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Direct role of furin in mammalian prosomatostatin processing.

ABSTRACT: We have previously reported that rat prosomatostatin (rPSS) undergoes conversion at Arg decreases and Lys decreases monobasic sites to SS-28 and PSS-(1-10) respectively in COS-7 cells, and have proposed furin or a related enzyme of the constitutive secretory pathway as the endoproteinase responsible. Here we have tested directly the ability of furin to cleave rPSS at the two monobasic sites as well as at the RXRK dibasic site of SS-14 conversion (a furin motif, except for Lys substituting for Arg at P1). Recombinant vaccinia virus (VV) vectors were used to co-express rPSS with graded doses of furin in COS-7 cells and LoVo colon carcinoma cells deficient in furin. PSS and cleavage products in cell extracts and media were characterized by HPLC analysis and C-terminal [SS-14-like immunoreactivity (SS-14 LI)] and N-terminal [PSS-(1-10) LI] directed radioimmunoassays. There was a dose-dependent increase in SS-28 production from rPSS by furin in COS-7 cells from 29% (control) to 58% (high-dose furin) associated with a progressive decrease in unprocessed PSS from > 60% to approximately 20% of total SS-14 LI. Significant SS-14 production occurred only at high levels of furin infection. Control LoVo cells infected with VV:rPSS exhibited production of approximately 21% SS-28, approximately 15% PSS-(1-10) and 3.5% SS-14. Infection of LoVo cells with VV:hfurin (hfurin = human furin) enhanced SS-28 production to 30-34%. SS-14 synthesis also increased to 25-40%, probably by conversion from SS-28. Overexpression of furin in COS-7 or LoVo cells failed to increase PSS-(1-10) production. These results show that furin is a candidate SS-28 convertase. Arginine is the preferred residue at the P1 site of furin cleavage. Furin does not process rPSS to PSS-(1-10), suggesting the existence of another monobasic convertase with a preference for Lys rather than Arg at P1. Such an enzyme could also explain the presence of endogenous SS-28-, PSS-(1-10)- and SS-14-producing activities in LoVo cells.

SUBMITTER: Galanopoulou AS

PROVIDER: S-EPMC1135796 | biostudies-other | 1995 Jul

REPOSITORIES: biostudies-other

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Project description:HIV-associated neurocognitive disorders (HAND) is a syndrome defined by neurocognitive deficits that are driven by viral neurotoxins, cytokines, free radicals, and proteases expressed in the brain. This neurological disease has also been linked to activation of Protease-Activated Receptors 1 and 2 (PAR1,2). These receptors are highly expressed in the central nervous system and are upregulated in HAND. Secretory basic-amino-acid-specific Proprotein Convertases (PCs), which cleave precursor proteins at basic residues, are also induced in HAND. They are vital for many biological processes including HIV-1 entry into cells. The cytoprotective role of Furin, PC5, and PACE4 has been linked to the presence of a potential PC-cleavage site R41XXXXR46? in PAR1. Furthermore, Furin binds PAR1 and both are trapped in the trans-Golgi-network (TGN) as inactive proteins, likely due to the intermediary trafficking role of phospho-Furin acidic cluster sorting protein 1 (PACS1). Nothing is known about PAR2 and its possible recognition by PCs at its putative R31XXXXR36? processing site. The present study implicates PACS1 in the retrograde trafficking of PAR1 to the TGN and demonstrates that the cytosolic extreme C-terminal tail of PAR1 contains an acidic phosphorylatable PACS1-sensitive domain. We further show the requirement of Asn47 in PAR1 for its Furin-dependent TGN localization. Our data revealed that Furin is the only convertase that efficiently cleaves PAR2 at Arg36?. N-glycosylation of PAR2 at Asn30 reduces the efficacy, but enhances selectivity of the Furin cleavage. Finally, in co-cultures comprised of human neuroblastoma SK-N-SH cells (stably expressing PAR1/2 and/or Furin) and HIV-1-infected primary macrophages, we demonstrate that the expression of Furin enhances neuronal cell viability in the context of PAR1- or PAR2-induced neuronal cytotoxicity. The present study provides insights into early stages of HIV-1 induced neuronal injury and the protective role of Furin in neurons co-expressing PAR1 and/or PAR2, as observed in HAND.

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Direct role of furin in mammalian prosomatostatin processing.

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