A peptidomics strategy to elucidate the proteolytic pathways that inactivate peptide hormones.

Proteolysis plays a key role in regulating the levels and activity of peptide hormones. Characterization of the proteolytic pathways that cleave peptide hormones is of basic interest and can, in some cases, spur the development of novel therapeutics. The lack, however, of an efficient approach to identify endogenous fragments of peptide hormones has hindered the elucidation of these proteolytic pathways. Here, we apply a mass spectrometry (MS) based peptidomics approach to characterize the intestinal fragments of peptide histidine isoleucine (PHI), a hormone that promotes glucose-stimulated insulin secretion (GSIS). Our approach reveals a proteolytic pathway in the intestine that truncates PHI at its C-terminus to produce a PHI fragment that is inactive in a GSIS assay, a result that provides a potential mechanism of PHI regulation in vivo. Differences between these in vivo peptidomics studies and in vitro lysate experiments, which showed N- and C-terminal processing of PHI, underscore the effectiveness of this approach to discover physiologically relevant proteolytic pathways. Moreover, integrating this peptidomics approach with bioassays (i.e., GSIS) provides a general strategy to reveal proteolytic pathways that may regulate the activity of peptide hormones.

[Peptide histidine-isoleucine and and its human analogue peptide histidine-methionine: localization, receptors and biological function]. / Peptyd histydyno-izoleucynowy i jego ludzki analog peptyd histydyno-metioninowy: wystepowanie, receptory i funkcja biologiczna.

Peptide histidine-isoleucine (PHI) and its human analogue peptide histidine-methionine (PHM) are members of a superfamily of structurally related peptides embracing, among others, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), peptide histidine-valine (PHV), and helodermin. All the peptides display a pleiotropic biological activity. PHI, PHM, PHV and VIP are co-synthesized from the same precursor and share high levels of structural and functional similarity. These peptides may act through common receptors and are widely distributed throughout the body tissues (the central nervous system, gastrointestinal tract, respiratory system, and reproductive system); however, their role remains largely unknown. Changes in the levels of the peptides in the course of different diseases suggest their possible importance and usefulness in diagnostics. Moreover, the neurotrophic and neuroprotective properties of PHI suggest, by analogy to VIP or PACAP, its therapeutic potential in many neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.

A C-terminally elongated form of PHI from porcine intestine.

A C-terminally elongated form of peptide histidine isoleucine amide (PHI) was isolated from porcine intestine based on its effect on cAMP production in IMR-32 cells. The structure was determined by amino acid sequence analysis of tryptic fragments and by mass spectrometry. The peptide has 42 amino acid residues like those described from human, rat and mouse, but the amino acid sequence of the C-terminal extension of pig PHI is unique. Unlike the other peptides, it has a C-terminal Ala and it differs at five positions from the human form and at six positions from the rat form, while the human and the rat forms differ by only two substitutions. To avoid confusion arising from different C-terminal residues, a unifying nomenclature is proposed: PHI-27 for the hormone and PHI-42 for the elongated product.

Sequence of a cDNA encoding chicken vasoactive intestinal peptide (VIP).

Mammalian pre-pro-vasoactive intestinal peptide (pre-proVIP) gives rise to the neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI). The cDNA encoding chicken VIP was cloned and sequenced. The region of chicken pre-proVIP homologous to the mammalian PHI region is not followed by an amidation signal. This unusual feature suggests that processing of the precursor may be different in the chicken.

Commonalities in vasoactive intestinal peptide and peptide N-terminal histidine C-terminal isoleucine stimulation of N-acetyltransferase activity in the rat pineal.

Exposure of adult rat pineal glands in organ culture to the polypeptides vasoactive intestinal polypeptide (VIP), and peptide N-terminal histidine C-terminal isoleucine (PHI) increases pineal serotonin N-acetyltransferase (NAT) activity and melatonin synthesis. The following research results are taken to indicate that VIP and PHI share common components of the NAT induction system: (1) The effects of the two peptides are additive at concentrations of 10 nM VIP and 100 nM PHI but not at higher peptide concentrations. (2) Pineals from newborns also respond to PHI with a dose dependent increase in NAT activity. NAT responses are additive at the same concentrations as seen with the adult pineals. (3) Light exposure affects the sensitivity of pineals to VIP and PHI stimulation in a similar manner; pineals taken after 3 hr of light are much less sensitive to PHI or VIP than those taken after 13 hr of light. (4) Pineals exposed for 48 hr to either PHI or VIP have a reduced NAT response to either agonist, which is reversible by culture in agonist-free media. (5) Neither VIP nor PHI stimulation of NAT activity is affected by concentrations of the VIP antagonists (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 (NAcTDGRF), L-8-K, VIP-Neurotensin Hybrid (VIPNET), or (4Cl-D-Phe6, Leu17)-VIP (4C1PLVIP), which affect VIP binding or function in other tissues.