No abstract

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves b-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in b-casein turnover when the assay temperature is raised from 37 to 45 8C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

No abstract

Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after ]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser 139 ,Ala-163 ]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.Bone remodeling is a constant process that involves bone resorption and rebuilding (for review, see Ref. 1). The resorption phase of this process is carried out by osteoclasts, which adhere to the surface of bone leading to the creation of an extracellular compartment termed the resorption pit. The resorption pit is maintained at an acidic pH, causing the dissolution of the mineral components of the underlying bone and exposure of the proteinaceous matrix to the action of proteolytic enzymes (2-6). The rebuilding phase of the remodeling process involves the recruitment of osteoblasts to the sites of prior bone resorption, where the layering of a new proteinaceous matrix occurs and becomes mineralized.Cathepsin K, a member of the papain cysteine protease family, has recently been implicated in the resorption of the bone matrix (7-12). The cDNA encoding this protease was cloned from human, rabbit, and mouse osteoclast libraries and expressed in baculovirus-infected insect cells by several independent groups as an inactive secreted proenzyme (7-13). Bossard (14) and Brömme (13) have demonstrated activation of the recombinant proenzyme in vitro by proteolytic degradation of the N-terminal 99-amino acid propeptide; however, different mechanisms leading to its activation were implicated.Activation of procathepsin K in vivo is likely to occur in the low pH environment of the resorption pit, via two possible mechanisms. The propeptide may be cleaved by another protease, such as cathepsin D as suggested by Brömme et al. or by an autocatalytic process, which is more consistent with the data presented by Bossard et al. (14).To elucidate the mechanism of activation of cathepsin K, we constructed a mutant in which the presumed active site Cys at position 139 was changed to Ser. The kinetics of activation of mutant and wild-type cathepsin K were studied in vitro.In this report we provide the following evidence for an autocatalytic activation mechanism. First, in vitro self-activation of wild-type procathepsin K occurs spontaneously at 4°C, pH 4 and is catalyzed by mature cathepsin K. Second, unlike wildtype enzyme, t...

No abstract

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups f lanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

No abstract