Bladder cancer detection using a peptide substrate of the 20S proteasome

Abstract

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 × 105 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.

A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome

“…Next, we evaluated the ability of substrate 1 to detect proteasome activity in biological samples. As previously reported, elevated 20S proteasome activity in human urine is often linked to a diagnosis of bladder cancer [21]. To verify whether substrate 1 could be used in such an assay, we recorded its cleavage intensity in healthy (n = 5) urine samples and bladder cancer urine (n = 8) samples.…”

Inhibition of human constitutive 20S proteasome and 20S immunoproteasome with novel N‐terminally modified peptide aldehydes and their antitumor activity

“…The selected internally quenched proteasome substrates, obtained in our previous researches via the combinatorial chemistry method, were converted into tetrapeptide aldehydes. Their primary structures, the observed values of [M + H] + peaks obtained by MALDI‐TOF mass spectrometry, and RP‐HPLC retention times are shown in Table .…”

“…The internally quenched peptide substrates, described by us previously, were converted successfully into corresponding peptide aldehydes composed of four amino acids. Most of them are capable of potently blocking the proteolytic subunits of human 20S proteasome.…”

“…Recently, we have presented three groups of novel, selective and sensitive, internally quenched peptide substrates of human cCP 20S, optimized at their primed and nonprimed positions . Each group included substrates presenting different specificity, either toward β 1c, β 2c, or β 5c subunits.…”

Emerging challenges in the design of selective substrates, inhibitors and activity‐based probes for indistinguishable proteases

“…Later, the Lesner group investigated the chymotrypsin‐like activity of the 20S proteasome subunit (β5) and identified the selective substrate ABZ‐Val‐Val‐Ser‐Tyr‐Ala‐Met‐Gly‐Tyr(3NO 2 )‐NH 2 and determined its kinetic parameters ( k cat / K m = 9.7 × 10 5 m −1 ·s −1 , k cat = 8 s −1 ). These were the first studies on the chymotrypsin‐like activity alone, leading to the optimization of the prime area of the substrate and avoiding non‐selective processing by other proteases with overlapping specificity .…”