Paul Cohen

Precursors for peptide hormones share common secondary structures forming features at the proteolytic processing sites

We have analyzed the amino acid sequences situated around the putative proteolytic cleavage sites in twenty different biosynthetic precursors of peptide hormones by processing enzymes. The prediction of the probability for forming secondary structures around the basic amino acids, constituting the cleavage sites, was made using the modified method of Chou and Fasman. The results indicate that the processing sequences which are cleaved in vivo, are in all cases located inside regions with high β‐turn formation probability or else immediately adjacent to these structures. The β‐turn forming region at the cleavage locus, is flanked on both sides by amino acid sequences with a high probability for forming highly ordered structures, either β‐sheet or α‐helix. These conformational features are not found in precursors around dibasic pairs, i.e. putative cleavage loci, but which are not cleaved in vivo and appear to be conserved. We hypothesize that β‐turns including the basic amino acids doublets, flanked by highly ordered secondary structures (either β‐sheet or α‐helix) may constitute a minimal requirement for the recognition by the endoproteases involved in the processing of these precursors.

Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules

An aminopeptidase of the B-type, with an apparent M(r) 72,000 and pI = 4.9, was isolated from rat testes and characterized. The enzyme was able to remove only Arg and/or Lys residues from L-amino acid beta-naphthylamide derivatives and from the N-terminus of several peptides. No cleavage occurred in the case of Arg-Pro bonds as found in bradykinin and substance P. The enzyme was sensitive to cysteinyl reagents and to aminopeptidase inhibitors, such as bestatin, amastatin and arphamenines A and B. The aminopeptidase activity, tested with L-Arg beta-naphthylamide and with Arg0-Met-enkephalin as substrates, was inhibited by o-phenanthroline, and restored by Zn2+ suggesting its metallopeptidase character. The partial characterization of an aminopeptidase-B activity in rat brain cortex identified a protein which is biochemically and immunologically related to the testis enzyme. By immunohistochemistry, the aminopeptidase-B was found to be particularly abundant in the seminiferous tubules at late stages of spermatogenesis and was clearly detected in a restricted area of elongated spermatids. Remarkably, the enzyme was observed to concentrate massively in the residual bodies. Since this aminopeptidase-B was able in vitro to trim out N-terminal Arg and/or Lys residues from peptides mimicking processing intermediates, it is proposed that this enzyme may be involved in propeptide and proprotein processing mechanisms in the course of spermatid differentiation.

Tritium labelling of 8-lysine vasopressin and its purification by affinity chromatography on sepharose bound neurophysins.

The antidiuretic hormone vasopressin is a nonapeptide containing one disulfide bridge and tyrosyl and phenylalanyl residues in position -2 and -3, respectively. 8-Lysine vasopressin (LVP) is found in pig and hippopotamus while 8-arginine vasopressin (AVP) occurs in several other mammals (man, ox, rat...). These pituitary polypeptides exert selective effects on the permeability of the distal part of the mammalian nephron and on the active sodium transport and passive permeability to water and other low molecular weight molecules of the amphibian skin and bladder epithelial cells. These effects have been shown to result from a specific activation of an adenyl cyclase located within the membrane of the target cells (for review, see for instance [ 1 ] ). Either LVP or AVP is associated with oxytocin, a related peptide, and carried in the post-hypophysis by a family of relatively small proteins, the neurophysins [2], with which they form non covalent complexes [3]. The isotopic labelling of polypeptidi¢ hormones has been successfully applied, in this laboratory, to oxytocin [4], angiotensine II [5] and other compounds [6]. The principle is an iodination of the tyrosyl (or histidyl) residue(s) followed by catalytic substitution of the peptide bound iodine with tritium. In this report we describe a similar procedure which produces, after purification by affinity chromatography on a column of bovine neurophysins immobilized on Agarose, a highly radioactive compound (10 Ci/mmole) which exhibits all the biological and biochemical properties of the native hormone. This, obviously, provides a very useful tool for the examination of the molecular mechanisms by which this important biological compound interacts with neurophysins [7] and biological membranes.

Proteolytic events in the post-translational processing of polypeptide hormone precursors

Selective and limited proteolysis is a key step in the post-translational modification of peptide hormone precursors. This process appears to involve a proteolytic machinery including highly specific endoproteases. Some of the enzyme systems possibly involved in the processing of pro-neuropeptides will be described and their mechanism of action discussed. Special emphasis will be on the following: i) the physico-chemical characteristics of proteolytic enzymes which are believed to be involved in the processing of some of these polypeptide hormone precursors; ii) the bio-specificity of these enzymes toward the substrates; iii) the importance of both secondary and tertiary structures of the cleavage domain in recognition by the selective proteases. These properties will be discussed in connection with the possible importance of the maturation enzymes in the in vivo regulation of hormone biosynthesis.

Biochemical Aspects of Neurosecretion: Neurophysin — Neurohypophyseal Hormone Complexes

Publisher Summary This chapter reviews two major aspects of the progress in the neurophysin field, namely, their molecular interactions with the neurohormones and their biosynthetic mechanisms. Both remain key problems for an understanding of neurosecretion at the molecular level. The chapter highlights the importance of these proteins in this brain function. Much is known about their interactions in vitro with the neurohormones. Analysis of radiocrystallographic data on the protein and its cocrystals with the nonapeptide hormones provides further insight into the structure of the neurophysin-hormone complexes. Although neurophysins are found in the circulatory system and are reported in several target organs of the neurohypophyseal hormones, no extragranular function of this complex protein is known. However, a number of observations stimulate a careful reevaluation of their possible biological significance apart from their intragranular complexing function. Also, there is preliminary evidence that the proneurophysins exists, at some stage, as glycosylated forms.

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets.

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.

Aminopeptidase B (EC 3.4.11.6)

Aminopeptidase B (EC 3.4.11.6) is a Zn(2+)-dependent exopeptidase which selectively removes arginine and/or lysine residues from the NH2-terminus of several peptide substrates including Arg0-Leu-enkephalin, Arg0-Met-enkephalin and Arg-1-Lys0-somatostatin-14. Analysis of its primary structure showed that aminopeptidase-B is structurally related to leukotriene A4 hydrolase, an important enzyme of the arachidonic acid pathway. This structural relationship is further supported by the capacity of aminopeptidase-B to hydrolyse leukotriene A4. Aminopeptidase-B is widely distributed in a number of tissues, including endocrine and non-endocrine cells. Moreover, in rat PC12 pheochromocytoma cells, the enzyme is secreted and associated with the external face of the plasma membrane. Together these data strongly argue in favour of a role of this bi-functional enzyme in the final stages of precursor processing mechanisms occurring either in the secretory pathway, at the plasma membrane, or at both locations.

An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues

The octacosapeptide sequence [Tyr18] pro-ocytocin/neurophysin (1-18)NH2 [pro-OT/Np(1-18)NH2] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in Lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly10-Lys11-Arg12+Ala13-Val-Leu-Asp-Leu-Tyr18 (NH2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly10-Lys11-Arg12 into OT-Gly10 was detected in the same granule Lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT/Np in vivo.

The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28 (1–12)

The products generated after addition of the ARG-LYS esteropeptidase activity purified from rat brain to synthetic somatostatin-28 were analyzed using radioimmunoassay, HPLC and amino acid analysis. In addition to somatostatin-14, both free arginine and free Lysine were identified together with somatostatin-28. The dipeptide ARG-LYS was not present, which indicates that three peptide bonds were hydrolyzed in order to achieve excision of the doublet. Since it is likely that the octacosapeptide is a precursor for both somatostatin-14 and somatostatin-28, these observations add further support to the hypothesis that the convertase is also involved in the in vivo processing of endogenous somatostatin-28.

Proteolytic enzymes in the post-translational processing of polypeptide hormone precursors.

Selective and limited proteolysis is a key step in the post-translational modification of peptide hormone precursors. This process appears to involve a proteolytic machinery including highly specific endoproteases. Some of the enzyme systems possibly involved in the processing of pro-neuropeptides will be described and their mechanism of action discussed. Special emphasis will be on the following: i) the physico-chemical characteristics of proteolytic enzymes which are believed to be involved in the processing of some of these polypeptide hormone precursors; ii) the bio-specificity of these enzymes toward the substrates; iii) the importance of both secondary and tertiary structures of the cleavage domain in recognition by the selective proteases. These properties will be discussed in connection with the possible importance of the maturation enzymes in the in vivo regulation of hormone biosynthesis.