Peptidomics of Zebrafish Brain in a 6-OHDA-Induced Neurodegeneration Model.

Bioactive peptides such as neuropeptides and peptide hormones are largely understood in their involvement in a variety of physiologic systems. In addition to the neuropeptides produced and processed by the classic secretory pathway, intracellular peptides (InPeps) have shown biological activity in studies involving different organisms. A model that has become attractive in many research fields is the zebrafish (Danio rerio), which has allowed correlating behavioral responses or physiological processes with underlying molecular pathways or signaling cascades, improving the understanding of homeostasis mechanisms of the central nervous system, as well as pathological processes such as neurodegenerative diseases.

Methods for Intracellular Peptidomic Analysis.

Peptides have broad biological significance among different species. Intracellular peptides are considered a particular class of bioactive peptides, whose generation is initiated by proteasomal degradation of cytosolic, nuclear, or mitochondrial proteins. To extract and purify intracellular peptides, which may apply for biological peptides in general, it is important to consider the initial source: tissue, cell, or fluid.

Quantitative Peptidomics Using Reductive Methylation of Amines.

A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (H), and reagents containing deuterium and C, it is possible to produce five isotopically distinct forms of the methylated peptides, which can be quantified by mass spectrometry.

Revealing Natural Intracellular Peptides in Gills of Seahorse .

The seahorse is a marine teleost fish member of the Syngnathidae family that displays a complex variety of morphological and reproductive behavior innovations and has been recognized for its medicinal importance. In the Brazilian ichthyofauna, the seahorse is among the three fish species most used by the population in traditional medicine. In this study, a protocol was performed based on fast heat inactivation of proteases plus liquid chromatography coupled to mass spectrometry to identify native peptides in gills of seahorse .

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized.

Hidden by the name: A new fluorescent pumpkin toadlet from the Brachycephalus ephippium group (Anura: Brachycephalidae).

Species of Brachycephalus has been having taxonomical issues due its morphological similarity and genetic conservatism. Herein, we describe a new species of Brachycephalus from the south Mantiqueira mountain range and semidecidual forests in the municipalities of Mogi das Cruzes, Campinas and Jundiaí, state of São Paulo, Brazil, based on an integrative approach. It can be distinguished from all species of the B.

Peptide Profile of Zebrafish Brain in a 6-OHDA-Induced Parkinson Model.

Parkinson's disease (PD) is a chronic neurodegenerative disorder mainly attributed to the progressive loss of dopaminergic neurons in the substantia nigra, which leads to uncontrolled voluntary movements causing tremors, postural instability, joint stiffness, and speech and locomotion difficulties, among other symptoms. Previous studies have shown the participation of specific peptides in neurodegenerative diseases. In this context, the present work analyzed changes in the peptide profile in zebrafish brain induced to parkinsonian conditions with 6-hydroxydopamine, using isotopic labeling techniques plus mass spectrometry.

The Relevance of Thimet Oligopeptidase in the Regulation of Energy Metabolism and Diet-Induced Obesity.

Thimet oligopeptidase (EC 3.4.24.

NFKF is a synthetic fragment derived from rat hemopressin that protects mice from neurodegeneration.

Previous studies suggested the pharmacological potential of rat hemopressin (PVNFKFLSH) and its shorter synthetic peptide NFKF, to protect from pilocarpine-induced seizures in mice. Orally administered NFKF was shown to be hundred times more potent than cannabidiol in delaying the first seizure induced by pilocarpine in mice. Here, using an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis we have shown that C57BL/6 J mice orally administrated with NFKF (500 μg/kg) presented better EAE clinical scores and improved locomotor activity compared to saline administrated control mice.

Thimet Oligopeptidase (EC 3.4.24.15) Key Functions Suggested by Knockout Mice Phenotype Characterization.

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1 exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN).

Effect of Protein Denaturation and Enzyme Inhibitors on Proteasomal-Mediated Production of Peptides in Human Embryonic Kidney Cells.

Peptides produced by the proteasome have been proposed to function as signaling molecules that regulate a number of biological processes. In the current study, we used quantitative peptidomics to test whether conditions that affect protein stability, synthesis, or turnover cause changes in the levels of peptides in Human Embryonic Kidney 293T (HEK293T) cells. Mild heat shock (42 °C for 1 h) or treatment with the deubiquitinase inhibitor b-AP15 led to higher levels of ubiquitinated proteins but did not significantly increase the levels of intracellular peptides.

Characterization of Intracellular Peptides from Zebrafish () Brain.

Peptides represent a large class of cell signaling molecules, and they are mainly produced by the classical secretory pathway or during protein degradation. The peptide profile of (zebrafish) shows a lack of information when compared with other consolidated animal models. The aim of this work was to characterize the peptide profile of zebrafish brain by using triplex reductive methylation of amines labeling and liquid chromatography coupled to electron spray mass spectrometry.

Effects of ocean acidification and salinity variations on the physiology of osmoregulating and osmoconforming crustaceans.

Survival, osmoregulatory pattern, oxygen consumption, energy spent on metabolism, ammonia excretion, type of oxidized energy substrate, and hepatosomatic index were evaluated in decapods (an osmoregulating crab, Callinectes danae, and an osmoconforming seabob shrimp, Xiphopenaeus kroyeri) exposed to carbon dioxide-induced water acidification (pH 7.3, control pH 8.0) and different salinities (20, 25, 30, 35, and 40‰) for 3 days.

Quantitative Peptidomics Using Reductive Methylation of Amines.

A number of different approaches have been used for quantitative peptidomics. In this protocol we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (H), and reagents containing deuterium and C, it is possible to produce five isotopically distinct forms of the methylated peptides which can be quantified by mass spectrometry.

Analysis of the Yeast Peptidome and Comparison with the Human Peptidome.

Peptides function as signaling molecules in species as diverse as humans and yeast. Mass spectrometry-based peptidomics techniques provide a relatively unbiased method to assess the peptidome of biological samples. In the present study, we used a quantitative peptidomic technique to characterize the peptidome of the yeast Saccharomyces cerevisiae and compare it to the peptidomes of mammalian cell lines and tissues.

Interferon-gamma activity is potentiated by an intracellular peptide derived from the human 19S ATPase regulatory subunit 4 of the proteasome.

Unlabelled: Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines.

Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington's Disease.

Huntington's disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation.

Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells.

A novel intracellular peptide derived from g1/s cyclin d2 induces cell death.

Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25-100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function.

Peptidomic analysis of the neurolysin-knockout mouse brain.

Unlabelled: A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein-protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated.

Neurolysin knockout mice generation and initial phenotype characterization.

The oligopeptidase neurolysin (EC 3.4.24.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity.

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides.

Alterations of the intracellular peptidome in response to the proteasome inhibitor bortezomib.

Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T) cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours), and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour.

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction.

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence.

The cysteine-rich protein thimet oligopeptidase as a model of the structural requirements for S-glutathiolation and oxidative oligomerization.

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.