G. Vanhoof

Proline motifs in peptides and their biological processing.

Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain in that the side‐chain is cyclized back onto the backbone amide position. Inside an a‐helix the possibility of making hydrogen bonds to the preceding turn is lost and a kink will be introduced. The conformational restrictions imposed by proline motifs in a peptide chain appear to imply important structural or biological functions as can be deduced from their often remarkably high degree of conservation as found in many proteins and peptides, especially cytokines, growth factors, G‐protein‐coupled receptors, V3 loops of the HIV envelope glycoprotein gpl20, and neuro‐ and vasoactive peptides. Only a limited number of peptidases are known to be able to hydrolyze proline adjacent bonds. Their activity is influenced by the isomeric state (cis‐trans) as well as the position of proline in the peptide chain. The three proline specific metallo‐peptidases (aminopeptidase P. car‐boxroeptidase P and prolidase) are activated by Mn2+, whereas the three serine type peptidases cleaving a post proline bond (prolyl oligopeptidase, dipep‐tidyl peptidase IV, and prolylcarboxypeptidase) share the sequential order of the catalytic Ser‐Asp‐His triade, which differentiates them from the chy‐motrypsin (His‐Asp‐Ser) and subtilisin (Asp‐His‐Ser) families. An endo or C terminal Pro‐Pro bond and an endo pre‐Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme.—Vanhoof, G., Goossens, F., De Meester, I., Hendrike, D., Schärpé, S. Proline motifs in peptides and their biological processing. FASEB J. 9, 736‐744 (1995)

Distribution of proline-specific aminopeptidases in human tissues and body fluids.

The proline-specific peptidases, aminopeptidase P (EC 3.4.11.9) and dipeptidyl peptidase IV (EC 3.4.14.5), were measured in human tissue homogenates and physiological fluids. All tissues examined contained measurable aminopeptidase P and dipeptidyl peptidase IV activities. High specific activities for both enzymes under study were found in benign prostatic hypertrophy. Normal prostate and prostatic adenocarcinoma had a much lower activity. This difference, however, is not reflected in the serum values of the patients. The most striking finding is the extremely high activity of dipeptidyl peptidase IV in prostatosomes, prostate-derived organelles, which occur freely in human seminal plasma, and which are important for enhancement of sperm forward motility.

Decreased serum dipeptidyl peptidase IV activity in major depression

It has been recently shown that severe depression is characterized by immune dysfunctions such as blunted mitogen-induced blast transformation, which is linked to interleukin-2 (IL-2) mechanisms, and to autoimmune responses. In order to explore one of the putative pathophysiological mechanisms underlying both factors, we have measured the predexamethasone and postdexamethasone serum dipeptidyl-peptidase IV (DPP IV) activity in depressed inpatients and normal controls. This enzyme is an important mediator of IL-2-related blast proliferation, and it may play a role in autoimmunity. We found significantly lower DPP IV levels in major depressives as compared with healthy controls, and melancholics exhibited significantly lower enzyme activity than minor depressives. There was a significant negative correlation between serum DPP IV activity and the severity of illness. However, we were unable to detect any significant relationships between DPP IV on the one hand, and mitogen-induced blast transformation, soluble IL-2 receptor accumulation in PHA culture supernatant, total number of leukocytes and lymphocytes, T lymphocytes, CD4+ and CD25+ cells, on the other. Men exhibited significantly higher serum DPP IV levels than women.

Pyrrolidides: synthesis and structure-activity relationship as inhibitors of dipeptidyl peptidase IV

Summary Dipeptidyl peptidase IV cleaves specifically the peptide bond at the carboxyl side of a proline at the penultimate N-terminal position of a peptide. It is thought to be important for the regulation of biologically active peptides. Moreover, it has been identified as an activation marker of T-lymphocytes (CD26). Pyrrolidides and thiazolidides are known as reversible inhibitors of DPP IV. Several homologues, unsaturated, open and 3-substituted analogues were synthesized in order to determine the structure-activity relationship of the P-1 site. l -Isoleucine was taken as P-2 amino acid. 1-( l -Isoleucyl)-3( S )-fluoropyrrolidine is about as active as the non-fluorinated compound and behaves as a competitive inhibitor. Other changes decrease or abolish the activity.

Proteases and their inhibitors: today and tomorrow

A major incentive in inhibitor research is that control of limited proteolysis constitutes a valuable pharmacological tool. Protease inhibitors have proved to be successful in influencing pathogenesis in many experimental models but a breakthrough to use in human therapy has mainly been restricted to aprotinin and angiotensin converting enzyme (ACE) inhibitors. However, the success of ACE inhibitors as pharmacological tools in hypertension has proved to be a strong stimulant for new protease inhibitor approaches to drug therapy. While emphasis in the search for next generations of ACE inhibitors may move from the circulation renin-angiotensin system to the local tissue systems, including heart, brain and genital tract, persistent and insightful design of renin inhibitors has already yielded highly specific molecules with potent activities in several in vivo models. The development of orally effective long-acting inhibitors will finally allow an evaluation to be made of their therapeutic profile with regard to the family of ACE inhibitors. The close relationship between renin and HIV-1 protease presents an exceptional opportunity for transfer of the knowledge acquired in renin inhibitor development during the past decade, to an accelerated generation of specific HIV-1 protease inhibitors as effective agents in treatment of AIDS. The self-assembly of 2 identical monomers into a symmetrical structure in HIV-1 protease is not only an elegant way to create an active enzyme while encoding a minimal amount of genetic information, but is also in concordance with the bilobular active-site found in mammalian aspartic proteases.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopeptidase P and dipeptidyl peptidase IV activity in human leukocytes and in stimulated lymphocytes

Human white blood cells were shown to contain high aminopeptidase P activity. The specific activities found in the high-speed supernatant of the extracts of granulocytes, lymphocytes and monocytes ranged from 30 to 70 units per mg protein. Culturing lymphocytes during 7 days in the presence of phytohaemagglutinin resulted in a 70-200% increase in the specific aminopeptidase P activity and a 200% increase in the specific activity of dipeptidyl peptidase IV. The time-course of the activity of both aminopeptidase P and dipeptidyl peptidase IV during the stimulation of human T-lymphocytes by phytohaemagglutinin indicates an involvement of these two enzymes in the proliferative process of these immunocompetent cells. Due to their substrate specificity their potential substrates must have the N-terminal Xaa-Pro sequence known to be present in several immunologically important polypeptides.

Exopeptidases in human platelets: an indication for proteolytic modulation of biologically active peptides

The determination in human platelets of four exopeptidases--aminopeptidase P, dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme--by means of fluorometric or liquid chromatography techniques was carried out. The results obtained show that the specific activities of dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme in intact and disrupted platelets are small compared to their specific activities in serum. However, for aminopeptidase P the specific activity of this enzyme is much higher in platelets than in serum. This suggests that circulating platelets may have a significant role as scavengers for circulating peptides containing bonds susceptible for aminopeptidase P.

Subregional mapping of the human lymphocyte prolyl oligopeptidase gene (PREP) to human chromosome 6q22

Prolyl oligopeptidase is a large monomeric proline specific serine endopeptidase, the activity of which correlates well with different stages of depression. We have subregionally mapped human lymphocytic prolyl oligopeptidase (PREP) by FISH using a cosmid probe. The probe mapped to the long arm of chromosome 6, and the signal clustered in band q22.

Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X-prolyl aminopeptidase (aminopeptidase P)

A novel human cDNA (XPNPEPL) encoding a protein of 623 amino acids exhibiting 44% sequence identity and 62% sequence similarity to pig kidney X-prolyl aminopeptidase (aminopeptidase P; EC 3.4.11.9) was obtained by reverse transcription/polymerase chain reaction of phytohemagglutinin-stimulated lymphocyte mRNA. Conserved sequences were found with the prokaryotic X-prolyl aminopeptidase encoding gene (pepP). The human gene translation product exhibits a high sequence homology to the Schizosaccharomyces pombe chromosome I hypothetical protein C22G7.01c and to the S. cerevisiae ORF y11029w. Northern blot analysis indicates an ubiquitous expression of the human XPNPEPL sequence.

Localization and characterization of aminopeptidase P in bovine adrenal medulla

Aminopeptidase P (EC 3.4.11.9) is demonstrated for the first time in the cytosolic fraction of chromaffin cells of the bovine adrenal medulla. The enzyme is inhibited by metal chelators and by sulfhydryl-reactive agents, which suggests that both a tightly bound metal ion and a cysteine residue are necessary for enzymatic activity. Aminopeptidase P might be important for the modulation of the biological activity of neuropeptides. Its occurrence in the adrenal chromaffin cells provides a useful tool for studying the function of this unique proline-specific peptidase in neuropeptide processing and secretion.