Structural and functional analysis of cystatin E reveales enzymologically relevant dimer and amyloid fibril states

Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer’s disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites towards papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions. ______________________________________ Cysteine proteases are key regulators in many physiological processes. By consequence, dysregulation of protease activity can have severe effects resulting in a variety of pathologies, including cancer and Alzheimer’s disease (1,2). Therefore, proteases must be regulated delicately and on different levels. Cystatins are inhibitors specifically controlling the activity of cysteine proteases. The cystatin superfamily shares a common fold that is characterized by a 5-stranded antiparallel -sheet that is enwrapping a central helix. It is further organized into three subfamilies: http://www.jbc. org/cgi/doi/10.1074/jbc.RA118.002154 The latest version is at JBC Papers in Press. Published on July 2, 2018 as Manuscript RA118.002154 by gest on July 5, 2018 hp://w w w . jb.org/ D ow nladed from Cystatin E transforms to multimers with distinct functions 2 (i) stefins, (ii) cystatins, and (iii) kininogens, fetuins and non-inhibitory cystatins (3,4). Family 1 cystatins (stefins) are mainly localized intracellularly and ubiquitously expressed in most cell types (3,5). They are potent inhibitors of papain (family C1) and the papain-like cathepsins with Ki values in the low nM range (6,7). The interaction of stefins with papain is mediated by a tripartite wedge-shaped structure formed by the Nterminus (Ser1-Val10, cystatin C numbering) and two hairpin loops (loop L1 and L2). Essentially, the N-terminus binds to the non-primed side, while the two adjacent hairpin loops occupy the primedsubstrate binding sites. Family 2 cystatins resemble the largest subfamily of the cystatin-fold with seven members identified so far. Contrasting the stefins, selected family 2 cystatins (C, E/M, F) harbor, in addition to their papain-binding site, a legumain binding site (810). Human legumain is a caspase-like cysteine protease (family C13) that mainly localizes to the endo-lysosomal system, where it plays an important function for the processing of antigens for presentation on the MHCII complex (11). On a pathophysiological level, legumain has been implicated in various disorders, including cancers and Alzheimer’s disease (12-14). Under these conditions legumain was found translocated to the nucleus, cytoplasm and extracellularly. Because of its strict specificity for cleaving after asparagine residues, it is synonymously referred to as the asparaginyl-endopeptidase (AEP) (15,16). This strict preference is exploited by the legumain inhibitory cystatins C, E and F, which use a conserved Asn39 residue, localized on a reactive center loop different from the papain inhibitory site to specifically bind to the legumain active site (9,17). Furthermore, the interaction with legumain involves an additional legumain exosite loop (LEL) inserted between cystatin strands 3 and 4. Complex formation leads to conformational stabilization of the pH-sensitive legumain at near neutral pH. Unlike family 1 cystatins, legumain inhibitory cystatins are secreted outside the cell and are in some cases glycosylated (10,18-20). While cystatin C is ubiquitously expressed in different human tissues, cystatin E/M is mainly localized to skin epithelia, emphasizing its role in cutaneous biology (5,10,21). Co-localization of human cystatin E (hCE) and legumain has been reported in hair follicles (22). Cystatins not only encode a high intrinsic variability because of their function as dual protease inhibitors but also because of their ability to transform to distinct oligomerization states upon conformational destabilization. Factors trigging this oligomerization include N-terminal truncation by proteolytic enzymes, acidic pH, heating and point mutations. These cause dimer formation via a domain swapping mechanism (23-25). Essentially, the N-terminal segment, comprising β1, α, β2 up to the L1-loop, of one monomer exchanges with that of a second monomer (26). Consequently, the papain inhibitory site becomes inaccessible, whereas the legumain inhibitory site remains intact. Cystatin C oligomerization leads to the formation of amyloid deposits in the brain at advanced age (25). A naturally occurring L68Q variant was identified in the cerebral fluid of patients suffering from hereditary cystatin C angiopathy (HCC, Iceland disease), which accelerates this process significantly (6,27). Similarly, N-terminally truncated cystatin C, lacking the first 10 amino acids of the native sequence, was isolated from cystatin C amyloid deposits (28). This truncation was associated with proteolytic processing by proteases released to the cerebrospinal fluid and similarly results in accelerated formation of amyloid depositions (29). Stefin B was also reported to form amyloid fibrils and is an Aβ-binding protein and therefore supposed to play a role in Alzheimer’s disease (30-32). Both legumain and cystatins became attractive drug targets due to their relevance in different types of cancer and dementia. Among the cystatins, the family two cystatins became especially interesting, because of their function as dual protease inhibitors and because they are secreted to the extracellular space, where legumain and cathepsins are similarly observed under pathophysiologic conditions. Cystatin E is the most potent physiologic legumain inhibitor, binding 100fold more tightly as compared to cystatin C (7). Thereby it is associated with a tumor suppressor function in prostate cancer, melanoma and oral carcinoma cells (33-35). Furthermore, cystatin E has been observed colocalized with legumain in the extracellular environment under normal and under pathophysiologic conditions (22,36). Notably, not only co-localization but also co-trafficking of by gest on July 5, 2018 hp://w w w . jb.org/ D ow nladed from Cystatin E transforms to multimers with distinct functions 3 legumain together with cystatin E from outside the cell to inside a cell has been reported (37). However, cystatin E’s physico-chemical properties including its propensity to form dimers and higher oligomers were hardly studied so far. Therefore, we set out to challenge the conformation of cystatin E by different triggering factors including N-terminal truncation, pH, and heating. Furthermore, we analyzed the effect thereof on its 3d structure and inhibitory function.