title: Induction and inhibition of CPAF activity during analysis of Chlamydia-infected cells.
abstract: Studies of the chlamydial protease CPAF have been complicated by difficulties in distinguishing bona fide intracellular proteolysis from in vitro proteolysis. This confounding issue has been attributed to CPAF activity in lysates from Chlamydia-infected cells. We compared three methods that have been used to inhibit in vitro CPAF-mediated proteolysis: (1) pre-treatment of infected cells with the inhibitor clasto-lactacystin, (2) direct cell lysis in 8 M urea and (3) direct lysis in hot 1% SDS buffer. We identified a number of experimental conditions that reduce the effectiveness of each method in preventing CPAF activity during lysate preparation. The amount of in vitro proteolysis in a lysate was variable and depended on factors such as the specific substrate and the time in the intracellular infection. Additionally, we demonstrated for the first time that artifactual CPAF activity is induced before cell lysis by standard cell detachment methods, including trypsinization. Protein analysis of Chlamydia-infected cells therefore requires precautions to inhibit CPAF activity during both cell detachment and lysate preparation, followed by verification that the cell lysates do not contain residual CPAF activity. These concerns about artifactual proteolysis extend beyond studies of CPAF function because they have the potential to affect the analyses of host and chlamydial proteins from Chlamydia-infected cells.
authors: Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697-2300, USA.
authors: Kirsten A
authors: KA
authors: Johnson
authors: Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697-2300, USA.
authors: Jennifer K
authors: JK
authors: Lee
authors: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA.
authors: Allan L
authors: AL
authors: Chen
authors: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA School of Medicine, UC Irvine, Irvine CA 92697-3950, USA.
authors: Ming
authors: M
authors: Tan
authors: Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697-2300, USA suetterc@uci.edu.
authors: Christine
authors: C
authors: Sütterlin
mesh_terms: D002689:Chlamydia / Q000737:chemistry*; D003584:Cytological Techniques / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism*; D004847:Epithelial Cells / Q000737:chemistry* / Q000382:microbiology*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007783:Lactones / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D007783:Lactones; D011480:Protease Inhibitors; D020543:Proteome; C098869:clasto-lactacystin beta-lactone; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D010450:Endopeptidases; C425923:CPA factor
keywords: proteolysis; proteolytic activity; substrates