Michel Chretien

Endorphins in Fetomaternal Physiology

Following the demonstration of specific opiate receptors in the nervous system of vertebrates and the isolation of a number of endogenous opiate ligands (β-endorphin, enkephalins, dynorphins, and neoendorphins) for these receptors, it was suggested that interactions of endogenous opiate peptides with opiate receptors may be involved in regulating some physiological processes. Initially, the analgesic activity of the endogenous opiates was investigated. However, it was soon noticed that various types of stress stimulate the release of endogenous opiates, suggesting that endogenous opiates may be important in the adaptation of the organism to various stressful situations. Endorphins were also found to modulate the release of prolactin (PRL) and the gonadotropin hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary in humans and experimental animals and to influence the reproductive behavior in animals.

PRO-OPIOMELANOCORTIN: A NEW CONCEPT ISSUED FROM ACTH AND LPH BIOSYNTHETIC STUDIES

We have investigated the biosynthesis of all portions of the precursor molecule related to ACTH/LPH/MSH/ endorphin in order to describe the important cellular events of maturation. Our approach which is based mainly on chemical characterization of the material gives more definitive results than the immunological approach. It has permitted us to demonstrate the presence of the complete N-terminal fragment as a main product of secretion in human, porcine and rat. They were characterized and results indicated (a) that rat N-terminus is a mixture of 2 molecules with one mutation at the N-terminus (Arg/Trp), thus could have two genes; (b) gamma-MSH region of human and porcine is identical to bovine; (c) the sequence of both porcine and human are quite homologous to the bovine species.

Discovery of the Proprotein Convertases and their Inhibitors

The members of the convertase family play a central role in the processing of various protein precursors ranging from hormones and growth factors to viral envelope proteins and bacterial toxins. The proteolysis of these precursors that occurs at basic residues is mediated by the proprotein convertases (PCs), namely: PC1, PC2, Furin, PACE4, PC4, PC5 and PC7. The proteolysis at non-basic residues is performed by subtilisin/kexin-like isozyme-1 (S1P/SKI-1) and the newly identified neural apoptosis-regulated convertase-1 (NARC-1/PCSK9). These proteases have key roles in many physiological processes and various pathologies including cancer, obesity, diabetes, neurodegenerative diseases and autosomal dominant hypercholesterolermia. Here we summarize the discovery of the proprotein convertases and their inhibitors, discuss their properties, roles, resemblance and differences

Testis-Specific Proprotein Convertase 4: Gene Structure, Optional Exons, and mRNA Isoforms

Proprotein convertase 4 (PC4) is a member of the recently discovered family of eukaryotic serine endoproteinases characterized by their structural similarity to bacterial subtilisin and to the yeast Kex2 gene product, as well as by their cleavage specificity after pairs of basic residues (1–8). This family also includes furin, PC1/PC3, PC2, PACE4, and PC5/PC6. Unlike the other convertases that have wide, albeit distinct, tissue distributions (1–4, 7, 8), PC4 expression is restricted to testicular germ cells (5, 6, 10).

The Possible Role of Plasma Kallikrein in Pro-Hormone and Pro-Enzyme Processing

It is now clear that a great number of proteins are originally synthesized as large polypeptide precursors containing within their sequence biologically active peptides. These “motifs” are usually sandwiched between either single or pairs of basic residues (Lazure et al., 1983). Post-translational processing of these precursors involves the participation of a number of enzymes which shape these biologically active segments into their final mature form(s). One of the least well understood maturation step concerns the protease(s) responsible for the “trypsin-like” limited proteolysis occuring at either pairs of basic residues or post-single basic residues. Recently, a number of candidate mammalian processing enzymes have been proposed. These include an aspartyl protease (Parish et al., 1986), metalloproteases (Clamagirand et al., 1987; Hutton et al., 1987), thiol protease (Fletcher et al., 1981) and serine proteases (Lindberg et al., 1984; Cromlish et al., 1986a,b; Seidah et al., 1986). So far, evidence for the participation of these candidate proteases in pro-protein processing has been indirect, based on their localization in secretory granules and/or their “in vitro” cleavage of either model substrates or pro-hormone precursors at sites identical to those cleaved “in vivo.”

Microsequence of Polypeptide Hormones: Its Usefulness to Monitor the Isolation of Novel Molecules

Novel peptides related to the N-terminal portion of proopiomelanocortin (POMC) (Chretien et al., 1979) have been isolated and characterized from human and porcine pituitary glands. Since the cysteine residues were previously found in other species, either by protein radioactive micro-sequencing or cDNA sequence, to be consistantly at positions 2, 8, 20, and 24, their purification was monitored with amino acid sequences of peptides labeled at their cysteine residues with [14C]iodoacetamide. This approach gave us, at all important steps of the isolation, clear indications of the presence of the desired peptides up to their final and complete purification. These results strongly suggest that chemical characterization, whenever possible, should be used to monitor the purification of proteins and polypeptides whose biological activity cannot be measured, as is the case for precursor molecules or some of their fragments. They also support a recent suggestion by Julliard et al. (1980) that at least some form of chemical characterization should be added to any purification carried out solely on the basis of the immunologic competence of the peptides sought.

ISOLATION AND NH 2 -TERMINAL SEQUENCE OF A HIGHLY CONSERVED PITUITARY AND HYPOTHALAMIC PROTEIN BELONGING TO A NEW SUPERFAMILY

During the course of purification of the N-terminal fragment of pro-opiomelanocortin we isolated and purified a 21,000 daltons (pI 4.9) novel protein from porcine anterior pituitaries and whole human pituitary glands whose amino acid composition revealed a high homology between these two species. The NH 2 -terminal sequence of the first 77 residues of the human and porcine homologues confirmed the homology since one conservative substitution was found at residue 12, namely an Ala for a Thr. A computer data bank search using a mutation data matrix and comparison with 338,327 segments of proteins revealed that this substance should be classified as belonging to a new protein superfamily. An antibody produced against a synthetic fragment of this molecule was raised and immunocytochemical staining revealed the presence of material in the anterior and posterior lobe of the pituitary and in the supraoptic nucleus of the hypothalamus. It was absent in the intermediate lobe. This novel pituitary and hypothalamus substance has partial sequence homology with duck pro-insulin, Rous sarcoma virus protein and secretin.