Dr Adrian Pierotti

E: A.Pierotti@gcal.ac.uk

• Processing and degradation of peptides and hormones by metalloproteases.
• Metalloprotease virulence factors in bacterial pathogens.

Research Group

• Maria Psatha B.Sc. (PhD Student)
• Lesley Morrison B.Sc. (PhD Student)
• Nicola Wilson B.Sc. (PhD Student)

Profile

Dr Pierotti received his Ph.D. at the MRC Brain Metabolism Unit (University of Edinburgh) where he worked on neuropeptide processing. He demonstrated differential processing and secretion of somatostatin-28 and -14 in the rat hypothalamus and median eminence [1,2].

Moving to New York to a post-doctoral fellowship at the Mount Sinai Medical Center with Prof. Jim Roberts and Dr Marian Orlowski, studies on neuropeptide processing were extended with the characterisation and subsequent cloning of Thimet Oligopeptidase (EC 3.4.24.15) from rat testis [3,4].

Neuropeptide processing and degrading enzymes were the theme of his second post-doctoral fellowship working with Prof. Paul Cohen in Paris. There a number of neuropeptide processing enzymes were characterised and one- the metalloprotease NRD-Convertase (EC 3.4.24.61) was cloned [5-8]. Returning to his native Glasgow in 1993, Dr Pierotti took up a lectureship at SBBS where he continues to work on Thimet Oligopeptidase and NRD-Convertase.

A. R. Pierotti & A.J Harmar.

Multiple forms of somatostatin-like immunoreactivity in the hypothalamus and amygdala of the rat: Selective localization of somatostatin-28 in the median eminence.

(1985) J. Endocrinol. 105: 383-389.

A. R. Pierotti, A.J. Harmar, L. Tannahill & G.W. Arbuthnott.

Different patterns of molecular forms of somatostatin are released by the rat median eminence and hypothalamus. (l985) Neurosci. Lett. 57: 2l5-220.

M. Orlowski, S. Reznik, J. Ayala & A. R. Pierotti.

Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides. (1989) Biochem. J. 261: 951-958.

A. Pierotti, K-W. Dong, M.J. Glucksman, M. Orlowski & J.L. Roberts.

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15. (1990) Biochemistry 29: 10323-10329.

J. Bourdais, A. R. Pierotti, H. Boussetta, N. Barre, G. Devilliers & P. Cohen.

Isolation and functional properties of an arginine selective endoprotease from rat intestinal mucosa: a putative prosomatostatin convertase. (1991) J. Biol. Chem. 266: 23386-23391

V. Chesneau, A. R. Pierotti, N. Barre C. Creminon, C. Tougard & P. Cohen.

Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues. (1994) J. Biol. Chem. 269: 2056-2061

A. R. Pierotti, A. Prat, V. Chesneau, F. Gaudoux, A-M. Leseney, T. Foulon & P. Cohen.

N-Arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes.

(1994) Proc. Natl. Acad. Sci. USA. 91 6078-6082

S. Cadel, A. R. Pierotti, T. Foulon, C. Creminon, N. Barre, D. Segretin & P. Cohen.

Aminopeptidase-B in the rat testis: isolation, functional properties and cellular localisation in the seminiferous tubules. (1995) Mol. Cell. Endocrinol. 110 149-160.

Research

The interests of my group are centred on the post-translational processing and degradation of hormones, proteins and peptides. Presently two metalloenzymes are under investigation by the group, Thimet Oligopeptidase and NRD-Convertase. The role of Matrix Metalloproteases, particularly Collagenases in the premature rupture of membranes is also under study.

Thimet Oligopeptidase

Thimet oligopeptidase (TOP: EC 3.4.24.15) is a thiol-dependent metallopeptidase, which can cleave and thereby modulate the activity of many neuropeptides. The enzyme is active in many endocrine tissues including testes, brain, and pituitary. In the median eminence it is postulated to play a role in the degradation of GnRH released from the hypothalamus and thus to modulate LH levels. In rat, the richest source of thimet oligopeptidase is the testes, with a specific activity 5 fold higher than that of brain.

In order to define the exact localisation of this enzyme within the rat and human testis, the distribution of TOP in the developing and adult gonad was examined in situ and in isolated cells. Ontogeny studies demonstrated that TOP is detectable by western blotting from 9 days with levels of expression increasing with the age of the animal. Immunolocalisation of the protein in the interstitium was positive from 9 days onwards but was negative within the seminiferous tubules before 35 days of age, whereas TOP mRNA was not detected within the testis until 35 days of age with subsequent stable expression levels up to 90 days. In the adult rat testis, a strong TOP immunoreactivity was observed within seminiferous tubules, in elongating and elongated spermatids and residual bodies. In the interstitial compartment, immunoreactivity was also observed in Leydig cells. Western and Northern blot analyses confirmed the distribution of expression observed using immunochemistry, however Leydig cells display a lower signal than expected from the immunohistochemical data. Analyses of various human tissue extracts showed that the testis displays the highest levels of TOP mRNA, with immunohistochemical experiments revealing that, as in the rat, the protein is principally expressed in elongated spermatids/residual bodies, and in Leydig cells.

The possible involvement of TOP in proteolytic events associated with the process of spermiogenesis is currently under investigation.

The mechanism whereby rat thimet oligopeptidase expression is regulated at the transcriptional level has been examined by reporter gene assay and electromobility shift assays after isolation of 1020bp of upstream sequence. Computer analysis predicts a number of potential transcription factor binding sites which were examined by deletion analysis and DNA binding studies. The promoter or its deletion fragments were fused to luciferase reporter gene vectors and introduced into various cell lines. Two regions of the promoter have been identified; a positive acting region (-901/-219) and a strong negative acting region (-219/-102).

Concomitantly, potential transcription factors interacting with the cis-acting elements of the promoter were studied by gel electrophoretic mobility shift assays. This has identified a number of transcription factor binding sites, the significance of which are presently under investigation.

NRD Convertase

Peptide hormones and neuropeptides are released from larger biosynthetic precursors (prohormones) by enzymatic cleavage at pairs of basic amino acids. N-Arginine Dibasic Convertase (NRD Convertase; NRD-C EC 3.4.24.61) a dibasic selective metalloendopeptidase that cleaves at the amino terminus of arginine residues has been isolated and cloned. The enzyme has been implicated in processing events but no biological role has been ascribed to it. Abundant in endocrine tissues, the highest levels are found in testis.

The mechanism whereby NRD-C expression is regulated at the transcriptional level has been examined by reporter gene assay and electrophoretic mobility shift assays following isolation of upstream sequences from both rat and human. Analysis of both promoters show that they are highly conserved containing a number of motifs which may correspond to transcription factor binding sites. The rat promoter has been cloned into a luciferase reporter vector and analysed in a number of cell lines. Full functionality of the promoter is observed with 5' deletions to 411bp upstream of the transcriptional start site in spermatid, prostate and pituitary cell lines. Further deletion to 101bp causes a complete loss of activity in spermatid and prostate lines. In contrast, GH3 pituitary cells display no reduction in promoter activity with deletion to 101bp of upstream sequence. A number of transcription factor binding sites have been identified by electrophoretic mobility shift assays in the region 411-101, however no differences in binding between the cell lines have been observed.

Collagenase

Bacteria isolated from vaginal swabs and prematurely ruptured amniotic membranes are being examined to determine whether proteases produced by these isolates contribute to the pathogenesis of bacterial vaginosis in pregnant females. In particular Bacteroides spp have been isolated and identified; half of the isolates contain Prevotella bivia. The protease profile of these bacteria was carried out and greater than 85% of isolated produce collagenase. The collagenase secreted by one of these P. bivia strains has been purified and characterised and is highly active against human placental collagen.

Recent Research Publications

T.J. Wu, A.R. Pierotti, M16 December, 2005g, G. Fink & J.L. Roberts. Endopeptidase EC 3.4.24.15: presence in the rat median eminence and hypophysial portal blood and its modulation of the leutinizing hormone surge. (1997) J. Neuroendocrinol 9 813-822.

C. Pineau, S. McCool, M. J. Glucksman, B. Jégou & A. R. Pierotti. Distribution of Thimet Oligopeptidase (E.C. 3.4.24.15) in human and rat testes. (1999) J. Cell Sci. 112 3455-3462.

A.G. Winter & A. R. Pierotti. Gene expression of the dibasic-pair cleaving enzyme NRD Convertase is differentially regulated in the GH3 pituitary and Mat-Lu prostate cell lines. (2000) Biochem. J. 351 755-764

S. McCool & A. R. Pierotti. Expression of the thimet oligopeptidase gene is regulated by positive and negative acting elements. (2000) DNA & Cell Biol. 19 729-738

Research Grants

J. McGroarty and A.R. Pierotti: An investigation of Virulence Factors of Bacteroides Species involved in Premature Rupture of Membranes. The Sir Jules Thorn Trust, £58,250, 1996-8

A.D. Corbett and A.R. Pierotti: Differential Gene Expression in Inflammatory Bowel Disease. The West of Scotland Ileostomy and Internal Pouch Support Group, £5,700, 1997-8

A.R.Pierotti: Development of a specific Quenched Fluorescent Substrate and inhibitor for NRD-Convertase. The Wellcome Trust, £45,561, 1997-2000