Bladder cancer detection using a peptide substrate of the 20S proteasome.
The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.
Faculty of Chemistry, University of Gdansk, Poland.
Natalia
N
Gruba
Faculty of Chemistry, University of Gdansk, Poland.
Magdalena
M
Wysocka
Faculty of Chemistry, University of Gdansk, Poland.
Magdalena
M
Brzezińska
Faculty of Chemistry, University of Gdansk, Poland.
Dawid
D
Dębowski
Faculty of Chemistry, Wroclaw University of Technology, Poland.
Marcin
M
Sieńczyk
Department of Electrochemistry, Institute of Chemistry, University of Bialystok, Poland.
Ewa
E
Gorodkiewicz
Department of Urology, J. Sniadecki Provincial Hospital of Bialystok, Poland.
Tomasz
T
Guszcz
Faculty of Chemistry, University of Gdansk, Poland.
Cezary
C
Czaplewski
Faculty of Chemistry, University of Gdansk, Poland.
Krzysztof
K
Rolka
Faculty of Chemistry, University of Gdansk, Poland.
Adam
A
Lesner
D062365:Aminobenzoates / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000652:urine*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000201:enzymology; D062367:ortho-Aminobenzoates
C000620395:2-aminobenzoic acid-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2)-NH2; D062365:Aminobenzoates; D005456:Fluorescent Dyes; D009842:Oligopeptides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D046988:Proteasome Endopeptidase Complex
bladder cancer biomarker; combinatorial chemistry; fluorescence assay; human 20S proteasome; intramolecularly quenched peptides