Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1371/journal.pntd.0008762 http://hdl.handle.net/11449/208090 |
Resumo: | Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmani-asis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery pro-grams, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening for-mat. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibi-tor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections. |
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Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania majorDeoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmani-asis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery pro-grams, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening for-mat. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibi-tor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.Chemistry Institute São Paulo State University—UNESPSchool of Pharmaceutical Sciences São Paulo State University—UNESPMolecular Biology and Genetic Engineering Center (CBMEG) Medicinal Chemistry Center (CQMED) Structural Genomics Consortium (SGC-UNICAMP) University of Campinas-UNICAMPDepartment of Structural and Functional Biology Institute of Biology University of Campinas—UNICAMPDepartment of Biochemistry and Tissue Biology Institute of Biology University of Campinas— UNICAMPChemistry Institute São Paulo State University—UNESPSchool of Pharmaceutical Sciences São Paulo State University—UNESPUniversidade Estadual Paulista (Unesp)Universidade Estadual de Campinas (UNICAMP)Silva, Suélen Fernandes [UNESP]Klippel, Angélica Hollunder [UNESP]Ramos, Priscila ZonziniSantiago, André da SilvaValentini, Sandro Roberto [UNESP]Bengtson, Mario HenriqueMassirer, Katlin BrauerBilsland, ElizabethCouñago, Rafael MiguezZanelli, Cleslei Fernando [UNESP]2021-06-25T11:06:11Z2021-06-25T11:06:11Z2020-10-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article1-31http://dx.doi.org/10.1371/journal.pntd.0008762PLoS Neglected Tropical Diseases, v. 14, n. 10, p. 1-31, 2020.1935-27351935-2727http://hdl.handle.net/11449/20809010.1371/journal.pntd.00087622-s2.0-8509421950015256654089001950000-0001-7831-1149Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengPLoS Neglected Tropical Diseasesinfo:eu-repo/semantics/openAccess2024-06-24T13:07:52Zoai:repositorio.unesp.br:11449/208090Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T19:39:51.020673Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
title |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
spellingShingle |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major Silva, Suélen Fernandes [UNESP] |
title_short |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
title_full |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
title_fullStr |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
title_full_unstemmed |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
title_sort |
Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of brugia malayi and leishmania major |
author |
Silva, Suélen Fernandes [UNESP] |
author_facet |
Silva, Suélen Fernandes [UNESP] Klippel, Angélica Hollunder [UNESP] Ramos, Priscila Zonzini Santiago, André da Silva Valentini, Sandro Roberto [UNESP] Bengtson, Mario Henrique Massirer, Katlin Brauer Bilsland, Elizabeth Couñago, Rafael Miguez Zanelli, Cleslei Fernando [UNESP] |
author_role |
author |
author2 |
Klippel, Angélica Hollunder [UNESP] Ramos, Priscila Zonzini Santiago, André da Silva Valentini, Sandro Roberto [UNESP] Bengtson, Mario Henrique Massirer, Katlin Brauer Bilsland, Elizabeth Couñago, Rafael Miguez Zanelli, Cleslei Fernando [UNESP] |
author2_role |
author author author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) Universidade Estadual de Campinas (UNICAMP) |
dc.contributor.author.fl_str_mv |
Silva, Suélen Fernandes [UNESP] Klippel, Angélica Hollunder [UNESP] Ramos, Priscila Zonzini Santiago, André da Silva Valentini, Sandro Roberto [UNESP] Bengtson, Mario Henrique Massirer, Katlin Brauer Bilsland, Elizabeth Couñago, Rafael Miguez Zanelli, Cleslei Fernando [UNESP] |
description |
Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmani-asis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery pro-grams, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening for-mat. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibi-tor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-10-01 2021-06-25T11:06:11Z 2021-06-25T11:06:11Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1371/journal.pntd.0008762 PLoS Neglected Tropical Diseases, v. 14, n. 10, p. 1-31, 2020. 1935-2735 1935-2727 http://hdl.handle.net/11449/208090 10.1371/journal.pntd.0008762 2-s2.0-85094219500 1525665408900195 0000-0001-7831-1149 |
url |
http://dx.doi.org/10.1371/journal.pntd.0008762 http://hdl.handle.net/11449/208090 |
identifier_str_mv |
PLoS Neglected Tropical Diseases, v. 14, n. 10, p. 1-31, 2020. 1935-2735 1935-2727 10.1371/journal.pntd.0008762 2-s2.0-85094219500 1525665408900195 0000-0001-7831-1149 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
PLoS Neglected Tropical Diseases |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
1-31 |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
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1808129103928229888 |