Substrate screening identifies a novel target sequence for the proteasomal activity regulated by ionizing radiation.
The screening for treatment-induced enzyme activities offers the opportunity to discover important regulatory mechanisms and the identification of potential targets for anti-cancer therapies. A novel screening technique was applied to screen substrate peptide sequences for proteolytic activities up- or down-regulated by ionizing radiation in tumor cells. One specific substrate sequence was cleaved in control cell extracts but to a smaller extent in irradiated cell extracts and investigated in detail. Based on protease-class-specific inhibitory studies and cleavage site analysis a potent warhead-inhibitor was synthesized and used to identify the proteasome as the protease of interest. The investigated sequence shows high homology to a regulatory site of nucleoporin 50, an element of the nuclear pore complex, and site specific cleavage of nucleoporin 50 was determined in vitro suggesting a novel link between the ionizing radiation-regulated proteasome and nuclear protein shuttling.
Broggini-Tenzer
Angela
A
Laboratory for Molecular Radiobiology, University Hospital Zurich, CH-8091 Zürich, Switzerland.
Hollenstein
Andreas
A
Pianowski
Zbigniew
Z
Wampfler
Andrea
A
Furmanova
Polina
P
Winssinger
Nicolas
N
Pruschy
Martin
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects*; D051379:Mice; D028861:Nuclear Pore Complex Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011839:Radiation, Ionizing; D013379:Substrate Specificity; D015854:Up-Regulation / Q000187:drug effects*
D028861:Nuclear Pore Complex Proteins; C105412:Nup50 protein, mouse; D019151:Peptide Library; D046988:Proteasome Endopeptidase Complex