Investigation of the P1′ and P2′ sites of IQF substrates and their selectivity toward 20S proteasome subunits

Radoslaw Gladysz; Natalia Malek; Wioletta Rut; Marcin Drag

doi:10.1515/hsz-2022-0261

Article

Investigation of the P1′ and P2′ sites of IQF substrates and their selectivity toward 20S proteasome subunits

Radoslaw Gladysz , Natalia Malek , Wioletta Rut and Marcin Drag

Published/Copyright: November 16, 2022

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From the journal Biological Chemistry Volume 404 Issue 2-3

Abstract

High levels of expression and activity of the 20S proteasome have been linked to many types of pathologies, including neoplasia, autoimmune disorders, neurodegenerative diseases and many more. Moreover, distinguishing between 20S proteasome catalytic subunits is neglected, although it may provide further insight into the development of pathologies. Several approaches have been developed to detect 20S proteasome activity, one of which is internally quenched fluorescent (IQF) substrates, which currently suffer from low efficiency and sensitivity. Previous reports focused on peptides including natural amino acids; therefore, in this report, we synthesized and analyzed IQF substrates with both natural and unnatural amino acids in the P1′ and P2′ positions to investigate their influences on selectivity toward 20S proteasome subunits. We found that elongation of the substrate by the P1′ and P2′ positions increased specificity in comparison to tetrapeptides. Moreover, we were able to obtain IQF substrates for the Ch-L subunit, which was characterized by higher selectivity than formerly used tetrapeptides. These findings may further contribute to the development of novel diagnostic tools for 20S proteasome-dependent disorders.

Keywords: 20s proteasome subunits; internally quenched fluorescence substrate; P1′ position; P2′ position; substrate specificity

Corresponding author: Marcin Drag, Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, ul. Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland, E-mail: Marcin.drag@pwr.edu.pl

Radoslaw Gladysz and Natalia Malek contributed equally to this work.

Funding source: National Science Center

Funding source: Ministry of Science and Higher Education

Acknowledgments

The work was created as a result of the research project No. 2017/25/B/ST5/00215 financed by the National Science Center.

Author contribution: M.D. designed the study, gained funding, and wrote the manuscript. R.G. and N.M. performed experiments, analyzed them, prepared figures, and contributed to manuscript writing. W.R. designed the study and contributed to experiments analysis. All authors read and approved the final manuscript.
Research funding: N.M. was a recipient of the scholarship for young researchers (no. 444/STYP/13/2018) funded by the Ministry of Science and Higher Education.
Conflict of interest statement: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
Data availability: This article contains supporting information. The data that support the findings of this study are available in the E-SCIENCE.PL platform, maintained by the Wroclaw Centre for Networking and Supercomputing of the Wroclaw University of Science and Technology. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2022-0261).

Received: 2022-08-23

Accepted: 2022-10-30

Published Online: 2022-11-16

Published in Print: 2023-02-23

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Supplementary Material

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Keywords for this article

20s proteasome subunits; internally quenched fluorescence substrate; P1′ position; P2′ position; substrate specificity