Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19INK4d

Calpains are a large family of Ca2+-dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21KIP1, for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of μ-calpain proteolysis of the p19INK4d protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: μ-calpain cleaves p19INK4d immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclin

Structural basis for the substrate specificity of bone morphogenetic protein 1/tolloid-like metalloproteases.

Procollagen C-peptidase, also known as bone morphogenetic protein 1 (BMP-1), is a multidomain, zinc endopeptidase of the astacin M12A family. BMP-1 is the prototype of a small group of proteases that have key roles in extracellular matrix formation and morphogenesis. BMP-1, its splice form mTLD, and the related proteases TLL-1 and TLL-2 are considered as promising drug targets for the treatment of excessive fibrosis and muscle wasting. We report here the crystal structures of the protease domains of human BMP-1 and the closely related Tolloid-like protease 1 (TLL-1). The crystal structures reveal an unexpected conformation of a cysteine-rich loop within the active site, and suggest that a flap movement is required in order to allow substrate binding. On the basis of these substantial differences between the BMP-1 and astacin active sites, a structural basis for their differing substrate specificities is proposed

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs

In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics.

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods