Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Starting from the sequences of oryzacystatin-1 and canecystatin-1, a hybrid clone was recently obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability.In this project the hybrid clone and its mutants will be analyzed by multidimensional high-resolution Nuclear Magnetic Resonance spectroscopy. Structural changes should be identified by the 15N-HSQC spectra and correlated with the changes of the inhibitory activity of the samples towards cathepsin B.
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