Subcellular Distribution of Prolyl Endopeptidase and Cation‐Sensitive Neutral Endopeptidase in Rabbit Brain

Dresdner, K P; Barker, Louis A.; Orłowski, Marian; Wilk, Sherwin

doi:10.1111/j.1471-4159.1982.tb05362.x

Cited by 60 publications

(43 citation statements)

References 16 publications

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“…A previous study identified exosome-like vesicles from airway epithelial cells and suggests the potential for these vesicles to harbor proteins critical in immune defense (39) but did not delineate the presence of proteases in these exosomelike vesicles. Previous studies have identified PE in the cytosol of a variety of cells, although the specific mechanism regarding its release has been poorly understood (40,41). Our results demonstrate localization of PE to exosomes in human airway epithelial cells, a mechanism bypassing the classical secretory pathway.…”

Section: Discussionsupporting

confidence: 54%

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes

Szul

Bratcher

Fraser³

et al. 2016

Am J Respir Cell Mol Biol

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Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PEcontaining exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.Keywords: protease; Toll-like receptor 4; exosome; cystic fibrosis; pulmonary Cell-to-cell communication via secreted soluble mediators is critical to the maintenance of cellular homeostasis in complex metazoans. Much of this intercellular communication is regulated through the classical secretory mechanism, in which proteins containing an endoplasmic reticulum (ER)-signaling sequence are actively released via the ER-toGolgi pathway (1). Despite the broad range of proteins entering this pathway, a number of actively secreted proteins lack a signal sequence for release and are released via alternative mechanisms, including exosomes (2).

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Section: Discussionsupporting

confidence: 54%

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes

Szul

Bratcher

Fraser³

et al. 2016

Am J Respir Cell Mol Biol

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“…This enzyme exhibits an apparent molecular mass of 87 kDa. The appearance of a heterogeneity in prolyl oligopeptidase activity was recognized previously in plasma (Soeda, 1984), in different cellular fractions (Dresdner et al, 1982;Dalmaz et al, 1986) and also recognized by Shirasawa (Shirasawa et al, 1994).…”

Section: Discussionsupporting

confidence: 52%

Development and Evaluation of Peptide‐Based Prolyl Oligopeptidase Inhibitors — Introduction of N‐Benzyloxycarbonyl‐Prolyl‐3‐Fluoropyrrolidine as a Lead in Inhibitor Design

Goossens

Vanhoof

Meester

et al. 1997

European Journal of Biochemistry

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The current study has been undertaken to develop new and biocompatible inhibitors for prolyl oligopeptidase, a highly specific endopeptidase, proposed to be involved, through its affinity for neuropeptides and kinins, in the processes of learning and memory and in the control of blood pressure. For in vitro evaluation of the inhibitors, human platelet prolyl oligopeptidase was purified to homogeneity and characterized. Northern blot analysis showed that mRNA coding for prolyl oligopeptidase was present in all tissues examined and only one transcript of 3.1 kb was detected. In addition to the human platelet enzyme, we also purified rat brain prolyl oligopeptidase, which proved to have the same characteristics as the human enzyme. In a series of tested peptides, bradykinin was found to be the best substrate. Based on this information, peptides bearing pseudopeptide bonds were generated and evaluated as inhibitors. The experiments clearly demonstrated that changes to the scissile peptide bond significantly decrease the affinity of prolyl oligopeptidase for the peptide derivatives. In our series of synthetic N-terminal blocked dipeptides, N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine was the most potent compound. Inhibition was reversible, but the inhibitor was bound tightly. Calculation of its K, according to Henderson [Henderson, J. P. (1972) Biochern. J. 127, 321 -3331 yielded a value of 0.8 nM. This compound was not cytotoxic in a cell culture system and inhibited the purified prolyl oligopeptidase from rat as well as from human origin. In vivo evaluation in male Whistar rats showed no acute toxicity. 5 h after administration, the most profound decrease in prolyl oligopeptidase activity was found in the thymus, brain, and testis. This study demonstrates that N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine is a potent inhibitor and a promising compound suitable to investigate the physiologic function of the enzyme in vitro and in vivo.

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“…When the bovine crude homogenate was resolved by centrifugation, the supernatant and particulate fractions were assayed for prolyl endopeptidase activity using the substrate ZGly-ProMCA. To date, prolyl endopeptidase activity had only been characterised from the cytosolic fraction of brain, although it had been suggested that a particulate prolyl endopeptidase activity may exist in small quantities (Dresdner et al, 1982;Camargo et al, 1984).…”

Section: Resultsmentioning

confidence: 99%

“…Although predominately a cytosolic enzyme, it has become increasingly clear that prolyl endopeptidase is found in both the soluble and particulate fractions of tissue homogenates. Dresdner et al (1982) noticed a sizable amount of prolyl endopeptidase activity to be associated with the particulate fraction in rabbit brain, but took that activity to be due to entrapped cytosolic activity, as the membrane fraction was not washed. Camargo et al (1984) found 10% of the prolyl endopeptidase activity in rabbit brain remained associated with the membrane fraction following salt washing, but was easily solubilised with detergents.…”

mentioning

confidence: 99%

Identification and Localization of a Synaptosomal Membrane Prolyl Endopeptidase from Bovine Brain

O'Leary

O’Connor

1995

European Journal of Biochemistry

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Prolyl endopeptidase, which has long been recognised for its importance in the degradation of several neuropeptides such as thyroliberin, luteinising hormone releasing hormone, angiotensin, substance P and neurotensin, has been widely characterised as a cytosolic enzyme. However, in this paper, we report the presence of a prolyl endopeptidase activity in the particulate fractions of bovine brain, which is distinct from that in the cytoplasm. This previously uncharacterised activity was found to reside in the synaptosoma1 membranes, a location which is highly significant for the inactivation of neuropeptides in brain. Following vigorous salt washing and osmotic shock, the prolyl endopeptidase activity was released from the membranes by treatment with the detergent Triton X-100, and was partially purified by gel filtration on a Sephacryl S-200HR column. This prolyl endopeptidase activity was shown to have a molecular mass (87 kDa) higher than the cytosolic prolyl endopeptidase but, from initial investigation, appears to demonstrate a similarly broad substrate specificity towards proline-containing neuropeptides. The partially purified enzyme was inhibited by certain thiol-protease inhibitors and was also found to be sensitive to the metal chelator 1 ,lO-phenanthroline.

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Subcellular Distribution of Prolyl Endopeptidase and Cation‐Sensitive Neutral Endopeptidase in Rabbit Brain

Cited by 60 publications

References 16 publications

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes

Development and Evaluation of Peptide‐Based Prolyl Oligopeptidase Inhibitors — Introduction of N‐Benzyloxycarbonyl‐Prolyl‐3‐Fluoropyrrolidine as a Lead in Inhibitor Design

Identification and Localization of a Synaptosomal Membrane Prolyl Endopeptidase from Bovine Brain

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