Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica

Detalhes bibliográficos
Autor(a) principal: Moreira, Rodrigo Alves
Data de Publicação: 2014
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UFG
Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/3899
Resumo: Cutaneous leishmaniasis is a neglected tropical disease that infects millions of people worldwide, representing a serious public health problem. The miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although the mechanism of action at the molecular level is poorly understood. Electron paramagnetic resonance (EPR) spectroscopy of the lipid spin lebel analog of stearic acid (5-DSA) and the maleimide derivative spin label (6-MSL) covalently bound to membrane proteins showed that the MT causes a large increase in the molecular dynamics of erythrocyte membranes (ghosts) and detergent resistant membranes (DRMs) prepared from erythrocyte membranes. In the vesicles of lipid raft constituents, it was shown that 20 mol% sphingomyelin could be replaced by 20 mol% MT with no change in the molecular dynamics. Furthermore, the effect of MT on DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-52μg/mL), our measurements in blood plasma indicated a hemolytic level of 2-5% and also showed that the MT concentration that changes the erythrocyte membrane fluidity to an extent that is detectable by EPR spectroscopy causes about 46% hemolysis. Subsequently, EPR studies performed with the same spin labels in the membrane of Leishmania (L.) amazonensis (promastigote) showed changes similar to those found in erythrocyte membranes. Cytotoxic effects on the parasites were also evaluated to investigate the relationships between the cytotoxic potential of MT and its ability to alter membrane fluidity. The EPR data showed that the minimum concentration of MT required to cause a change in the parasite membrane occurred near the values of MT concentration which inhibits 50 % of cell growth (IC50); thus, there is a correlation between the cytotoxicity and changes in the membrane. Although these III membrane alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼ 70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.
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spelling Alonso, Antôniohttp://lattes.cnpq.br/5013069863616789Alonso, AntônioUliana, Silvia Reni BortolinUrbano, Ricardo RodriguesLoyola, Patrícia Resende Alo NagibCarvalho, Sheila Gonçalves do Coutohttp://lattes.cnpq.br/2384497784331636Moreira, Rodrigo Alves2015-01-16T17:37:28Z2014-06-04MOREIRA, Rodrigo Alves. Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica. 2014. 139 f. Tese (Doutorado em Fisica) - Universidade Federal de Goiás, Goiânia, 2014.http://repositorio.bc.ufg.br/tede/handle/tede/3899Cutaneous leishmaniasis is a neglected tropical disease that infects millions of people worldwide, representing a serious public health problem. The miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although the mechanism of action at the molecular level is poorly understood. Electron paramagnetic resonance (EPR) spectroscopy of the lipid spin lebel analog of stearic acid (5-DSA) and the maleimide derivative spin label (6-MSL) covalently bound to membrane proteins showed that the MT causes a large increase in the molecular dynamics of erythrocyte membranes (ghosts) and detergent resistant membranes (DRMs) prepared from erythrocyte membranes. In the vesicles of lipid raft constituents, it was shown that 20 mol% sphingomyelin could be replaced by 20 mol% MT with no change in the molecular dynamics. Furthermore, the effect of MT on DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-52μg/mL), our measurements in blood plasma indicated a hemolytic level of 2-5% and also showed that the MT concentration that changes the erythrocyte membrane fluidity to an extent that is detectable by EPR spectroscopy causes about 46% hemolysis. Subsequently, EPR studies performed with the same spin labels in the membrane of Leishmania (L.) amazonensis (promastigote) showed changes similar to those found in erythrocyte membranes. Cytotoxic effects on the parasites were also evaluated to investigate the relationships between the cytotoxic potential of MT and its ability to alter membrane fluidity. The EPR data showed that the minimum concentration of MT required to cause a change in the parasite membrane occurred near the values of MT concentration which inhibits 50 % of cell growth (IC50); thus, there is a correlation between the cytotoxicity and changes in the membrane. Although these III membrane alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼ 70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.A leishmaniose cut ˆanea ´e uma doenc¸a tropical negligenciada que afetamilh˜oes de pessoas em todo mundo, representando um grave problema de sa´ude p´ublica. A miltefosina (MT) ´e um alquilfosfolip´ıdio aprovado inicialmente para tratamento de cˆancer metast ´atico e tamb´em foi licenciada para o tratamento da leishmaniose visceral, embora seu mecanismo de ac¸ ˜ao a n´ıvel molecular permanec¸a pouco compreendido. A espectroscopia de ressonˆancia paramagn´etica eletr ˆonica (RPE) do marcador de spin lip´ıdico an´alogo do ´acido este´arico (5-DSA) e do marcador de spin derivado do maleimido (6-MSL) ligado covalentemente `as prote´ınas de membrana demonstraram que a MT provoca um grande aumento na dinˆamica molecular das membranas de eritr ´ocito (ghosts) e membranas resistentes `a extrac¸ ˜ao por detergente (DRMs) preparadas a partir de membranas de eritr ´ocito. A t ´ecnica tamb´em demonstrou que em ves´ıculas formadas com lip´ıdios constituintes de rafts, 20 mol% de esfingomielina pode ser substitu´ıdo por 20 mol% de MT, sem qualquer alterac¸ ˜ao em termos de dinˆamica molecular. Al ´em disso, o efeito da MT em DRMs foi mais pronunciado do que em ghosts de eritr ´ocito, consistente com a hip´otese da MT ser um modulador de raft. Na concentrac¸ ˜ao de MT presente no plasma sangu´ıneo durante o tratamento de leishmaniose (31-52 μg/mL), as nossas medidas realizadas diretamente no sangue total indicaram um n´ıvel de 2-5% de hem´olise e tamb´em mostrou que a concentrac¸ ˜ao de MT capaz de alterar a fluidez da membrana de eritr ´ocito a n´ıvel detect ´avel pela espectroscopia de RPE causou cerca de 46% de hem´olise. Posteriormente, estudos de RPE realizados com os mesmos marcadores de spin na membrana de Leishmania (L.) amazonensis (forma promastigota) demonstraram alterac¸ ˜oes similares `as encontradas nas membranas de eritr ´ocito. Os efeitos citot ´oxicos sobre os parasitas tamb´em foram avaliados para investigar as relac¸ ˜oes entre os potenciais citot ´oxicos da MT e sua capacidade de alterar I a fluidez da membrana. Os dados de RPE demonstraram que a concentrac¸ ˜ao m´ınima necess´aria de MT para causar alterac¸ ˜ao na membrana do parasito ocorreu pr ´oximo dos valores de concentrac¸ ˜ao que inibe 50% do crescimento celular (IC50), existindo, portanto, uma correlac¸ ˜ao entre as alterac¸ ˜oes na membrana e a citotoxicidade. Embora o marcador de spin lip´ıdico tamb´em tenha detectado alterac¸ ˜oes na fluidez da membrana, os experimentos de RPE indicaram que a MT atua predominantemente no componente prot ´eico da membrana. A lise celular tamb´em foi detectada atrav´es da an´alise dos sobrenadantes das amostras centrifugadas onde foram encontrados fragmentos de membranas marcadas e tamb´em prote´ınas citoplasm´aticas. Usando um m´etodo para a r ´apida incorporac¸ ˜ao da MT na membrana, os efeitos de citotoxidade foram medidos imediatamente ap´os o tratamento e na mesma faixa de concentrac¸ ˜oes de MT que causam a inibic¸ ˜ao do crescimento celular. A citotoxicidade, estimada atrav´es da contagem microsc´opica das c´elulas vivas e mortas, indicou ∼ 70% de morte celular na concentrac¸ ˜ao de MT em que a espectroscopia de RPE detectou uma alterac¸ ˜ao significativa na dinˆamica da membrana. Ap´os o primeiro impacto no n´umero de parasitas vi ´aveis, o processo de morte e crescimento celular continuou durante as primeiras quatro horas de incubac¸ ˜ao. Os espectros de RPE de prote´ınas de membrana marcadas com o 6-MSL foram consistentes com conformac¸ ˜oes de prote´ına mais expandidas e expostas ao solvente, sugerindo uma ac¸ ˜ao t´ıpica dos detergentes. Nossa interpretac¸ ˜ao ´e que a MT pode formar estruturas semelhantes das micelas ao redor das cadeias polipept´ıdicas, expandindo as prote´ınas e que as prote´ınas com maior hidrofobicidade poderiam induzir a penetrac¸ ˜ao de grupos hidrof´ılicos da MT no interior da membrana, causando sua ruptura e consequentemente a morte da c´elula.Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-16T17:25:01Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Rodrigo Alves Moreira - 2014.pdf: 6048324 bytes, checksum: 289d0c70e7ed1db107b03924d35a81de (MD5)Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-16T17:37:28Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Rodrigo Alves Moreira - 2014.pdf: 6048324 bytes, checksum: 289d0c70e7ed1db107b03924d35a81de (MD5)Made available in DSpace on 2015-01-16T17:37:28Z (GMT). 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dc.title.por.fl_str_mv Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
dc.title.alternative.eng.fl_str_mv Interaction of miltefosine with the lipid and protein components of the erythrocyte and Leishmania membranes studied by electron paramagnetic resonance
title Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
spellingShingle Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
Moreira, Rodrigo Alves
Miltefosina
RPE
Marcador de spin
Membrana do eritrócito
Raft
Leishmania
Miltefosine
EPR
Spin label
Erythrocyte membrane
CIENCIAS EXATAS E DA TERRA::FISICA
title_short Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
title_full Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
title_fullStr Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
title_full_unstemmed Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
title_sort Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
author Moreira, Rodrigo Alves
author_facet Moreira, Rodrigo Alves
author_role author
dc.contributor.advisor1.fl_str_mv Alonso, Antônio
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/5013069863616789
dc.contributor.referee1.fl_str_mv Alonso, Antônio
dc.contributor.referee2.fl_str_mv Uliana, Silvia Reni Bortolin
dc.contributor.referee3.fl_str_mv Urbano, Ricardo Rodrigues
dc.contributor.referee4.fl_str_mv Loyola, Patrícia Resende Alo Nagib
dc.contributor.referee5.fl_str_mv Carvalho, Sheila Gonçalves do Couto
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/2384497784331636
dc.contributor.author.fl_str_mv Moreira, Rodrigo Alves
contributor_str_mv Alonso, Antônio
Alonso, Antônio
Uliana, Silvia Reni Bortolin
Urbano, Ricardo Rodrigues
Loyola, Patrícia Resende Alo Nagib
Carvalho, Sheila Gonçalves do Couto
dc.subject.por.fl_str_mv Miltefosina
RPE
Marcador de spin
Membrana do eritrócito
Raft
Leishmania
topic Miltefosina
RPE
Marcador de spin
Membrana do eritrócito
Raft
Leishmania
Miltefosine
EPR
Spin label
Erythrocyte membrane
CIENCIAS EXATAS E DA TERRA::FISICA
dc.subject.eng.fl_str_mv Miltefosine
EPR
Spin label
Erythrocyte membrane
dc.subject.cnpq.fl_str_mv CIENCIAS EXATAS E DA TERRA::FISICA
description Cutaneous leishmaniasis is a neglected tropical disease that infects millions of people worldwide, representing a serious public health problem. The miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although the mechanism of action at the molecular level is poorly understood. Electron paramagnetic resonance (EPR) spectroscopy of the lipid spin lebel analog of stearic acid (5-DSA) and the maleimide derivative spin label (6-MSL) covalently bound to membrane proteins showed that the MT causes a large increase in the molecular dynamics of erythrocyte membranes (ghosts) and detergent resistant membranes (DRMs) prepared from erythrocyte membranes. In the vesicles of lipid raft constituents, it was shown that 20 mol% sphingomyelin could be replaced by 20 mol% MT with no change in the molecular dynamics. Furthermore, the effect of MT on DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-52μg/mL), our measurements in blood plasma indicated a hemolytic level of 2-5% and also showed that the MT concentration that changes the erythrocyte membrane fluidity to an extent that is detectable by EPR spectroscopy causes about 46% hemolysis. Subsequently, EPR studies performed with the same spin labels in the membrane of Leishmania (L.) amazonensis (promastigote) showed changes similar to those found in erythrocyte membranes. Cytotoxic effects on the parasites were also evaluated to investigate the relationships between the cytotoxic potential of MT and its ability to alter membrane fluidity. The EPR data showed that the minimum concentration of MT required to cause a change in the parasite membrane occurred near the values of MT concentration which inhibits 50 % of cell growth (IC50); thus, there is a correlation between the cytotoxicity and changes in the membrane. Although these III membrane alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼ 70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.
publishDate 2014
dc.date.issued.fl_str_mv 2014-06-04
dc.date.accessioned.fl_str_mv 2015-01-16T17:37:28Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
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dc.identifier.citation.fl_str_mv MOREIRA, Rodrigo Alves. Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica. 2014. 139 f. Tese (Doutorado em Fisica) - Universidade Federal de Goiás, Goiânia, 2014.
dc.identifier.uri.fl_str_mv http://repositorio.bc.ufg.br/tede/handle/tede/3899
identifier_str_mv MOREIRA, Rodrigo Alves. Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica. 2014. 139 f. Tese (Doutorado em Fisica) - Universidade Federal de Goiás, Goiânia, 2014.
url http://repositorio.bc.ufg.br/tede/handle/tede/3899
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dc.publisher.none.fl_str_mv Universidade Federal de Goiás
dc.publisher.program.fl_str_mv Programa de Pós-graduação em Fisica (IF)
dc.publisher.initials.fl_str_mv UFG
dc.publisher.country.fl_str_mv Brasil
dc.publisher.department.fl_str_mv Instituto de Física - IF (RG)
publisher.none.fl_str_mv Universidade Federal de Goiás
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