tailieunhanh - Báo cáo Y học: Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A. | Eur. J. Biochem. 269 2747-2754 2002 FEBS 2002 doi Substrate specificity of human kallikrein 2 hK2 as determined by phage display technology Sylvain M. Cloutier1 Jair Ribeiro Chagas2 Jean-Pierre Mach3 Christian M. Gygi1 Hans-Jurg Leisinger1 and David Deperthes1 1 Urology Research Unit Department of Urology Lausanne Switzerland 2Centro Interdisciplinar de Investigacao Bioquimica Universidade de Mogi das Cruzes Brazil 3Institute of Biochemistry University of Lausanne Switzerland Human glandular kallikrein 2 hK2 is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development however there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein derived from green fluorescent protein that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position which is further enhanced by a Ser in PT position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2 which include protein C inhibitor semenogelins and fibronectin. Moreover three new putative hK2 protein substrates shown elsewhere to

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