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

Magdalena Wysocka,Artur Giełdoń,Natalia Gruba,Michalina Michalska,Adam Lesner,Anita Romanowska

doi:10.3390/ijms21072396

Magdalena Wysocka, Artur Giełdoń + Show 4 more

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https://doi.org/10.3390/ijms21072396

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Abstract

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ–Dap(O2(Cbz))–Dap(GO1)–Dap(O2(Cbz))–Arg–ANB–NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 μM, kcat = 245 s−1, and kcat/Km = 7.61 × 107 M−1 s−1. This process was practically halted when a selective inhibitor of the β2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10−11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.

Highlights

The 20S proteasome has a tube-like structure composed of four rings formed by 28 protein subunits [1,2]
The cleavage specificity of each catalytically active β subunit is defined by the structure of its S1 binding pocket, the chemical properties of the amino acid residue located at position 45 of the proteasome structure [4]
We developed a new 20S proteasome fluorescent peptidomimetic probe with superior kinetic parameters, yielding 7.61 × 107 M−1 s−1

Summary

Introduction

The 20S proteasome has a tube-like structure composed of four rings formed by 28 protein subunits [1,2]. All α subunits have structural functions, whereas only three of the β subunits (β1, β2, and β5) have active centers (catalytic sites) [3]. In each of these β subunits, a threonine (Thr) residue is positioned at the N-terminal. This amino acid is crucial for the proteolytic activity of the enzyme. The cleavage specificity of each catalytically active β subunit is defined by the structure of its S1 binding pocket, the chemical properties of the amino acid residue located at position 45 of the proteasome structure [4]. The β5 subunit, with Met in the substrate pocket [5,6], cleaves peptide bonds on the carboxyl side of hydrophobic amino acids (aliphatic and aromatic)

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