The von Hippel-Lindau protein pVHL suppresses renal tumorigenesis in part by promoting degradation of hypoxia-inducible HIF-alpha transcription factors1, and additional mechanisms have been proposed2. pVHL also stabilizes plant homeodomain (PHD) protein Jade-1, which is a candidate renal tumor suppressor that may correlate with renal cancer risk3-5. We show here that Jade-1 binds the oncoprotein β-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type β-catenin, but not a cancer-causing form of β-catenin. While β-TrCP ubiquitinates only phosphorylated β-catenin6, Jade-1 ubiquitinates both phosphorylated and non-phosphorylated β-catenin and therefore regulates canonical Wnt signaling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of β-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL down-regulates β-catenin in a Jade-1-dependent manner and inhibits Wnt signaling, supporting a role for Jade-1 and Wnt signaling in renal tumorigenesis. The pVHL tumor suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH 2 -terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-GlyAsp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.Lysyl oxidase (protein-lysine 6-oxidase, EC 1.4.3.13) is a copper-dependent amine oxidase that oxidatively deaminates the ⑀-amino group of specific peptidyl lysine and hydroxylysine residues of collagen and of lysine in elastin. The resultant peptidyl aldehydes can spontaneously condense with other vicinal peptidyl aldehydes or with unreacted ⑀-amino groups to form inter-and intramolecular cross-linkages stabilizing the fibrous forms of these connective tissue structural proteins (1). Evidence has been presented that the inhibition of lysyl oxidase action toward collagen molecules results in the accumulation and ultimate proteolytic degradation of soluble collagen monomers, thus preventing the formation of insoluble collagen fibers (2). The participation of this enzyme is critical, therefore, to the development and repair of structurally sound connective tissues as in the respiratory, cardiovascular, and skeletal systems of the body.Recent studies of the pathway by which lysyl oxidase is produced in arterial smooth muscle cells revealed that the protein is translated as a 46-kDa preproenzyme. Following signal peptide cleavage, the proenzyme undergoes N-glycosylation apparently within the propeptide region, and the resulting 50-kDa proenzyme is then secreted into the extracellular space. The secreted proenzyme is then proteolytically converted to the functional catalyst derived from the COOH-terminal sequence of the propr...

The family of WD-repeat proteins comprises over 30 different proteins that share a highly conserved repeating motif [Neer, E. J., Schmidt, C. J., Nambudripad, R., & Smith, T. F. (1994) Nature 371, 297-300]. Members of this family include the signal-transducing G protein beta subunit, as well as other proteins that regulate signal transduction, transcription, pre-mRNA splicing, cytoskeletal organization, and vesicular fusion. The crystal structure of one WD-repeat protein (G beta) has now been solved (Wall et al., 1995; Sondek et al, 1996) and reveals that the seven repeating units form a circular, propeller-like structure with seven blades each made up of four beta strands. It is very likely that all WD-repeat proteins form a similar structure. If so, it will be possible to use information about important surface regions of one family member to predict properties of another. If WD proteins form structures similar to G beta, their hydrodynamic properties should be those of compact, globular proteins, and they should be resistant to cleavage by trypsin. However, the only studied example of a WD-repeat protein, G beta, synthesized in vitro in a rabbit reticulocyte lysate, is unable to fold into a native structure without its partner protein G gamma. The non-WD-repeat amino terminal alpha helix of G beta does not inhibit folding because G beta does not fold even when this region is removed. It is not known whether all WD-repeat proteins are unable to fold when synthesized in an in vitro system. We synthesized seven members of the family in a rabbit reticulocyte lysate, determined their Stokes radius, sedimentation coefficient, and frictional ratio, and assayed their stability to trypsin. Our working definition of folding was that the proteins from globular, trypsin-resistant structures because, except for G beta gamma, their functions are not known or cannot be assayed in reticulocyte lysates. We chose proteins that include amino and carboxyl extensions as well as proteins that are made up entirely of WD-repeats. We show that unlike G beta, several proteins with WD-repeats are able to fold into globular proteins in a rabbit reticulocyte lysate. One protein, beta Trcp, formed large aggregates like G beta, suggesting that it may also require a partner protein. Despite the presence of many potential tryptic cleavage sites, all of the proteins that did fold gave stable large products on tryptic proteolysis, as predicted on the basis of the structure of G beta. These studies suggest that other WD-repeat proteins are likely to form propeller structures similar to G beta.