Stefin A displaces the occluding loop of cathepsin B only by as much as required to bind to the active site cleft.

Cathepsin B (EC 3.4.22.1) is one of the most versatile human cysteine cathepsins. It is important for intracellular protein degradation under normal conditions and is involved in a number of pathological processes. The occluding loop makes cathepsin B unique among cysteine cathepsins. This ∼ 20 residue long insertion imbedded into the papain-like protease scaffold restricts access to the active site cleft and endows cathepsin B with its carboxydipeptidase activity. Nevertheless, the enzyme also exhibits endopeptidase activity and is inhibited by stefins and cystatins. To clarify the structural properties of the occluding loop upon the binding of stefins, we determined the crystal structure of the complex between wild-type human stefin A and wild-type human cathepsin B at 2.6 Å resolution. The papain-like part of cathepsin B structure remains unmodified, whereas the occluding loop residues are displaced. The part enclosed by the disulfide bridge containing histidines 110 and 111 (i.e. the 'lasso' part) is rotated by ∼ 45° away from its original position. A comparison of the structure of the unliganded cathepsin B with the structure of the proenzyme, its complexes with chagasin and stefin A shows that the magnitude of the shift of the occluding loop is related to the size of the binding region. It is smallest in the procathepsin structures and increases in the series of complexes with stefin A and chagasin, although it has no impact on the binding constant. Hence, cathepsin B can dock inhibitors and certain substrates regardless of the size of the binding region.

Expression and activity profiling of selected cysteine cathepsins and matrix metalloproteinases in synovial fluids from patients with rheumatoid arthritis and osteoarthritis.

Cysteine cathepsins and matrix metalloproteases are considered to play important roles in the development of arthritic diseases. Their accumulation in synovial fluid of primarily rheumatoid arthritis patients is also well documented. However, a detailed comparison between the protease levels and activities between rheumatoid arthritis samples and osteoarthritis samples has never been made. Here, we report that both cysteine cathepsins B and S and matrix metalloproteases-1, -3 and -13 are detected in patient synovial fluid samples with significantly higher levels detected in rheumatoid arthritis patients. Among the proteases, cathepsin S was found to be significantly elevated, consistent with its critical role in the immune response. These results suggest that cysteine cathepsins have a major role in inflammation at least in rheumatoid arthritis. In addition to proteases, interleukin-6 was detected at significant levels in most samples, suggesting that proinflammatory cytokines might be in-volved in the stimulation of expression of these proteases during inflammation.

Autocatalytic processing of procathepsin B is triggered by proenzyme activity.

Cathepsin B (EC 3.4.22.1) and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not yet been clarified. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin (Z-Arg-Arg-NH-MEC), suggesting that procathepsin B is catalytically active. The activity-based probe DCG-04, which is an E-64-type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming the zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. On the basis of these results, a model for autocatalytic activation of cysteine cathepsins is proposed, involving propeptide dissociation from the active-site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences because cathepsin zymogens were often found secreted in various pathological states.