Strategies for the Identification of Bioactive Neuropeptides in Vertebrates

Abstract

Neuropeptides exert essential functions in animal physiology by controlling e.g., reproduction, development, growth, energy homeostasis, cardiovascular activity and stress response. Thus, identification of neuropeptides has been a very active field of research over the last decades. This review article presents the various methods used to discover novel bioactive peptides in vertebrates. Initially identified on the basis of their biological activity, some neuropeptides have also been discovered for their ability to bind/activate a specific receptor or based on their biochemical characteristics such as C-terminal amidation which concerns half of the known neuropeptides. More recently, sequencing of the genome of many representative species has facilitated peptidomic approaches using mass spectrometry and in silico screening of genomic libraries. Through these different approaches, more than a hundred of bioactive neuropeptides have already been identified in vertebrates. Nevertheless, researchers continue to find new neuropeptides or to identify novel functions of neuropeptides that had not been detected previously, as it was recently the case for nociceptin.

Keywords: bioactiity; de novo; identification; neuropeptide; peptidomic approach; review.

FIGURE 1

Examples illustrating the 3 categories of neuropeptide precursors. (A) Illustration of monofonctional precursors with single copy of the peptide of interest as it is the case for CCK, UII, NPY, and SST. (B) Illustration of monofonctional precursors with several copies of the peptide of interest as it is the case for TRH. (C) Illustration of multifonctional precursors which express several different bioactive peptides as it is the case with POMC. ACTH: adrenocorticotropic hormone. CCK: cholecystokinin. POMC: pro-opiomelanocortin. CLIP: corticotropin-like intermediate lobe peptide. LPH: lipotropic hormone. MSH: melanocyte-stimulating hormone. NPY: neuropeptide Y. SP: signal peptide. SST: somatostatin. TRH: thyrotropin-releasing hormone. UII: urotensin II. aa: aminoacid. Adapted from Neveu (2012).

FIGURE 2

Neuropeptide biosynthesis. The signal recognition particle (SRP) is fixed to the complex formed by the peptide, the ribosome and the mRNA (❶) and then binds to its receptor on the endoplasmic reticulum membrane (❷). This allows the peptide to enter the reticulum through the translocon so that peptide synthesis can continue (❸). The signal peptide (red circles) is then cleaved off by an endopeptidase (❹). Once synthesized, the neuropeptide will pass through the Golgi apparatus to undergo post-translational modifications (❺) before being secreted in a regulated manner (❻).

FIGURE 3

Schematic representation summarizing the various strategies of identification of bioactive peptides. Examples of the peptides identified through the various strategies are presented.

FIGURE 4

Strategy of identification of pituitary adenylate cyclase-activating polypeptide (PACAP). The team of Akira Arimura extracted peptides from 5000 ovine hypothalamic fragments and separated them according to their charge and hydrophobicity. A highly basic peptide different from any other known hypophysiotropic neurohormone was found to activate cAMP in cultured rat anterior pituitary cells. Characterization revealed a 38-amino acid peptide with 68% identity with VIP that they named pituitary adenylate cyclase-activating polypeptide or PACAP (Arimura, 2007). cAMP: cyclic adenosine monophosphate.

FIGURE 5

Representation of the various steps of peptidomic studies. Each step can be adapted to the specificity of the peptide, the complexity of the matrix and the information needed (e.g., identification, quantification). HPLC: high pressure liquid chromatography.

FIGURE 6

Correlation between the expression level of various peptides and the mechanisms occurring during cerebellar cortex development. (A) Variation of five peptide concentrations during cerebellar development. (B) Temporal evolution of the cellular processes involved in cerebellar development, i.e., proliferation, migration, differentiation and cell death. Peptide concentrations are high during the developmental processes on which they act. P: postnatal day. PACAP: pituitary adenylate cyclase-activating polypeptide.

FIGURE 7

Representation of the workflow used to filter the peptides identified by de novo sequencing. Sequences with a PEAKS score higher than 70 were compared to UniProt/SwissProt database in order to exclude peptides originating from already known precursors. Remaining sequences were filtered by occurrence among samples (>7/10) and regulation during development (>2). This workflow led to the selection of 11 sequences which correspond to putative peptides.