Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.
BACKGROUND:Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this d...
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ftdoajarticles:oai:doaj.org/article:22d50a4e755e42308af3ecd1ce199c9f 2023-05-15T15:16:40+02:00 Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. Juliana Ide Aoki Adriano Cappellazzo Coelho Sandra Marcia Muxel Ricardo Andrade Zampieri Eduardo Milton Ramos Sanchez Audun Helge Nerland Lucile Maria Floeter-Winter Paulo Cesar Cotrim 2016-09-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0004972 https://doaj.org/article/22d50a4e755e42308af3ecd1ce199c9f EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC5015992?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0004972 https://doaj.org/article/22d50a4e755e42308af3ecd1ce199c9f PLoS Neglected Tropical Diseases, Vol 10, Iss 9, p e0004972 (2016) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2016 ftdoajarticles https://doi.org/10.1371/journal.pntd.0004972 2022-12-31T00:12:16Z BACKGROUND:Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS:After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. CONCLUSIONS/SIGNIFICANCE:This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 10 9 e0004972 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Juliana Ide Aoki Adriano Cappellazzo Coelho Sandra Marcia Muxel Ricardo Andrade Zampieri Eduardo Milton Ramos Sanchez Audun Helge Nerland Lucile Maria Floeter-Winter Paulo Cesar Cotrim Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
BACKGROUND:Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS:After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. CONCLUSIONS/SIGNIFICANCE:This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine ... |
format |
Article in Journal/Newspaper |
author |
Juliana Ide Aoki Adriano Cappellazzo Coelho Sandra Marcia Muxel Ricardo Andrade Zampieri Eduardo Milton Ramos Sanchez Audun Helge Nerland Lucile Maria Floeter-Winter Paulo Cesar Cotrim |
author_facet |
Juliana Ide Aoki Adriano Cappellazzo Coelho Sandra Marcia Muxel Ricardo Andrade Zampieri Eduardo Milton Ramos Sanchez Audun Helge Nerland Lucile Maria Floeter-Winter Paulo Cesar Cotrim |
author_sort |
Juliana Ide Aoki |
title |
Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. |
title_short |
Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. |
title_full |
Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. |
title_fullStr |
Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. |
title_full_unstemmed |
Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. |
title_sort |
characterization of a novel endoplasmic reticulum protein involved in tubercidin resistance in leishmania major. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2016 |
url |
https://doi.org/10.1371/journal.pntd.0004972 https://doaj.org/article/22d50a4e755e42308af3ecd1ce199c9f |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 10, Iss 9, p e0004972 (2016) |
op_relation |
http://europepmc.org/articles/PMC5015992?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0004972 https://doaj.org/article/22d50a4e755e42308af3ecd1ce199c9f |
op_doi |
https://doi.org/10.1371/journal.pntd.0004972 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
10 |
container_issue |
9 |
container_start_page |
e0004972 |
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1766346962001461248 |