Structural Basis of Reduction-dependent Activation of Human Cystatin F

Abstract

Cystatins are important natural cysteine protease inhibitors targeting primarily papain-like cysteine proteases, including cathepsins and parasitic proteases like cruzipain, but also mammalian asparaginyl endopeptidase. Mammalian cystatin F, which is expressed almost exclusively in hematopoietic cells and accumulates in lysosome-like organelles, has been implicated in the regulation of antigen presentation and other immune processes. It is an unusual cystatin superfamily member with a redox-regulated activation mechanism and a restricted specificity profile. We describe the 2.1 Å crystal structure of human cystatin F in its dimeric "off" state. The two monomers interact in a fashion not seen before for cystatins or cystatin-like proteins that is crucially dependent on an unusual intermolecular disulfide bridge, suggesting how reduction leads to monomer formation and activation. Strikingly, core sugars for one of the two N-linked glycosylation sites of cystatin F are well ordered, and their conformation and interactions with the protein indicate that this unique feature of cystatin F may modulate its inhibitory properties, in particular its reduced affinity toward asparaginyl endopeptidase compared with other cystatins.

Internalization of Exogenous Cystatin F Supresses Cysteine Proteases and Induces the Accumulation of Single-chain Cathepsin L by Multiple Mechanisms

“…Molecular modeling of monomeric cystatin F (PDB:2CH9) with CatL was previously described elsewhere (11) using the stefin B-papain complex (PDB: 1STF). Surface display and highlighting of putative AEP interaction sites were performed in Pymol.…”

“…Cystatin F is the only family member to form a di-sulfide linked dimeric structure (10), a configuration that renders the molecule inactive because of mutual steric hindrance of one protease inhibitory site by the other subunit (9, 11). Although the dimer can be rendered active in vitro by reduction (12), high levels of reducing agent are required and the monomeric form generated can inhibit some but not all physiological targets of cystatin F (9, 12).…”

Diverse regulatory roles for lysosomal proteases in the immune response

“…We recently showed that cystatin F, which is principally found in immune cells, could inhibit cathepsin C [65]. Cystatin F is made initially as an inactive disulphide-linked dimer [66][67][68]. Reduction generates an inhibitor able to target some endopeptidases but we found that carbohydrate-driven lysosomal targeting [69] resulted in monomerisation and activation by proteolysis, which extended the protease target range to enzymes such as cathepsin C [65].…”

Analysis of inflammatory and lipid metabolic networks across RAW264.7 and thioglycolate-elicited macrophages

“…Some of these genes are related to immune response; for example, interleukin 12b (Il12b) (46-fold upregulated at 4 h) and interleukin 12 receptor ␤ 1. Cystatin F (Cst7), which has been implicated in the regulation of antigen presentation and other immune processes ( 34,35 ), is highly upregulated only in TGEM cells (18-fold upregulated at 24 h ). Besides, Il12a, Cxcl1, Cxcl3, and vascular cell adhesion molecule 1 are also highly upregulated in TGEM cells only.…”