Prolyl carboxypeptidase and its substrates

Ondertitel:a potential role in metabolic disorders and the cardiovascular system

Prolyl carboxypeptidase (PRCP) is a serine protease with a function in metabolic disorders and the cardiovascular system because of its role as angiotensin II- and III-, des-Arg9-bradykinin-, ?-MSH 1-13- and (pyr)-apelin-13-cleaving enzyme.During this doctoral research, the physiological role of PRCP was further explored. To guarantee a solid supply, we produced recombinant human PRCP by use of the baculovirus insect cell expression system. Using this in-house produced recombinant enzyme, it was confirmed that compound 8o is a potent, reversible and selective inhibitor of PRCP, although the earlier reported potency (IC50 = 1 nM) could not be confirmed.Compound 8o was used to further explore the role of PRCP in peptide turnover in human umbilical vein (HUVEC) and aortic (HAoEC) endothelial cells. The selective inhibitors compound 8o, DX600 and KYP-2047 were used to study the contribution of PRCP, angiotensin converting enzyme 2 (ACE2) and prolyl oligopeptidase (PREP). For the first time, the role of PRCP was investigated by use of a selective inhibitor. In HUVEC, the C-terminal cleavage of (pyr)-apelin-13 was mediated only by PRCP, while in HAoEC, also ACE2 contributed to this cleavage. PREP was not involved in the cleavage of (pyr)-apelin-13 and the studied enzymes did not contribute to the C-terminal cleavage of ?-MSH 1-13 in endothelial cells. The C-terminal cleavage of both angiotensin II and III was PRCP-dependent in HUVEC and HAoEC, while the C-terminal cleavage of des-Arg9-bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. To unravel where exactly PRCP exerts its function in this peptide turnover, we studied the subcellular location of PRCP in endothelial cells. We did confirm lysosomal location of PRCP, while we did not observe its location at the cell membrane, contrary to an earlier report. Additionally, pro-inflammatory stimulation of endothelial cells leads to the secretion of PRCP in the extracellular environment.In the context of its role in metabolic disorders, PRCP activity was measured in human subcutaneous and visceral adipose tissue and corresponding serum samples and muscle tissue of obese, type 2 diabetic and non-diabetic, and lean men. For the first time, PRCP activity was detected in adipose tissue. Moreover, the PRCP activity was significantly higher in adipose tissue in comparison with the activity in muscle tissue, in both lean and obese individuals. The serum PRCP activity was significantly higher in obese type 2 diabetic individuals in comparison with lean and obese non-diabetic individuals.Additionally, the serum PRCP activity was positively correlated with biochemical parameters important in the diagnosis of diabetes.

Jaar van publicatie:2021

Trefwoorden:Doctoral thesis

Toegankelijkheid:Closed