Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P)

Stefan Kunz,Laura Cendron,Antonella Pasquato,Nabil Georges Seidah,Joel Ramos Da Palma

doi:10.1074/jbc.m115.677757

Abstract

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.

Highlights

Proteolytic processing by endoproteases represents a major post-translational modification and is essential to regulate and diversify protein functions
Our recent studies on the mechanisms underlying SKI-1/site-1 protease (S1P) maturation revealed that immature forms retaining fragments of the prodomain are already enzymatically active [14], which appears very different from the maturation of the prototypic basic proprotein convertases (PCs) furin
Partial or Complete Deletion of the N-terminal Prodomain Impairs SKI-1/S1P Autocatalytic Cleavage Both in cis and in trans—Previous studies indicated that in bacterial subtilases [30] and in SKI-1/S1P [14], the prodomain is crucial for folding, maturation, and acquisition of catalytic activity of the enzyme [14, 30], similar to other PCs

Summary

Experimental Procedures

Antibodies—mAb 83.6 mouse anti-LCMV glycoprotein was produced and purified as described previously [19]. Determination of in Vitro Activities of Soluble SKI-1/S1P—To assess the in vitro activities of the soluble SKI-1/S1P BTMD variants used in this study, constructs were expressed in HEK293T cells, and enzymatic activity was detected in conditioned medium via a reliable enzymatic assay as described previously [22]. Region optimization was performed by analysis of discrete optimized protein energy profiles of the produced three-dimensional models structural alignments with the templates and based in protein secondary structure prediction. Nonredundant protein sequences that contained both a signal peptide and a fully conserved catalytic triad where kept for further analysis, resulting in a total of 143 sequences from unique species This protein sequence data set was used to construct a multiple sequence alignment using the MAFFT V.017 [28] algorithm from inside the Geneious 7.19 software [29]. The multiple sequence alignment was used to map residue conservation in the SKI-1/S1P homology-based models

Results

Secondary structure assignment of the recombinant proAC construct

Discussion