms has provided baseline information to indicate that muscle responses to FaRPs are mediated by a G-protein-coupled receptor, and that the signal transduction pathway for contraction involves the second messengers cAMP and protein kinase C.

Multivariate Statistical Analysis Applied to an IL6 Signal Transduction Model in Hepatocytes

This paper introduces the application of linear multivariate statistical techniques, including partial least squares (PLS), canonical correlation analysis (CCA) and reduced rank regression (RRR), into the area of Systems Biology. This new approach aims to extract the important proteins embedded in complex signal transduction pathway models.The analysis is performed on a model of intracellular signalling along the janus-associated kinases/signal transducers and transcription factors (JAK/STAT) and mitogen activated protein kinases (MAPK) signal transduction pathways in interleukin-6 (IL6) stimulated hepatocytes, which produce signal transducer and activator of transcription factor 3 (STAT3).A region of redundancy within the MAPK pathway that does not affect the STAT3 transcription was identified using CCA. This is the core finding of this analysis and cannot be obtained by inspecting the model by eye. In addition, RRR was found to isolate terms that do not significantly contribute to changes in protein concentrations, while the application of PLS does not provide such a detailed picture by virtue of its construction.This analysis has a similar objective to conventional model reduction techniques with the advantage of maintaining the meaning of the states prior to and after the reduction process. A significant model reduction is performed, with a marginal loss in accuracy, offering a more concise model while maintaining the main influencing factors on the STAT3 transcription.The findings offer a deeper understanding of the reaction terms involved, confirm the relevance of several proteins to the production of Acute Phase Proteins and complement existing findings regarding cross-talk between the two signalling pathways.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased similar to6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappa B pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.

Limnonectins: A new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis)

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating “shotgun” cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog – the Fujian large-headed frog, Limnonectes fujianensis – and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 µM) and are devoid of haemolytic activity at concentrations up to 160 µM. Thus the “shotgun” cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data

Ran Is a Potential Therapeutic Target for Cancer Cells with Molecular Changes Associated with Activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK Pathways

Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We nowinvestigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.

Expression of C5a receptor in mouse brain: Role in signal transduction and neurodegeneration

In this study we explored the potential role of the complement derived anaphylatoxin C5a and the expression of its receptor in mouse brain. Using in situ hybridization, we found that C5a receptor messenger RNA is expressed in mouse brain. In response to intraventricular kainic acid injection, there was marked increase in the C5a receptor messenger RNA expression, particularly in hippocampal formation and cerebral cortex. C5a ligand-binding autoradiography confirmed the functional expression and elevation of the C5a receptor post-lesioning. The expression of Cia receptor messenger RNA in brain was confirmed by northern blot hybridization of total RNA from neuronal and glial cells in vitiro. Based on these findings we explored the role of C5a in mechanisms of signal transduction in brain cells. Treatment of primary cultures of mouse astrocytes with human recombinant C5a resulted in the activation of mitogen-activated extracellular signal-regulated protein kinase. This response appeared to be mediated by the C5a receptor since astrocyte cultures derived from C5a receptor knockout mice were not responsive to the treatment. Understanding the regulation of C5a receptor in brain and mechanisms by which pro-inflammatory C5a modulates specific signal transduction pathways in brain cells is crucial to studies of inflammatory mechanisms in neurodegeneration. (C) 1998 IBRO. Published by Elsevier Science Ltd.

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.