The role of basic amino acid surface clusters on the collagenase activity of cathepsin K.

Cathepsin K is a highly potent collagenase in osteoclasts and is responsible for bone degradation. We have previously demonstrated that its unique collagenolytic activity is modulated by glycosaminoglycans that form high molecular weight complexes with the protease. However, mutational analysis of a specific glycosaminoglycan-cathepsin K binding site only led to a 60% reduction of the collagenolytic activity suggesting additional glycosaminoglycan binding sites or other determinants controlling this activity.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter.

Background: To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein.

Structure-activity analysis of cathepsin K/chondroitin 4-sulfate interactions.

In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L.

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex.

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination.

Production and activation of recombinant papain-like cysteine proteases.

Papain-like cysteine proteases are the most numerous family of the cysteine protease class. They are expressed throughout the animal and plant kingdoms as well as in viruses and bacteria. More recently, this protease family has drawn attention as a potential pharmaceutical drug target in diseases characterized by excessive extracellular matrix degradation such as in osteoporosis, arthritis, vascular diseases, and cancer.