Cargando…
Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range
[Image: see text] The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is,...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
|
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10399199/ https://www.ncbi.nlm.nih.gov/pubmed/37459182 http://dx.doi.org/10.1021/acs.biochem.3c00139 |
_version_ | 1785084219065630720 |
---|---|
author | Yoon, Michael C. Phan, Von Podvin, Sonia Mosier, Charles O’Donoghue, Anthony J. Hook, Vivian |
author_facet | Yoon, Michael C. Phan, Von Podvin, Sonia Mosier, Charles O’Donoghue, Anthony J. Hook, Vivian |
author_sort | Yoon, Michael C. |
collection | PubMed |
description | [Image: see text] The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range. |
format | Online Article Text |
id | pubmed-10399199 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-103991992023-08-04 Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range Yoon, Michael C. Phan, Von Podvin, Sonia Mosier, Charles O’Donoghue, Anthony J. Hook, Vivian Biochemistry [Image: see text] The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range. American Chemical Society 2023-07-17 /pmc/articles/PMC10399199/ /pubmed/37459182 http://dx.doi.org/10.1021/acs.biochem.3c00139 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Yoon, Michael C. Phan, Von Podvin, Sonia Mosier, Charles O’Donoghue, Anthony J. Hook, Vivian Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range |
title | Distinct Cleavage
Properties of Cathepsin B Compared
to Cysteine Cathepsins Enable the Design and Validation of a Specific
Substrate for Cathepsin B over a Broad pH Range |
title_full | Distinct Cleavage
Properties of Cathepsin B Compared
to Cysteine Cathepsins Enable the Design and Validation of a Specific
Substrate for Cathepsin B over a Broad pH Range |
title_fullStr | Distinct Cleavage
Properties of Cathepsin B Compared
to Cysteine Cathepsins Enable the Design and Validation of a Specific
Substrate for Cathepsin B over a Broad pH Range |
title_full_unstemmed | Distinct Cleavage
Properties of Cathepsin B Compared
to Cysteine Cathepsins Enable the Design and Validation of a Specific
Substrate for Cathepsin B over a Broad pH Range |
title_short | Distinct Cleavage
Properties of Cathepsin B Compared
to Cysteine Cathepsins Enable the Design and Validation of a Specific
Substrate for Cathepsin B over a Broad pH Range |
title_sort | distinct cleavage
properties of cathepsin b compared
to cysteine cathepsins enable the design and validation of a specific
substrate for cathepsin b over a broad ph range |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10399199/ https://www.ncbi.nlm.nih.gov/pubmed/37459182 http://dx.doi.org/10.1021/acs.biochem.3c00139 |
work_keys_str_mv | AT yoonmichaelc distinctcleavagepropertiesofcathepsinbcomparedtocysteinecathepsinsenablethedesignandvalidationofaspecificsubstrateforcathepsinboverabroadphrange AT phanvon distinctcleavagepropertiesofcathepsinbcomparedtocysteinecathepsinsenablethedesignandvalidationofaspecificsubstrateforcathepsinboverabroadphrange AT podvinsonia distinctcleavagepropertiesofcathepsinbcomparedtocysteinecathepsinsenablethedesignandvalidationofaspecificsubstrateforcathepsinboverabroadphrange AT mosiercharles distinctcleavagepropertiesofcathepsinbcomparedtocysteinecathepsinsenablethedesignandvalidationofaspecificsubstrateforcathepsinboverabroadphrange AT odonoghueanthonyj distinctcleavagepropertiesofcathepsinbcomparedtocysteinecathepsinsenablethedesignandvalidationofaspecificsubstrateforcathepsinboverabroadphrange AT hookvivian distinctcleavagepropertiesofcathepsinbcomparedtocysteinecathepsinsenablethedesignandvalidationofaspecificsubstrateforcathepsinboverabroadphrange |