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

  • Radoslaw Gladysz , Natalia Malek , Wioletta Rut und Marcin Drag ORCID logo EMAIL logo
Veröffentlicht/Copyright: 16. November 2022
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 404 Heft 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:
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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

References

Borissenko, L. and Groll, M. (2007). Diversity of proteasomal missions: fine tuning of the immune response. Biol. Chem. 388: 947–955, https://doi.org/10.1515/bc.2007.109.Suche in Google Scholar

Egerer, K., Kuckelkorn, U., Rudolph, P.E., Rückert, J.C., Dörner, T., Burmester, G.-R., Kloetzel, P.-M., and Feist, E. (2002). Circulating proteasomes are markers of cell damage and immunologic activity in autoimmune diseases. J. Rheumatol. 29: 2045–2052.Suche in Google Scholar

Gruba, N., Wysocka, M., Brzezińska, M., Debowski, D., Rolka, K., Martin, N.I., and Lesner, A. (2016a). Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome. Anal. Biochem. 508: 38–45, https://doi.org/10.1016/j.ab.2015.08.019.Suche in Google Scholar PubMed

Gruba, N., Wysocka, M., Brzezińska, M., Dębowski, D., Sieńczyk, M., Gorodkiewicz, E., Guszcz, T., Czaplewski, C., Rolka, K., and Lesner, A. (2016b). Bladder cancer detection using a peptide substrate of the 20S proteasome. FEBS J. 283: 2929–2948, https://doi.org/10.1111/febs.13786.Suche in Google Scholar PubMed

Hoffmann, O., Heubner, M., Anlasik, T., Winterhalter, M., Dahlmann, B., Kasimir-Bauer, S., Kimmig, R., Wohlschlaeger, J., and Sixt, S.U. (2011). Circulating 20S proteasome in patients with non-metastasized breast cancer. Anticancer Res. 31: 2197–2201.Suche in Google Scholar

Krishnan, K.M. and Williamson, K.C. (2018). The proteasome as a target to combat malaria: hits and misses. Transl. Res. J. Lab. Clin. Med. 198: 40–47, https://doi.org/10.1016/j.trsl.2018.04.007.Suche in Google Scholar PubMed PubMed Central

Maly, D.J., Leonetti, F., Backes, B.J., Dauber, D.S., Harris, J.L., Craik, C.S., and Ellman, J.A. (2002). Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore. J. Org. Chem. 67: 910–915, https://doi.org/10.1021/jo016140o.Suche in Google Scholar PubMed

Opoku-Nsiah, K.A. and Gestwicki, J.E. (2018). Aim for the core: suitability of the ubiquitin-independent 20S proteasome as a drug target in neurodegeneration. Transl. Res. J. Lab. Clin. Med. 198: 48–57, https://doi.org/10.1016/j.trsl.2018.05.002.Suche in Google Scholar PubMed PubMed Central

Orlowski, M. and Wilk, S. (2000). Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex. Arch. Biochem. Biophys. 383: 1–16, https://doi.org/10.1006/abbi.2000.2036.Suche in Google Scholar PubMed

Poreba, M., Szalek, A., Rut, W., Kasperkiewicz, P., Rutkowska-Wlodarczyk, I., Snipas, S.J., Itoh, Y., Turk, D., Turk, B., Overall, C.M., et al.. (2017). Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families. Sci. Rep. 7: 43135, https://doi.org/10.1038/srep43135.Suche in Google Scholar PubMed PubMed Central

Rut, W. and Drag, M. (2016). Human 20S proteasome activity towards fluorogenic peptides of various chain lengths. Biol. Chem. 397: 921–926, https://doi.org/10.1515/hsz-2016-0176.Suche in Google Scholar PubMed

Rut, W., Poręba, M., Kasperkiewicz, P., Snipas, S.J., and Drąg, M. (2018). Selective substrates and activity-based probes for imaging of the human constitutive 20S proteasome in cells and blood samples. J. Med. Chem. 61: 5222–5234, https://doi.org/10.1021/acs.jmedchem.8b00026.Suche in Google Scholar PubMed

Tanaka, K., Mizushima, T., and Saeki, Y. (2012). The proteasome: molecular machinery and pathophysiological roles. Biol. Chem. 393: 217–234, https://doi.org/10.1515/hsz-2011-0285.Suche in Google Scholar PubMed

Voss, C., Scholz, C., Knorr, S., Beck, P., Stein, M.L., Zall, A., Kuckelkorn, U., Kloetzel, P.-M., Groll, M., Hamacher, K., et al.. (2014). α-Keto phenylamides as P1′-extended proteasome inhibitors. ChemMedChem 9: 2557–2564, https://doi.org/10.1002/cmdc.201402244.Suche in Google Scholar PubMed

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

© 2022 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Highlight: Structure and Function of the Peroxisomal Translocon
  3. Highlight: structure and function of the peroxisomal translocon
  4. Super-resolution microscopy and studies of peroxisomes
  5. Good things come to those who bait: the peroxisomal docking complex
  6. Determining the targeting specificity of the selective peroxisomal targeting factor Pex9
  7. Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes
  8. Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer
  9. Dynamics of the translocation pore of the human peroxisomal protein import machinery
  10. Distinct conformational and energetic features define the specific recognition of (di)aromatic peptide motifs by PEX14
  11. Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase
  12. Comparison of human PEX knockout cell lines suggests a dual role of PEX1 in peroxisome biogenesis
  13. Research Articles/Short Communications
  14. Proteolysis
  15. Investigation of the P1′ and P2′ sites of IQF substrates and their selectivity toward 20S proteasome subunits
  16. Novel Techniques
  17. Microwave characterization of aqueous amino acid solutions using the multifrequency WGM resonator technique
  18. Corrigendum
  19. Corrigendum to: determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay
https://doi.org/10.1515/hsz-2022-0261
Schlagwörter für diesen Artikel
20s proteasome subunits; internally quenched fluorescence substrate; P1′ position; P2′ position; substrate specificity

Artikel in diesem Heft

  1. Frontmatter
  2. Highlight: Structure and Function of the Peroxisomal Translocon
  3. Highlight: structure and function of the peroxisomal translocon
  4. Super-resolution microscopy and studies of peroxisomes
  5. Good things come to those who bait: the peroxisomal docking complex
  6. Determining the targeting specificity of the selective peroxisomal targeting factor Pex9
  7. Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes
  8. Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer
  9. Dynamics of the translocation pore of the human peroxisomal protein import machinery
  10. Distinct conformational and energetic features define the specific recognition of (di)aromatic peptide motifs by PEX14
  11. Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase
  12. Comparison of human PEX knockout cell lines suggests a dual role of PEX1 in peroxisome biogenesis
  13. Research Articles/Short Communications
  14. Proteolysis
  15. Investigation of the P1′ and P2′ sites of IQF substrates and their selectivity toward 20S proteasome subunits
  16. Novel Techniques
  17. Microwave characterization of aqueous amino acid solutions using the multifrequency WGM resonator technique
  18. Corrigendum
  19. Corrigendum to: determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay
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