Stephen P. Matthews

Multistep Autoactivation of Asparaginyl Endopeptidase in Vitro and in Vivo

Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently discovered lysosomal cysteine protease that specifically cleaves after asparagine residues. How this unusually specific lysosomal protease is itself activated is not fully understood. Using purified recombinant pro-enzyme, we show that activation is autocatalytic, requires sequential removal of C- and N-terminal pro-peptides at different pH thresholds, and is bimolecular. Removal of the N-terminal propeptide requires cleavage after aspartic acid rather than asparagine. Cellular processing, either of exogenously added AEP precursor or of pulse-labeled endogenous precursor, introduces at least one further cleavage to yield the final mature lysosomal enzyme. We also provide evidence that in living cells, there is clear compartmental heterogeneity in terms of AEP activation status. Moreover, we show that human monocyte-derived dendritic cells harbor inactive proforms of AEP that become activated upon maturation of dendritic cells with lipopolysaccharide.

Diverse regulatory roles for lysosomal proteases in the immune response

The innate and adaptive immune system utilise endocytic protease activity to promote functional immune responses. Cysteine and aspartic proteases (cathepsins) constitute a subset of endocytic proteases, the immune function of which has been described extensively. Although historically these studies have focused on their role in processes such as antigen presentation and zymogen processing within the endocytic compartment, recent discoveries have demonstrated a critical role for these proteases in other intracellular compartments, and within the extracellular milieu. It has also become clear that their pattern of expression and substrate specificities are more diverse than was first envisaged. Here, we discuss recent advances addressing the role of lysosomal proteases in various aspects of the immune response. We pay attention to reports demonstrating cathepsin activity outside of its canonical endosome/lysosome microenvironment.

Asparaginyl endopeptidase : case history of a class II MHC compartment protease

Summary:  Although the endpoint of the class II antigen‐processing pathway is well characterized, the processing events that lead to the production of class II major histocompatibility complex (MHC)/peptide complexes are not. It is generally assumed that protease action on native antigen substrates leads to unfolding and capture of either long or short peptides. Whether specific protease activities are needed for presentation of particular T‐cell epitopes is largely unknown. Here, we review our recent studies that aim to identify the processing enzymes that initiate processing of different antigens. We suggest a general strategy that can potentially identify preferred relationships between substrates and processing enzymes in vitro and suggest ways in which these relationships can be tested in vivo. We draw heavily on the example of asparaginyl endopeptidase, which is involved in both productive and destructive processing of different antigen substrates. Overall, while there is undoubtedly redundancy in class II MHC antigen processing, the contributions of individual enzymes can be clearly dissected.

Role of CCL11 in Eosinophilic Lung Disease during Respiratory Syncytial Virus Infection

ABSTRACT Respiratory syncytial virus (RSV) is a major viral pathogen of infants and the elderly. Significant morbidity is caused by an overexuberant mixed lung cell infiltrate, which is thought to be driven by chemokines. One of the main chemotactic mediators responsible for the movement of eosinophils is CCL11 (eotaxin). Using a mouse model of eosinophilic bronchiolitis induced by RSV, we show here that treatment in vivo with a blocking antibody to CCL11 greatly reduces lung eosinophilia and disease severity. In addition, anti-CCL11 caused a striking inhibition of CD4-T-cell influx and shifted cytokine production away from interleukin-5 without reducing the resistance to viral replication. These results suggest that in addition to influencing eosinophil diapedesis and survival, anti-CCL11 has an action on T cells. These studies strengthen the case for anti-CCL11 treatment of Th2-driven diseases.

Creation versus Destruction of T Cell Epitopes in the Class II MHC Pathway

Abstract: Proteases perform two key roles in the class II MHC antigen processing pathway. They initiate removal of the invariant chain chaperone for class II MHC and they generate peptides from foreign and self proteins for eventual capture and display to T cells. How a balance is achieved between generation of suitable peptides versus their complete destruction in an aggressive proteolytic environment is not known. Nor is it known in most cases which proteases are actually involved in antigen processing. Our recent studies have identified asparagine endopeptidase (AEP or legumain) as an enzyme that contributes to both productive and destructive antigen processing in the class II MHC pathway. The emerging consensus seems to be that individual proteolytic enzymes make clear and non‐redundant contributions to antigen processing.

Asparagine endopeptidase is required for normal kidney physiology and homeostasis

Although protein recapture and catabolismi is known as a key function of kidney proximal tubular cells (PTCs), to date, no single protease has been shown to be required. Asparagine endopeptidase (AEP) is an unusually specific endosomal and lysosomal cysteine protease, expressed at high levels in the PTCs of the mammalian kidney. We report that mice lacking AEP accumulate a discrete set of proteins in their PTC endosomes and lysosomes, which indicates a defect in the normal catabolism of proteins captured from the filtrate. Moreover, the mice develop progressive kidney pathology, including hyperplasia of PTCs, interstitial fibrosis, development of glomerular cysts, and renal pelvis dilation. By 6 mo of age, the glomerular filtration rate in AEP‐null mice dropped by almost a factor of 2, and the mice developed proteinuria. We also show that EGF receptor levels are significantly higher in AEP‐null PTCs, which likely explains the hyperplasia, and we show that chemical inhibition of AEP activity suppresses down‐regulation of the EGF receptor in vitro. Thus, AEP is required for normal protein catabolism by PTCs, and its loss induces proliferative and other abnormalities in the murine kidney, at least in part through defective regulation of the EGF receptor.—Miller, G., Matthews, S. P., Reinheckel, R., Fleming, S., Watts, C. Asparagine endopeptidase is required for normal kidney physiology and homeostasis. FASEB J. 25, 1606–1617 (2011). www.fasebj.org

Distinct Protease Requirements for Antigen Presentation In Vitro and In Vivo

Asparagine endopeptidase (AEP) or legumain is a potentially important Ag-processing enzyme that introduces limited cleavages that trigger unfolding and class II MHC binding of different Ag substrates. AEP is necessary and sufficient for optimal processing and presentation of the tetanus toxin C fragment (TTCF) Ag in vitro, but its importance has not been tested in vivo. Surprisingly, virtually normal T cell and Ab responses to TTCF were mounted in AEP-deficient mice when examined 10 d after immunization. This was the case when TTCF was emulsified with CFA, adsorbed onto alum, or expressed within live Salmonella typhimurium. In addition, the dominant Ab and T cell determinants recognized in TTCF were essentially unchanged in AEP-deficient mice. These data are explained, at least in part, by the much lower levels of AEP expressed in primary murine APCs compared with immortalized B cell lines. Even so, the initial in vivo kinetics of TTCF presentation were slower in AEP-deficient mice and, as expected, boosting AEP levels in primary APCs enhanced and accelerated TTCF processing and presentation in vitro. Thus, AEP remains the protease of choice for TTCF processing; however, in its absence, other enzymes can substitute to enable slower, but equally robust, adaptive immune responses. Moreover, clear relationships between Ags and processing proteases identified from short-term in vitro processing and presentation studies do not necessarily predict an absolute in vivo dependency on those processing enzymes, not least because they may be expressed at strikingly different levels in vitro versus in vivo.

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

Background: Cystatin F is a protease inhibitor normally found within the endocytic pathway, but can be secreted. Results: Secreted cystatin F can be internalized thereby inhibiting multiple targets and causing the accumulation of cathepsin L. Conclusion: Cystatin F inhibits the CatL convertase AEP and stabilizes CatL protein levels. Significance: Secreted cystatin F can be activated in trans expanding its inhibitory potential beyond its site of synthesis. Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169–179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.

The PDK1–Rsk Signaling Pathway Controls Langerhans Cell Proliferation and Patterning

Langerhans cells (LC), the dendritic cells of the epidermis, are distributed in a distinctive regularly spaced array. In the mouse, the LC array is established in the first few days of life from proliferating local precursors, but the regulating signaling pathways are not fully understood. We found that mice lacking the kinase phosphoinositide-dependent kinase 1 selectively lack LC. Deletion of the phosphoinositide-dependent kinase 1 target kinases, ribosomal S6 kinase 1 (Rsk1) and Rsk2, produced a striking perturbation in the LC network: LC density was reduced 2-fold, but LC size was increased by the same magnitude. Reduced LC numbers in Rsk1/2−/− mice was not due to accelerated emigration from the skin but rather to reduced proliferation at least in adults. Rsk1/2 were required for normal LC patterning in neonates, but not when LC were ablated in adults and replaced by bone marrow–derived cells. Increased LC size was an intrinsic response to reduced LC numbers, reversible on LC emigration, and could be observed in wild type epidermis where LC size also correlated inversely with LC density. Our results identify a key signaling pathway needed to establish a normal LC network and suggest that LC might maintain epidermal surveillance by increasing their “footprint” when their numbers are limited.

Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability.

Summary Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.