Role of Prohormone Convertases in Pro-Neuropeptide Y Processing:  Coexpression and in Vitro Kinetic Investigations

Abstract

Proneuropeptide Y (ProNPY) undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which is further processed successively by carboxypeptidase-like and peptidylglycine alpha-amidating monooxygenase enzymes. To investigate whether prohormone convertases are involved in ProNPY processing, a vaccinia virus derived expression system was used to coexpress recombinant ProNPY with each of the prohormone convertases PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated neuroendocrine and constitutive prototype cell lines, respectively. The analysis of processed products shows that only PC1/3 generates NPY in NIH 3T3 cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells. The convertases furin and PACE4 are unable to process ProNPY in either cell line. Moreover, comparative in vitro cleavage of recombinant NPY precursor by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2 are involved in specific cleavage of the dibasic site. Kinetic studies demonstrate that PC1/3 cleaves ProNPY more efficiently than PC2. The main difference between the cleavage efficiency is observed in the Vmax values whereas no major difference is observed in Km values. In addition the cleavage by PC1/3 and PC2 of two peptides reproducing the dibasic cleavage site with different amino acid sequence lengths namely (20-49)-ProNPY and (28-43)-ProNPY was studied. These shortened ProNPY substrates, when recognized by the enzymes, are more efficiently cleaved than ProNPY itself. The shortest peptide is not cleaved by PC2 while it is by PC1/3. On the basis of these observations it is proposed, first, that the constitutive secreted NPY does not result from the cleavage carried out by ubiquitously expressed enzymes furin and PACE4; second, that PC1/3 and PC2 are not equipotent in the cleavage of ProNPY; and third, substrate peptide length might discriminate PC1/3 and PC2 processing activity.

Obesity Induces Hypothalamic Endoplasmic Reticulum Stress and Impairs Proopiomelanocortin (POMC) Post-translational Processing

“…Our results suggest that ER stress does not mediate changes in PC2 mRNA because neither DIO nor pharmacological induction of ER stress altered PC2 mRNA. However, it is difficult to measure PC2 mRNA expression specifically in POMC neurons because the ARC contains a heterogeneous mixture of POMC and NPY/ AgRP cells that have opposing actions on food intake are both regulated by PC2 at the intracellular level (67)(68)(69). Thus, we also analyzed PC2 promoter activity in N43/5 POMC-positive cells and found no effect of tunicamycin, which further supports the idea that ER stress does not alter PC2 mRNA.…”

Specificity of Prohormone Convertase 2 on Proenkephalin and Proenkephalin-related Substrates

“…Studies of recombinant PC2 cleavage on natural substrates include proglucagon (18,19), cholecystokinin-33 (20), and prodynorphin (21). Comparative work on both enzymes includes reports on the cleavage of proneuropeptide Y (22) and proinsulin (23,24). Taken together, this work supported the idea that both prohormone convertases prefer paired basic cleavage sites containing a P4 basic residue and can cleave at single basic residues given the presence of additional amino-terminal basic residues.…”

Neuropeptide Y: the universal soldier

“…However, with the discovery and cloning of the Y4 and Y5 receptors, the classical Y1 agonist [Leu 31 , Pro 34 ]NPY was found to bind significantly to these two receptor subtypes and thus to be less selective than previously thought. New compounds became available and among them, [D-Arg 25 ]NPY, [D-His 26 ]NPY and Des-AA [11][12][13][14][15][16][17][18] [Cys 7,21 , D-Lys 9 (Ac),D-His 26 ,Pro 34 ]NPY demonstrated high affinities for the Y1 receptor and inhibited forskolin-stimulated cyclic AMP (cAMP) production at subnanomolar concentrations [40]. Therefore, these peptides seem to be highly selective for the NPY Y1 receptor.…”