Harold A. Chapman

Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency

Pycnodysostosis, an autosomal recessive osteochondrodysplasia characterized by osteosclerosis and short stature, maps to chromosome 1q21. Cathepsin K, a cysteine protease gene that is highly expressed in osteoclasts, localized to the pycnodysostosis region. Nonsense, missense, and stop codon mutations in the gene encoding cathepsin K were identified in patients. Transient expression of complementary DNA containing the stop codon mutation resulted in messenger RNA but no immunologically detectable protein. Thus, pycnodysostosis results from gene defects in a lysosomal protease with highest expression in osteoclasts. These findings suggest that cathepsin K is a major protease in bone resorption, providing a possible rationale for the treatment of disorders such as osteoporosis and certain forms of arthritis.

Regulation of Integrin Function by the Urokinase Receptor

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration.

Cellular migration is critically dependent on an interplay between forces of attachment and detachment. Recent studies show that the serine protease urokinase and its major inhibitor and receptor regulate the adhesive properties of integrins, at least in part through initiation of cellular signals. These new functions for an old protease system imply intricate connections between proteolysis and adhesion that operate at the cell surface to regulate migration.

Essential Role for Cathepsin S in MHC Class II–Associated Invariant Chain Processing and Peptide Loading

Destruction of li by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complexes to the cell surface. The cysteine protease cathepsin S is highly expressed in spleen, lymphocytes, monocytes, and other class II-positive cells, and is inducible with interferon-gamma. Specific inhibition of cathepsin S in B lymphoblastoid cells prevented complete proteolysis of li, resulting in accumulation of a class II-associated 13 kDa li fragment in vivo. Consequently, the formation of SDS-stable complexes was markedly reduced. Purified cathepsin S, but not cathepsin B, H, or D, specifically digested li from alpha beta li trimers, generating alpha beta-CLIP complexes capable of binding exogenously added peptide in vitro. Thus, cathepsin S is essential in B cells for effective li proteolysis necessary to render class II molecules competent for binding peptides.

Cathepsin S Required for Normal MHC Class II Peptide Loading and Germinal Center Development

Major histocompatibility complex (MHC) class II molecules acquire antigenic peptides after degradation of the invariant chain (Ii), an MHC class II-associated protein that otherwise blocks peptide binding. Antigen-presenting cells of mice that lack the protease cathepsin S fail to process Ii beyond a 10 kDa fragment, resulting in delayed peptide loading and accumulation of cell surface MHC class II/10 kDa Ii complexes. Although cathepsin S-deficient mice have normal numbers of B and T cells and normal IgE responses, they show markedly impaired antibody class switching to IgG2a and IgG3. These results indicate cathepsin S is a major Ii-processing enzyme in splenocytes and dendritic cells. Its role in humoral immunity critically depends on how antigens access the immune system.

Proteases involved in MHC dass II antigen presentation

Summary: Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen‐presenting cells must be broken down into antigenic peptides. Second, class II tnolecules are synthesized with their peptide‐binding site blocked by invariant chain (li), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified tnice deficietit in single lysosomal proteases has allowed us to determine their role in these processes, Cathepsins (Cat) B and D. previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immtine responses and for T‐cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co‐exist in the same antigen‐presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications.

Cathepsins and compartmentalization in antigen presentation

Intracellular trafficking and cell surface expression of MHC class II molecules is a tightly regulated process and is to a large extent, determined by the fate of the class II chaperone, the invariant chain. Inhibition of endosomal proteases critical to invariant chain proteolysis reveals marked shunting of class II complexes to lysosomal compartments. Regulation of endosomal protease activity by expression of cystatin C directs class II cell surface expression during maturation of dendritic cells. These studies highlight the taut interactions between class-II-invariant-chain complexes and endosomal proteases during MHC class II maturation.

Endosomal proteolysis and MHC class II function

Newly synthesized MHC class II alpha and beta chains associate with a protein chaperone, the invariant chain, which promotes the proper assembly of MHC class II complexes and their trafficking through cells and prevents their untimely loading with peptides. Efficient loading of MHC class II heterodimers with antigenic peptides requires concurrent proteolytic processing of both the invariant chain and endocytosed proteins. Recent studies have elucidated the critical roles of specific cysteine proteases, especially cathepsins S and L, in degrading the invariant chain and regulating the convergence of processed antigen and MHC class II dimers competent for peptide loading.

Expression of cathepsins B and S in the progression of prostate carcinoma

Cathepsins B and S (CatB, CatS) are lysosomal cysteine proteases which, among other functions, appear to play a role in cancer progression in different tumor models due to their matrix‐degrading properties. To investigate their possible involvement in the development of prostate carcinoma, we immunohistochemically analyzed CatB and CatS in 38 primary human prostatic adenocarcinomas, as well as concomitant high‐grade prostatic intra‐epithelial neoplasia, nodular hyperplasia and normal tissue. CatB expression was observed in 28 (74%) and CatS in 32 (84%) carcinomas, being concomitant in 24 cases (63%). High‐grade intra‐epithelial neoplasia expressed CatB in 20/23 cases (87%), and a similar result was obtained for CatS, with expression of both coinciding in 18 cases (78%). In non‐neoplastic tissue, strong expression of both proteases was observed in macrophages, inflamed glands and transitional metaplasia, whereas atrophic glands and basal cells of normal glands displayed intense CatB positivity. We conclude that CatB and CatS are often expressed together in neoplastic prostatic cells from pre‐invasive to invasive and clinically detectable stages, suggesting a putative role in local invasion, though other functions cannot be ruled out.

Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H‐2DM

Major histocompatibility complex (MHC) class II molecules bind and present to CD4+ T cells peptides derived from endocytosed antigens. Class II molecules associate in the endoplasmic reticulum with invariant chain (Ii), which (i) mediates the delivery of the class II–Ii complexes into the endocytic compartments where the antigenic peptides are generated; and (ii) blocks the peptide‐binding site of the class II molecules until they reach their destination. Once there, Ii must be removed to allow peptide binding. The bulk of Ii–class II complexes reach late endocytic compartments where Ii is eliminated in a reaction in which the cysteine protease cathepsin S and the accessory molecule H‐2DM play an essential role. Here, we here show that Ii is also eliminated in early endosomal compartments without the intervention of cysteine proteases or H‐2DM. The Ii‐free class II molecules generated by this alternative mechanism first bind high molecular weight polypeptides and then mature into peptide‐loaded complexes.