DrosophilaSerpin 4 Functions as a Neuroserpin-Like Inhibitor of Subtilisin-Like Proprotein Convertases

Abstract

The proteolytic processing of neuropeptide precursors is believed to be regulated by serine proteinase inhibitors, or serpins. Here we describe the molecular cloning and functional expression of a novel member of the serpin family, Serine protease inhibitor 4 (Spn4), that we propose is involved in the regulation of peptide maturation in Drosophila. The Spn4 gene encodes at least two different serpin proteins, generated by alternate splicing of the last coding exon. The closest vertebrate homolog to Spn4 is neuroserpin. Like neuroserpin, one of the Spn4 proteins (Spn4.1) features a unique C-terminal extension, reminiscent of an endoplasmic reticulum (ER) retention signal; however, Spn4.1 and neuroserpin have divergent reactive site loops, with Spn4.1 showing a generic recognition site for furin/SPC1, the founding member of the intracellularly active family of subtilisin-like proprotein convertases (SPCs). In vitro, Spn4.1 forms SDS-stable complexes with the SPC furin and directly inhibits it. When Spn4.1 is overexpressed in specific peptidergic cells of Drosophila larvae, the animals exhibit a phenotype consistent with disrupted neuropeptide processing. This observation, together with the unique combination of an ER-retention signal, a target sequence for SPCs in the reactive site loop, and the in vitro inhibitory activity against furin, strongly suggests that Spn4.1 is an intracellular regulator of SPCs.

Comparative Analysis and Expression of Neuroserpin in <i>Xenopus laevis</i>

“…In contrast, the region around the P1 site of the Drosophila Spn4.1 protein consists of the basic amino acids arginine (P3)-arginine (P2)-lysine (P1)-arginine (P1′), which suggests that Spn4.1 targets serine proteases with a basic recognition site. This prediction was confirmed by several groups that have found in vitro complex formation of Spn4.1 with furin, a member of the family of subtilisin-like proprotein convertases, with a consensus arginine (P4) – X (P3) – arginine/lysine (P2) – arginine (P1) cleavage site [18, 26, 27]. …”

“…To this end, we performed RT-PCR and amplified, cloned and sequenced a specific product of 1,248 bp. Alignment of the deduced amino acid sequence with the mammalian and chicken neuroserpin sequences, and with the Drosophila Spn4.1 amino acid sequence (the closest Drosophila homologue to neuroserpin [18]), revealed an overall identity of 64, 73 and 32%, respectively (fig. 1A).…”

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia

“…Spn4, the fly homolog of the mammalian neuroserpin, seems to be an intracellular regulator of the subtilisin-like proprotein convertase furin (29). Ectopic expression of Spn4 failed to suppress the CHMP2B Intron5 phenotype.…”

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

“…However, the engineered PC target sequence is recognized by all PC family members and therefore such inhibitors are of limited use as therapeutics or research tools. Of particular note, protein protease inhibitors of the serpin family have been reported to be natural inhibitors of PCs, although their physiologic targets have not been defined (15)(16)(17)(18). Serpins inhibit their target proteases by a distinctive suicide substrate mechanism that leads to the formation of a stable serpin-proteinase covalent inhibitory complex (19,20).…”