Publications (2)2.93 Total impact
Article: Cell cycle-dependent caspase-like activity that cleaves p27(KIP1) is the beta(1) subunit of the 20S proteasome.[show abstract] [hide abstract]
ABSTRACT: We previously described a caspase-like activity, which we termed KIPase that is implicated in the turnover of the mammalian cell cycle regulator p27(KIP1). KIPase cleaves a tetra-peptide substrate, Ac-DPSD-AMC, which mimics the target site in p27(KIP1), and inhibitors based on this tetra-peptide are ineffective against other known caspases. Here we describe the purification and characterization of KIPase, and trace its activity to the beta(1) subunit of the 20S proteasome. Further analyses revealed that the activity of the beta(1) subunit is up-regulated as cells enter the cell cycle without concomitant change in the levels of the proteasome beta(1), beta(2) or beta(5) subunits. To our knowledge, this is the first description of cell cycle regulation of the caspase-like activity of the 20S proteasome.Archives of Biochemistry and Biophysics 11/2007; 466(2):186-93. · 2.93 Impact Factor
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ABSTRACT: In this chapter 1 describe the PCR-coupled subtractive hybridization technique of representational difference analysis of cDNA (cDNA RDA). cDNA RDA is based on the representational difference analysis (RDA) method previously described by Lisitsyn et al., and can be used to identify genes whose expression is modified between two populations of cells. cDNA RDA is relatively inexpensive to perform and requires no prior knowledge of genome sequence data. The combining of PCR with a subtractive methodology results in a highly effective and extremely sensitive technique with application to very low amounts of starting material. The procedure can be divided into three main phases: PCR generation of amplicons representative of the starting populations of RNA molecules being compared; the two-step subtractive hybridization of these representations, leading to the enrichment of amplified fragments of differentially expressed genes and the sequential depletion of sequences common to both populations; and the purification, cloning, and sequencing of the resulting difference products.Methods in molecular medicine 02/2004; 94:49-66.