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UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
CENTRO DE BIOTECNOLOGIA
PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA CELULAR E MOLECULAR
RENATA MARIA SOARES TERRA
ANÁLISE CONFORMACIONAL DA MELITINA POR DINÂMICA
MOLECULAR E CARACTERIZAÇÃO DOS EFEITOS DO PEPTÍDEO
NA FUNÇÃO PLAQUETÁRIA
Porto Alegre
2006
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RENATA MARIA SOARES TERRA
ANÁLISE CONFORMACIONAL DA MELITINA POR DINÂMICA
MOLECULAR E CARACTERIZAÇÃO DOS EFEITOS DO PEPTÍDEO
NA FUNÇÃO PLAQUETÁRIA
Dissertação submetida ao programa de pós-
graduação em Biologia Celular e Molecular, Centro
de Biotecnologia, da Universidade Federal do Rio
Grande do Sul como requisito parcial para
obtenção do título de Mestre.
Orientador Prof. Dr. Jorge Almeida Guimarães
Porto Alegre
2006
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RENATA MARIA SOARES TERRA
ANÁLISE CONFORMACIONAL DA MELITINA POR DINÂMICA MOLECULAR E
CARACTERIZAÇÃO DOS EFEITOS DO PEPTÍDEO NA FUNÇÃO PLAQUETÁRIA
Dissertação submetida ao Programa de Pós-graduação em Biologia Celular e
Molecular (PPGBCM) do Centro de Biotecnologia – UFRGS como requisito parcial
para a obtenção do título de Mestre.
BANCA EXAMINADORA
Dr. Jorge Almeida Guimarães (UFRGS) – orientador
Dr. Jamil Assreuy (UFSC)
Dr. Rafael Roesler (UFRGS)
Dr. Robson de Queiroz Monteiro (UFRJ)
Dra. Célia Regina Ribeiro da Silva Carlini (UFRGS) - Suplente
4
Aos meus pais e irmão,
meus grandes motivadores e amigos
5
Agradecimentos
Agradeço ao meu orientador, Dr. Jorge Almeida Guimarães, pelo exemplo
maravilhoso de cientista e pessoa, pela oportunidade de realizar este trabalho, pelo
incentivo constante e carinho.
Aos professores Dr. Hugo Verli, Dra. Célia Carlini e Dr. Carlos Termignoni
pelos valiosos conhecimentos passados ao longo desse período.
Ao Antônio Pinto pela cumplicidade no laboratório e, é claro, pelo amor,
suporte e alegria que traz a minha vida.
Aos meus excelentes colegas e grandes amigos Markus Berger, Kátia Moura,
José Reck Jr., Walter Beys e Lucélia Santi por serem pessoas tão maravilhosas,
transformando o laboratório em nossa segunda casa.
Aos amigos do Laboratótio de Bioquímica Farmacológica e Grupo de
Bioinformática Estrutural, Simone Kobe, Fabiano Pasin, Rafael Caceres, Tiago
Charão, Ana Veiga, Hermes Amorim, Fernanda Oliveira e Camila Becker pelos
ótimos momentos que passamos.
Aos demais amigos dos laboratórios 217 (CBiot-UFRGS) e 204
(Departamento de Biofísica-UFRGS), em especial German Wassermann, Deiber
Severo e Diogo Demartini.
Aos professores do Programa de Pós-graduação em Biologia Celular e
Molecular e aos membros da minha comissão de acompanhamento Dr. Luiz Augusto
Basso e Dr. Paulo Augusto Netz.
Aos secretários da pós-graduação Luciano Saucedo e Silvia Centeno.
Aos professores Dra. Célia Carlini, Dr. Jamil Assreuy, Dr. Robson Monteiro e
Dr. Rafael Roesler por gentilmente aceitarem o convite para comporem esta banca.
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“Pode-se viver no mundo uma vida
magnífica, quando se sabe trabalhar e
amar: trabalhar pelo que se ama e amar
aquilo em que se trabalha.”
Liev Tólstoi
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Resumo
Acidentes envolvendo abelhas africanizadas são freqüentemente relatados,
particularmente na América do Sul. As picadas de abelhas causam reações
localizadas e sistêmicas e os sintomas do envenenamento incluem náuseas,
vômitos, hemólise, falência renal e coagulação intravascular disseminada. Durante
muito tempo todas as reações tóxicas eram atribuídas à presença de uma
fosfolipase A2, mesmo sendo o veneno uma mistura complexa de substâncias. A
melitina, o componente mais abundante e tóxico do veneno de abelha, é um
peptídeo de 26 aminoácidos com a habilidade de interagir e danificar membranas
celulares. A melitina é também capaz de modular muitas proteínas, aumentando a
diversidade de atividades biológicas do peptídeo. Até recentemente, acreditava-se
que a estrutura tri-dimensional biologicamente ativa do peptídeo, que possui
conformação em hélice, era um tetrâmero.
Neste trabalho avaliamos a conformação da melitina e seus estados
oligoméricos em solução por dinâmica molecular e a interferência da melitina na
função plaquetária. Aqui está demonstrado que a melitina possui uma conformação
randômica em condições fisiológicas e que sua estrutura tri-dimensional sofre
alterações de acordo com as condições ambientais. Ainda, foi demonstrada uma
nova atividade biológica do peptídeo melitina. O peptídeo é capaz de induzir a
8
agregação plaquetária de forma dose-dependente e de interagir diretamente com a
superfície de plaquetas. A correlação entre a conformação da melitina e suas
atividades biológicas é discutida.
Os resultados aqui apresentados podem ser valiosos no entendimento do
papel da melitina nas coagulopatias induzidas por veneno de abelha. O estudo
estrutural mostrado aqui pode ser aplicado para explicar as diferentes atividades do
peptídeo.
9
Abstract
Accidents involving africanized bees are frequently reported, particularly in
South America. Bee stings cause localized and systemic reactions and the symptoms
of envenomation include nausea, vomiting, hemolysis, kidney failure and
disseminated intravascular coagulation. For a long time, all toxic reactions were
ascribe to the presence of a phospholipase A2, despite of being the venom a
complex mixture of substances. Melittin, the most abundant and the major toxic
component of bee venom, is a 26 amino acid peptide with the ability to interact and
disrupt cell membranes. Melittin is also able to modulate many proteins, enhancing
the wide range of the peptide biological activities. The biologically active
tridimensional structure of the peptide, which has a helical conformation, has been
described until now as a tetramer.
In this work we evaluated the conformation of melittin and its oligomeric states
in solution by molecular dynamics simulations and performed studies of melittin effect
on platelet function. Here we demonstrate that melittin has a random conformation
under physiological conditions and its tridimensional structure changes under
different environmental conditions. Moreover, here we describe a new biological
activity of melittin. The peptide is able to induce platelet aggregation in a dose-
10
dependent manner and can interact directly with the platelet surface. The correlation
between melittin conformation and biological activity is discussed.
Our results might contribute to elucidate the role of melittin in bee venom
induced coagulopathies. The structural data gathered in this work may explain the
different activities of the peptide.
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Sumário
AGRADECIMENTOS..................................................................................................5
RESUMO.....................................................................................................................7
ABSTRACT.................................................................................................................9
SUMÁRIO .................................................................................................................11
INTRODUÇÃO..........................................................................................................13
ABELHAS E SUA IMPORTÂNCIA MÉDICA........................................................................13
COMPONENTES DO VENENO DA ABELHA APIS MELLIFERA .............................................16
Aminas Biogênicas.............................................................................................17
Componentes enzimáticos.................................................................................18
Peptídeos bioativos............................................................................................19
PEPTÍDEO MELITINA ..................................................................................................20
OBJETIVOS..............................................................................................................23
RESULTADOS..........................................................................................................24
1. ANÁLISE CONFORMACIONAL DA MELITINA POR DINÂMICA MOLECULAR24
1.1 DINÂMICA MOLECULAR NO ESTUDO DE ESTRUTURA E CONFORMAÇÃO DE
PROTEÍNAS
..............................................................................................................24
12
1.2 ARTIGO 1: TERRA, RMS; GUIMARÃES, JA; VERLI, H. STRUCTURAL AND
FUNCTIONAL BEHAVIOR OF BIOLOGICALLY ACTIVE MONOMERIC MELITTIN
. J. MOL. GRAPH.
MODEL. (2006)........................................................................................................25
2. CARACTERIZAÇÃO DOS EFEITOS DA MELITINA NA FUNÇÃO
PLAQUETÁRIA ........................................................................................................33
2.1 FUNÇÃO E AVALIAÇÃO DE FUNÇÃO PLAQUETÁRIA ..............................................33
2.2 ARTIGO 2: TERRA, RMS; PINTO AFM; BERGER, M; DE OLIVEIRA, SK; JULIANO,
MA; JULIANO, L; GUIMARÃES, JA. MELITTIN-INDUCED PLATELET SIGNALING AND
AGGREGATION
. MANUSCRITO EM PREPARAÇÃO...........................................................36
CONCLUSÕES.........................................................................................................62
REFERÊNCIAS BIBLIOGRÁFICAS.........................................................................64
CURRICULUM VITAE – RENATA MARIA SOARES TERRA..................................70
13
Introdução
Abelhas e sua importância médica
As abelhas melíferas (Apis mellifera mellifera, Apis mellifera lingustica, Apis
mellifera carnica e Apis mellifera iberica) foram trazidas para as Américas por
imigrantes europeus. Porém, o fato de abelhas africanas (Apis mellifera scutellata)
produzirem maior quantidade de mel despertou o desejo de se introduzir esta nova
espécie no continente americano. Como espécie isolada, estas abelhas não
conseguiram manter-se na região. Entretanto, em 1957, no Brasil, durante um
experimento com abelhas africanas, alguns indivíduos fugiram, hibridizando-se com
abelhas européias e dando origem ao que hoje conhecemos como abelhas
africanizadas (Sherman, 1995). Estima-se que este processo de africanização tenha
se espalhado cerca de 320 km por ano, norte e sul, atingindo o estado americano do
Texas em 1990 e espalhando-se pela costa oeste até o estado da Califórnia,
Estados Unidos (Sherman, 1995). Apesar da miscigenação, as abelhas mantiveram
muitas das características das abelhas africanas, especialmente o caráter migratório,
14
a agitabilidade e a agressividade, fato que lhes rendeu o apelido de “abelhas
assassinas” (do inglês, killer bees) (Sherman, 1995). Pouco se conhece a respeito
da dinâmica da hibridização, contudo uma análise de DNA mitocondrial sugere que o
processo tenha ocorrido principalmente pela migração das fêmeas (Sherman, 1995).
A característica comportamental destas abelhas faz com que elas estejam
freqüentemente envolvidas em ataques massivos a seres humanos e animais,
tornando esse tipo de envenenamento um problema de saúde pública. Os ataques
ocorrem em resposta a um estímulo ameaçador e são fatais para as abelhas
envolvidas, porém bastante efetivos para a colônia (Sherman, 1995; Vetter et al.,
1999). Pequenos acidentes com abelhas da espécie Apis mellifera usualmente
causam dor intensa localizada, pequeno edema e eritema imediato (Ewan, 1998). No
entanto, reações alérgicas são bastante comuns, podendo o paciente ser
sensibilizado após poucas picadas ou mesmo em um único evento (Ewan, 1998). As
reações alérgicas podem ser classificadas como imediatas ou tardias e locais ou
sistêmicas, subdividindo-se as últimas em leves, moderadas ou graves (Ewan,
1998). As reações localizadas causam prurido intenso e edema em algumas horas e
eventualmente são seguidos de infecção secundária (Ewan, 1998; Sherman, 1995;
Steen et al., 2005). As reações sistêmicas, subdivididas em três graus de
severidade, têm seus sintomas descritos na Tabela 1, assim como seu tratamento e
uma possível indicação de terapia de dessensibilização. Reações sistêmicas são
capazes de ocasionar o óbito do paciente atingido, principalmente se o mesmo não
receber tratamento adequado na primeira hora após o acidente (Sherman, 1995).
A imunoterapia, ou terapia de dessensibilização, é indicada para pacientes
que sofreram reação alérgica sistêmica severa (Ewan, 1998). A imunoterapia
convencional deve ser realizada em centros especializados onde um aparato para
15
ressuscitação deve estar disponível, uma vez que é elevado o risco de choque
anafilático (Ewan, 1998; Sherman, 1995; Steen et al., 2005). Os pacientes recebem
pequenas doses de veneno semanalmente durante três meses até uma dose
máxima de 100 µg de veneno (equivalente a 2 picadas). A manutenção é realizada
com injeções mensais da dose máxima por um período de até três anos (Ewan,
1998). A despeito de se acreditar que um paciente com histórico de reação alérgica
sistêmica tenha o processo exacerbado em um novo contato, não existem métodos
capazes de prever o grau da resposta em novos eventos (Ewan, 1998).
Paradoxalmente, um paciente com reação sistêmica pode sofrer apenas de reações
locais em um novo contato com o veneno, tornando o emprego da terapia uma
decisão a ser cuidadosamente julgada.
Tabela 1
Classificação de reações alérgicas sistêmicas a picadas de abelha,
sintomatologia e conduta clinica desejável.
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Além da anafilaxia, o veneno de abelhas pode ocasionar uma série de outras
reações sistêmicas imediatas ou tardias. Ataques massivos - de dezenas a centenas
de picadas, e em alguns casos, milhares - têm elevada toxicidade, podendo
ocasionar a falência de diversos órgãos (Franca et al., 1994; Grisotto et al., 2006;
Kolecki, 1999; Vetter et al., 1999). Reações tóxicas incluem hemólise com
conseqüente hematúria, rabdomiólise, trombocitopenia, coagulação intravascular
disseminada, dano cardíaco, hepático e renal, hipercalemia, hiperglicemia e
hipertensão (Franca et al., 1994; Gawlik et al., 2004; Grisotto et al., 2006; Kolecki,
1999; Sherman, 1995; Steen et al., 2005; Vetter et al., 1999). Achados bioquímicos
demonstram um aumento nos níveis das enzimas creatina quinase e lactato
desidrogenase assim como de aminotransferases hepáticas em pacientes
envenenados, demonstrando importante lesão tecidual (Grisotto et al., 2006; Kolecki,
1999; Vetter et al., 1999). Casos graves podem ocasionar morte do paciente em um
período de 4 horas a 12 dias e a mesma ocorre usualmente em decorrência de
falência renal por subprodutos de hemólise e miólise ou parada cardíaca pela
toxicidade do veneno (Vetter et al., 1999).
Componentes do veneno da abelha Apis mellifera
O veneno de abelhas, assim como de outros animais, é uma complexa
mistura de substâncias bioativas. A composição dos venenos de diferentes espécies
de Apis parece não variar, entretanto pequenas diferenças quantitativas de seus
componentes são observadas (Vetter et al., 1999). Dentre os componentes do
veneno, duas proteínas têm maior relevância: o peptídeo hemolítico melitina,
17
compondo cerca de 50% do peso seco do veneno e a enzima fosfolipase A2,
representando 10-12% da massa do veneno bruto (Habermann, 1972; Peiren et al.,
2005; Vetter et al., 1999). Aminas biogênicas como histamina, dopamina e
norepinefrina, assim como peptídeos ativos – apamina e o peptídeo degranulador de
mastócitos (MCD) – e componentes enzimáticos – hialuronidase, serino-protease
com domínio CUB e uma fosfatase ácida, também compõem esta complexa mistura
(Habermann, 1972; Peiren et al., 2005; Vetter et al., 1999). Em recente abordagem
proteômica, três novas proteínas foram encontradas no veneno de Apis mellifera
carnica, sendo que para duas delas a identificação foi possível (Peiren et al., 2005).
A primeira proteína foi identificada como contendo domínio PDGF (fator de
crescimento derivado de plaquetas)/VEGF (fator de crescimento endotelial vascular)
e a segunda mostrou homologia com uma proteína hipotética similar a MRJP (major
royal jelly protein) (Peiren et al., 2005). Estas proteínas são de baixa abundância no
veneno e sua participação na cinética do envenenamento é desconhecida (Peiren et
al., 2005).
Aminas Biogênicas
O papel das aminas biogênicas no veneno e nos quadros clínicos observados
após o envenenamento ainda é bastante obscuro. Estas substâncias parecem ter
seus efeitos mascarados e até mesmo estimulados por outros componentes
protéicos (Grisotto et al., 2006; Habermann, 1972). A dopamina e a norepinefrina
foram descritas in vivo nos reservatórios de veneno de abelhas, porém sua presença
no veneno secretado e liofilizado não foi observada (Habermann, 1972). Entretanto,
acredita-se que estas catecolaminas tenham um efeito importante nas alterações
18
hemodinâmicas renais (Grisotto et al., 2006). Dentre as aminas, a histamina é a
substância de maior relevância. Representando de 0,1-1,5% do peso seco do
veneno, estima-se que uma única picada possa injetar cerca de 2 µg de histamina,
representando 14 µg de histamina exógena /Kg em um caso de envenenamento
massivo (quando estima-se 500 picadas em um indivíduo de 70 Kg) (Grisotto et al.,
2006). Alterações cardiovasculares podem ser observadas em humanos saudáveis
na dose de 1 µg de histamina /Kg, atribuindo-se a ela um papel fundamental na
hipotensão observada pós-envenenamento (Grisotto et al., 2006). Além disso, existe
a presença de peptídeo degranulador de mastócitos (MCD) que é capaz de liberar
histamina endógena (Grisotto et al., 2006).
Componentes enzimáticos
A fosfolipase A2, dentre as enzimas que compõem o veneno de abelha, é a
proteína mais estudada e talvez a enzima de maior importância. A identificação da
atividade fosfolipásica fez com que durante muitos anos se creditassem todos os
efeitos biológicos do veneno a esta enzima (Habermann, 1972). A fosfolipase é a
proteína com maior potencial imunogênico e, como tal, vem sendo usada em
estratégias para imunização e dessensibilização (Jones et al., 1999; Vetter et al.,
1999). A atividade catalítica da enzima é potencializada pelo peptídeo melitina, outro
componente importante do veneno (Mingarro et al., 1995). Assim como outras
fosfolipases de venenos animais, a enzima possui atividade neurotóxica através de
ligação receptor-específica (Nicolas et al., 1997). Necrose muscular pode ser
observada após a injeção da enzima pura, evidenciando sua atividade miotóxica,
demonstrada como sendo diretamente relacionada à atividade catalítica (Ownby et
19
al., 1997). A ocorrência de rabdomiólise em pacientes que sofreram ataques
massivos por abelhas parece estar relacionada à fosfolipase e sua capacidade de
alterar membranas celulares (Grisotto et al., 2006). A modulação da função
plaquetária pela fosfolipase parece ter efeito bimodal e dose-dependente (Ouyang e
Huang, 1984). Distúrbios na coagulação foram observados in vitro; a fosfolipase A2
de veneno de abelha é capaz de alterar todas as fases da coagulação (via
intrínseca, extrínsica e comum) (Petroianu et al., 2000). Demonstrou-se que a
enzima é capaz de reduzir a atividade dos fatores da coagulação, trombina, fator V e
fator VIII, e esta capacidade pode servir de parâmetro para o monitoramento do
envenenamento (Petroianu et al., 2000).
Outras três enzimas parecem ser componentes importantes dos venenos de
abelha, apesar da literatura a respeito das mesmas ser bastante escassa. Duas
dessas proteínas são importantes alergenos, sendo a hialuronidase a enzima com
segundo maior potencial imunológico do veneno de abelha. Verificou-se que 78%
dos pacientes alérgicos apresentam IgE específica para fosfolipase e 71% para
hialuronidase (Markovic-Housley et al., 2000). A terceira enzima é uma serino-
protease que apresenta sítio catalítico tipo tripsina e a presença de um domínio CUB
(Garcia, 2006; Winningham et al., 2004). Este domínio lhe confere a capacidade de
fazer interações proteína-proteína, sendo sua ligação à IgE recentemente
comprovada (Winningham et al., 2004).
Peptídeos bioativos
A apamina é um peptídeo neurotóxico de apenas 18 aminoácidos
diferenciando-se de neurotoxinas clássicas de serpentes e escorpiões que possuem
20
cerca de 60 resíduos (Habermann, 1972). A apamina tem dose letal (DL
50
) de 4
mg/Kg em camundongos e doses altas (1 mg/Kg) são capazes de produzir
movimentos descoordenados e involuntários após 15 minutos, culminando em
convulsões (Habermann, 1972). O peptídeo é um bloqueador específico de um
subtipo de canal de potássio ativado por cálcio (SK
Ca
) presente em neurônios e, em
baixas doses, parece aumentar a resposta cognitiva, o aprendizado e ter uma
atividade anti-depressiva (van der Staay et al., 1999).
Outra importante proteína do veneno é o denominado peptídeo degranulador
de mastócitos (MCD). O MCD é o principal componente do veneno capaz de induzir
a liberação de histamina (Habermann, 1972). O peptídeo é praticamente atóxico,
com uma dose letal (DL
50
) >40 mg/Kg em camundongos, todavia é capaz de
produzir cianose em doses acima de 0,5 mg/Kg (Habermann, 1972). O MCD tem
uma atividade dupla na inflamação, apresentando atividades pró- e anti-inflamatórias
dependendo da dose e tem importante potencial imunogênico (Buku, 1999).
Recentemente mostrou-se que na degranulação de mastócitos ocorre a liberação de
carboxipeptidase A e outras proteases que oferecem um efeito protetor contra o
envenenamento por abelhas e serpentes (Metz et al., 2006; Rivera, 2006).
Peptídeo melitina
A melitina é o componente majoritário do veneno da abelha Apis mellifera,
representando cerca de 50% do peso seco do mesmo (Habermann, 1972). É um
peptídeo anfipático, em hélice, composto de 2.9 KDa, com a seqüência
GIGAVLKVLTTGLPALISWIKRKRQQ e uma carga global de +4, concentrada na
porção carboxi-terminal. Sua estrutura tridimensional tem sido estudada por diversas
21
técnicas como cristalografia de raios X, ressonância magnética nuclear (RMN),
dicroísmo circular e dinâmica molecular (Bello et al., 1982; Glattli et al., 2006; Lam et
al., 2001; Lin e Baumgaertner, 2000; Terwilliger e Eisenberg, 1982; Wang e
Polavarapu, 2003). A estrutura terciária do peptídeo altera-se em função do meio,
formando uma hélice perfeita quando em contato com membranas (Glattli et al.,
2006; Qiu et al., 2005; Sengupta et al., 2005; Yang et al., 2001).
A atividade hemolítica da melitina é seu efeito biológico mais característico,
sendo usado para a identificação do peptídeo em frações de veneno (Tosteson et
al., 1985). Esta atividade é modulada pela presença de colesterol em eritrócitos,
sendo a capacidade hemolítica do peptídeo aumentada em células depletadas de
colesterol (Raghuraman e Chattopadhyay, 2005). O peptídeo ainda é capaz de
interagir e extravasar o conteúdo de leucócitos e mastócitos (Habermann, 1972).
Além disso, a capacidade lítica da melitina não se restringe às células humanas e
animais uma vez que o peptídeo apresenta uma capacidade antibacteriana e
antifúngica (Asthana et al., 2004; Habermann, 1972). As características líticas da
melitina podem ser explicadas pela sua elevada capacidade de interagir e
desestabilizar membranas naturais ou artificiais (Dempsey, 1990; Habermann,
1972). A inserção em membranas provoca a formação de poros e a permeabilização
das mesmas, causando rompimento celular (Dempsey, 1990; Yang et al., 2001).
A melitina apresenta atividade tóxica frente a diversos tipos de células. O
peptídeo é uma conhecida cardiotoxina (Okamoto et al., 1995), efeito este
relacionado a um aumento no influxo de cálcio. O fluxo de íons pode ser modulado
através da interação da melitina com canais iônicos, efeito observado também em
uma diversidade de outros tecidos (Baker et al., 1995; Shorina et al., 2004; Voss et
al., 1995). Igualmente, a melitina é capaz de modular proteínas G (Fukushima et al.,
22
1998), podendo afetar uma série de receptores acoplados a estas proteínas em
maneira similar ao peptídeo do veneno de vespas – mastoparan (Higashijima et al.,
1990; Higashijima et al., 1988).
A modulação de proteínas pela melitina confere a ela uma série de outras
atividades biológicas interessantes. Fosfolipases são moduladas pelo peptídeo que
aumenta a atividade catalítica da fosfolipase A2 do veneno de abelha assim como
de uma fosfolipase D humana e ainda tem efeito bimodal sobre uma fosfolipase A2
humana (Koumanov et al., 2003; Mingarro et al., 1995; Saini et al., 1999). A proteína
ligadora de cálcio calmodulina também tem sua atividade regulada por melitina e a
estrutura tridimensional do complexo vem sendo estudada por diferentes
abordagens (Hait et al., 1985; Kaetzel e Dedman, 1987; Kataoka et al., 1989; Scaloni
et al., 1998). A geração de ácido araquidônico e seus metabólitos pelo peptídeo é
explicada através da modulação de cicloxigenases e lipoxigenases (Salari et al.,
1985). Outra característica interessante é a capacidade do peptídeo de simular
superfícies ou fosfolipídios de membrana. Acredita-se que a melitina é capaz de
suportar a atividade do fator IX da coagulação na ativação do fator X fornecendo a
superfície necessária para uma eficiente atividade proteolítica (Blostein et al., 2000).
23
Objetivos
A presente dissertação teve como objetivo geral a caracterização estrutural e
biológica da melitina. Especificamente, os objetivos deste trabalho são:
1. Caracterização conformacional da melitina por dinâmica molecular.
Avaliação dos estados de oligomerização do peptídeo em solução
Avaliação do efeito do pH sobre a estrutura tridimensional (3D) do
peptídeo
Avaliação do efeito da força iônica sobre a estrutura 3D do peptídeo
Avaliação do efeito do solvente sobre a estrutura 3D do peptídeo
2. Identificação dos efeitos da melitina sobre a função plaquetária
Avaliação da capacidade pró-agregante do peptídeo
Avaliação da secreção de adenosina tri-fosfato como efeito do peptídeo
Avaliação das características morfológicas das plaquetas tratadas com
o peptídeo
Avaliação dos efeitos do peptídeo sobre a adesão plaquetária
24
Resultados
1. Análise conformacional da melitina por dinâmica
molecular
1.1 Dinâmica molecular no estudo de estrutura e conformação de
proteínas
Há quase trinta anos, a primeira simulação de dinâmica molecular foi
publicada utilizando o inibidor pancreático da tripsina bovina como alvo de estudo
(McCammon et al., 1977). O emprego desta técnica, apesar de bastante limitada na
época, ajudou na desmistificação de que proteínas eram estruturas estáticas
(Karplus, 2002). O entendimento de que as estruturas protéicas são dinâmicas vem
auxiliando o estudo de sistemas biológicos complexos e a caracterização molecular
das proteínas é valiosa. As simulações de dinâmica molecular de proteínas, assim
como de outros sistemas, podem ajudar a descrever detalhadamente a mobilidade e
flexibilidade das moléculas em função do tempo (Karplus, 2002; Karplus e
25
McCammon, 2002). A validação dos modelos obtidos deve ser verificada por
comparação a resultados experimentais, porém, a técnica vem sendo também
amplamente utilizada para o refinamento de dados obtidos por outras metodologias
como cristalografia de raios-x e ressonância magnética nuclear (Karplus e
McCammon, 2002).
Nos últimos anos, as simulações de dinâmica molecular foram popularizadas
e reconhecidas pela comunidade científica e os avanços na capacidade
computacional permitem hoje uma descrição complexa da dinâmica das moléculas.
Em consulta ao banco de dados do portal ISI WEB of Knowledge
(http://portal.isiknowledge.com/), quando os termos “molecular dynamics” e “proteins”
são consultados, cerca de 7600 artigos são encontrados, sendo 743 trabalhos
publicados no ano de 2006.
1.2 Artigo 1: Terra, RMS; Guimarães, JA; Verli, H. Structural and
functional behavior of biologically active monomeric melittin. J.
Mol. Graph. Model. (2006)
O trabalho aqui apresentado é uma análise detalhada da conformação do
peptídeo do veneno de abelha melitina por simulações de dinâmica molecular.
Aspectos conformacionais são avaliados em função dos graus de oligomerização do
peptídeo, assim como seu comportamento em solução sob diferentes condições
físico-químicas. Os resultados obtidos neste trabalho permitiram um entendimento
global do comportamento da melitina em solução e ajudaram em uma melhor
compreensão da diversidade de suas funções biológicas. O artigo foi submetido ao
26
Journal of Molecular Graphics and Modelling e aceito para publicação no dia 26 de
junho de 2006.
+ Models
JMG-5593; No of Pages 6
Structural and functional behavior of biologically active
monomeric melittin
Renata M.S. Terra
a
, Jorge A. Guimara
˜
es
a,
*
, Hugo Verli
a,b
a
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500,
CP 15005, Porto Alegre 91500-970, RS, Brazil
b
Faculdade de Farma
´
cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752,
Porto Alegre 90610-000, RS, Brazil
Received 27 February 2006; received in revised form 26 June 2006; accepted 26 June 2006
Abstract
Melittin is a well-known water-soluble toxic peptide present in bee venom of Apis mellifera, capable of interacting with and disrupting cell
membranes thus producing many effects on living cells. Additionally, melittin induces activation of phospholipases and calmodulin upon
interaction with cellular membranes. The conformation and aggregation state adopted by melittin in solution depends on several factors including
the peptide concentration, ionic strength, pH and the nature of the ions in the aqueous medium. Such conformational dependence on the peptide
environment gives new insights over the currently available 3D structures of melittin and, ultimately, over its biologically functional unit. Basedon
crystallographic data, the melittin tetramer has been proposed as its bioactive form. Contrarily to such data, we show in this work the results
obtained from molecular dynamics simulations, which clearly indicate that the tetrameric organization of melittin is not stable under biological
conditions dissociating after 2.5 ns through a 10 ns trajectory. We found that the tetrameric form of melittin is stable only in conditions of high pH
and high peptide concentration in the molecular dynamics simulations. Moreover, when in plasma melittin appears to be a random coil monomer,
folding only upon interaction with biological membranes. In summary, these findings elucidate several properties of melittin structure and
dynamics, projecting significant implications in the study of its biological function.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Melittin; Molecular dynamics; Bee venom; Apis mellifera; Peptide folding; Melittin biological unit
1. Introduction
Melittin is a well-known water-soluble toxic peptide present
in bee venom of Apis mellifera, comprising about 50% of its dry
weight. This peptide is able to disrupt membranes, producing
many effects on living cells [1,2]. Like other amphiphilic a-
helical peptides, melittin has an antibacterial activity, induces
voltage-gated channel formation and can also produce micelli-
zation of phospholipids bilayers due to its membrane-interacting
effect [1,3,4]. However, the major effect of melittin on
erythrocytes is to cause lyses, as its binding to cell membranes
results in the release of hemoglobin to the extracellular medium
[3]. The molecular mechanism underlying melittin interaction
with biological membranes and lipid bilayers is not well
understood. Moreover, it seems that different molecular
mechanisms could generate different actions of the peptide
[3]. Interestingly, melittin interaction with certain proteins in the
cell, such as phospholipases and calmodulin, thus inducing their
functional modulation, has been also described [5–7].
This helical amphipatic peptide consists of 26 amino acid
residues, comprising the sequence: GIGAVLKVLTTGLPALIS-
WIKRKRQQ, and a total net charge of +5, four of which
(KRKR) are in the C-terminal portion. Its conformation and
structure has been studied by different approaches, including
X-ray crystallography, nuclear magnetic resonance (NMR)
spectroscopy, circular dichroism (CD), and molecular dynamics
(MD) simulations [8–16]. Melittin is soluble in both water and
methanol, and its monomeric structure is described as shaping
like a bent rod due to the presence of a proline residue in position
14 [9]. The peptide’s crystallographic structure was deposited in
the Protein Data Bank (PDB) under accession number 2MLT
[10]. According to this data melittin crystallizes as a dimer,
www.elsevier.com/locate/JMGM
Journal of Molecular Graphics and Modelling xxx (2006) xxx–xxx
* Corresponding author. Tel.: +55 51 3316 6068; fax: +55 51 3316 7309
˜
es).
1093-3263/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jmgm.2006.06.006
Please cite this article as: Renata M.S. Terra et al., Structural and functional behavior of biologically active monomeric melittin, Journal of
Molecular Graphics and Modelling (2006), doi:10.1016/j.jmgm.2006.06.006.
and the proposed biologically active unit has been described as a
tetramer [10,11]. Contrarily to crystallographic data, biochem-
ical studies show that the helical content and aggregation state
adopted by the peptide depends, in a complex way, on several
factors including the peptide concentration, ionic strength, pH
and the nature of the ions in the aqueous medium [2,8]. Such
apparent contradiction between structural and biochemical data
has not been explained or correlated to conformational and
functional data by any molecular model so far.
A recent publication [17] presented a molecular dynamics
simulation of the melittin tetramer as the bioactive form of this
peptide. The authors describe the tetrameric form as a
‘channel’, defined between two melittin dimers, which
collapse during a water drying transition. Surprisingly, using
the same methodology we were unable to reproduce these
computational results. We believe that this is due to the fact that
computational models should be built in a way that the system
factors that are essential to a precise quantification of the
property of interest must be both adequately considered and
described, as well as sufficiently sampled [18].Thecorrect
representation of these factors can be inferred by how well a
molecular dynamics simulation reproduces known quantities
[19]. Regarding melittin description by molecular dynamics,
an adequate model should necessarily be capable of reprodu-
cing its conformational dependence of pH, salt and peptide
concentrations.
In this work we used the crystallographic structure (PDB
2MLT) as the starting point for molecular dynamics simulations
of melittin. We investigate the conformation of the peptide on
both monomeric and oligomeric forms under different solvents,
peptide concentrations, ionic strength and pH conditions. We
describe here the conformational equilibrium assumed by the
peptide in solution, a result that reproduces and correlates, for
the first time, the peptide’ structural properties with its
biochemical data so far reported.
2. Methods
2.1. Software
Energy minimization calculations, molecular dynamics
simulations and trajectory analysis were done using the
GROMACS simulation suite [20]. Molecular visualization
was done in Swiss PBD viewer (SPDBV) environment [21] and
the secondary structure content analyses were performed with
PROCHECK [22].
2.2. Molecular dynamics simulations
Melittin in its tetrameric form was retrieved from Protein
Data Bank under code 2MLT. This oligomerization state was
kept for simulation of the tetramer, the chains AB were isolated
for simulation of the dimer, and the chain A was isolated for
simulation of the monomeric form of the peptide. The
monomeric, dimeric, and tetrameric forms of melittin were
further solvated in a cubic box using periodic boundary
conditions and using single point charge (SPC) water model
[23] or Gromos96 methanol model [24] for water and methanol
modeling, respectively. The ionic strength was adjusted by
addition of chloride or sulfate ions, while the peptide
concentration was obtained by varying the box size. Different
protonation states of melittin under pH 7.0 and 11.0 were
adjusted manually according to the standard pK
a
values of the
amino acids side chains. Consequently, at pH 11.0 lysine side
chains were deprotonated. The final systems composed by
peptide, water and ions comprise up to 28,000 atoms. The Lincs
and Settle methods [25,26] were applied to constrain covalent
bonds lengths, allowing an integration step of 2 fs after an
initial energy minimization using the Steepest Descents
algorithm. Electrostatic interactions were calculated with the
generalized reaction-field method [27], with Coulomb and
Lennard-Jones cut-off adjusted at 16 A
˚
. The simulations were
performed under constant-pressure (1 atm) and constant-
temperature (310 K), using Gromos96 or OPLSAA force
fields. The dielectric constant was treated as e =1. The
monomeric, dimeric and tetrameric systems were heated
slowly, from 50 to 310 K, in steps of 5 ps. At each step the
reference temperature was increased by 50 K, allowing a
progressive thermalization of the molecular systems. The
simulation was then extended up to 10 ns. As several
simulations were performed, the total conformational sampling
of the peptide was about 0.1 ms.
3. Results and discussion
3.1. Simulations of oligomeric melittin
In order to elucidate the oligomeric organization of melittin
in biological solutions we performed unrestrained molecular
dynamics simulations of a melittin tetramer in three different
conditions: (1) 23.5 mM melittin, 120 mM chloride ions, pH
7.0; (2) 54.6 mM melittin, 273 mM chloride ions, pH 7.0; (3)
23.5 mM melittin, 120 mM chloride ions, pH 11.0. Observing
R.M.S. Terra et al. / Journal of Molecular Graphics and Modelling xxx (2006) xxx–xxx2
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JMG-5593; No of Pages 6
Please cite this article as: Renata M.S. Terra et al., Structural and functional behavior of biologically active monomeric melittin, Journal of
Molecular Graphics and Modelling (2006), doi:10.1016/j.jmgm.2006.06.006.
Fig. 1. Molecular dynamics simulation of oligomeric melittin. (a) Melittin crystallographic conformation (PDB 2MLT). (b) Melittin tetrameric conformation after
2 ns of molecular dynamics simulation in 120 mM chloride ions and pH 7.0; the distance in nm between dimers AC and BD is shown in dotted lines. (c) Snapshot of
melittin dimeric conformation after 4 ns of molecular dynamics simulation.
the simulation condition closest to the plasmatic medium
(condition 1), the monomers that compose the tetrameric
structure present a progressive re-orientation, irreversibly
splitting into two dimers within 2.5 ns of the trajectory (see
Supplementary data). The initial conformation of tetrameric
melittin can be observed in Fig. 1a and the resulting tetrameric
form unfolded into two dimers (AC and BD) is seen in Fig. 1b.
The distances between dimers increases drastically, as shown in
Fig. 2a, becoming larger than 3.5 nm after the first 1.5 ns. Such
behavior allowed us to conclude that the formation of the
‘channel’ as proposed in a previous work [17] on this system,
seems to be an artifact.
In fact, the stability of the tetrameric form of melittin (and of
its ‘channel’) in unrestrained simulations was obtained only
through the modulation of the medium physical–chemical
conditions. Simulation under condition 2 presents a 2.3-fold
increase in both melittin and salt concentration, while condition
3 presents an increase in pH from 7.0 to 11.0. Under conditions
2 and 3 the initial tetrameric conformation (Fig. 1a) could not
be set apart (Fig. 2b) during the trajectories indicating a stable
tetrameric form. It must be emphasized that even the lowest
melittin concentration (23.5 mM) tested (simulation conditions
1 and 3) is at least eight-fold higher than its concentration in
crude bee venom. This observation is of extreme importance
regarding any mechanism proposed for explaining the peptide’s
biological activity, which should take into consideration both
the conformation and oligomerization status of the peptide
when acting in a physiological medium. As clearly indicated
here, a lower plasmatic concentration would induce an even
faster unfolding of the melittin tetramer.
Since the previous work on melittin simulation used the
OPLSAA force field to describe the tetrameric form as a
‘channel’ [17] and in order to avoid any force field dependent
results, we also simulated the melittin tetramer using such force
field under both low (23.5 mM) and high (56.6 mM) peptide
concentrations. The conformational behavior of melittin
tetramer was very similar in both Gromos96 and OPLSAA
force fields, being stable only at high peptide concentrations.
However, under OPLSAA a partial unfolding of its constituent
peptide units was observed (Fig. 2c). Our data demonstrate that
the ‘channel’ between the dimers in the crystallographic
structure remains stable only under extreme and non-
physiological conditions (Fig. 2b), being thus inappropriate
to attribute a biological role for such melittin structure. In
agreement with these observations, a recent work [29] reported
that aggregation of melittin in a tetrameric form was only
observed in high salt aqueous solutions.
Considering the fact that the melittin tetramer disassembles
into two dimers under conditions close to that of a physiological
medium, we also performed molecular dynamics simulations of
the crystallographic dimeric form of the peptide. The
parameters of pH, melittin and salt concentration chosen for
such molecular dynamics simulation were the same as the
simulation condition 1. Doing so, we observed that the dimeric
form of melittin also dissociates in solution, with the distance
between monomers changing from 1.3 to 4.5 nm in the first
2.5 ns of the trajectory (Fig. 1c). This result led us to the
conclusion that, in plasma, melittin should not be in a
tetrameric nor a dimeric oligomerization state, but is probably
present as a monomeric peptide.
3.2. Simulations of monomeric melittin
The data obtained by molecular simulations of oligomeric
melittin indicate that the aggregation state of the peptide is
highly dependent on the environment conditions, i.e. peptide
concentration and/or pH. The effect of physical–chemical
parameters of the medium on the structure of melittin can be
easily noticed in the monomeric form of the peptide. For
example, the helical content of melittin increased from 12% to
65% in response to a salt concentration changing from near zero
to 1 M NaCl, as reported by others [8,16]. Thus, in addition to
the oligomerization state, the peptide secondary structure is
also dependent on solution conditions.
Considering the lack of stability of the peptide oligomeric
states under ‘physiological’ conditions and aiming to correlate
these data to the peptide’s helical content, we investigated the
behavior of monomeric melittin under a number of different
R.M.S. Terra et al. / Journal of Molecular Graphics and Modelling xxx (2006) xxx–xxx 3
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JMG-5593; No of Pages 6
Please cite this article as: Renata M.S. Terra et al., Structural and functional behavior of biologically active monomeric melittin, Journal of
Molecular Graphics and Modelling (2006), doi:10.1016/j.jmgm.2006.06.006.
Fig. 2. Plot of the distances between dimers of melittin against molecular dynamics simulation time (upper) and snapshots of tetrameric conformation after 4 ns of
molecular dynamics simulations (lower). (a) Condition 1, 23.5 mM melittin, 120 mM Cl
À
, pH 7.0, Gromos96 force field. (b) Condition 2, 56.6 mM melittin, 273 mM
Cl
À
, pH 7.0, Gromos96 force field. (c) Condition 3, 56.6 mM melittin, 273 mM Cl
À
, pH 7.0, OPLSAA force field.
conditions. An overview of melittin conformations after 10 ns
of molecular dynamics simulations under different simulation
parameters, is presented in Fig. 3.
In agreement with biochemical observations, the simula-
tions of a melittin monomer in presence of 50 mM chloride ions
indicated a significant decrease in its helical character (Fig. 3b),
a behavior also observed under an equivalent concentration of
sulfate ions (Fig. 3c). On the other hand, the increase in ionic
strength, using either chloride or sulfate ions, enhance the helix
content of melittin in an ion-nature independent manner
(Fig. 3d and e). In addition, a curious behavior is observed at
50 mM SO
4
2À
, in which the helical character is partially
transformed in a b-sheet structure. Such conformational
modification is reproducible and can be reverted under higher
ionic strength.
The pH has a critical effect on the stabilization of the helical
structure of melittin, as reported by other authors [8]. Again,
molecular dynamics simulations correctly reproduce experi-
mental data: loss of the helical character is observed when the
peptide is exposed to pH 7.0 (Fig. 3c), being the secondary
structure almost completely recovered upon increasing the pH
medium to 11.0 (Fig. 3f). These data are illustrated in Fig. 4 by
R.M.S. Terra et al. / Journal of Molecular Graphics and Modelling xxx (2006) xxx–xxx4
+ Models
JMG-5593; No of Pages 6
Please cite this article as: Renata M.S. Terra et al., Structural and functional behavior of biologically active monomeric melittin, Journal of
Molecular Graphics and Modelling (2006), doi:10.1016/j.jmgm.2006.06.006.
Fig. 3. Monomeric melittin conformations after 10 ns of molecular dynamics simulations. (a) Melittin crystallographic structure (PDB 2MLT). (b) Melittin in water
pH 7.0, Cl
À
50 mM. (c) Melittin in water pH 7.0, SO
4
2À
50 mM. (d) Melittin in water pH 7.0, Cl
À
1.2 M. (e) Melittin in water pH 7.0, SO
4
2À
1.2 M. (f) Melittin in
water pH 11.0, SO
4
2À
50 mM. Secondary structure prediction by PROCHECK [23] is presented in black.
Fig. 4. Ellipticity maps [27] of monomeric melittin as a function of time and aminoacid number. (a) 26.5 mM melittin, 50 mM Cl
À
, pH 7.0. (b) 26.5 mM melittin,
1.2 M Cl
À
, pH 7.0.
means of the ellipticity parameter [28] as a function of both
time and amino acid sequence.
Besides the critical parameters of ionic strength and pH,
some solvents, especially methanol, have been considered in
the study of melittin structure and conformation. Methanol is an
interesting solvent for investigation, since it was the solvent
used to obtain the NMR structure of melittin [9], which shows
the peptide as a completely folded helix. However, contrarily to
this a 10 ns molecular dynamic simulation in presence of
methanol presented an unfolded structure on the peptide N-
terminal portion (data not shown). Conversely, the C-terminal
helix remained stable over the entire simulation. Similar results
were obtained in previous work, in which a completely
unfolded melittin was seen after a 120 ps molecular dynamics
simulation in methanol [15]. Regarding the molecular basis for
the difference between NMR and molecular dynamics findings,
a possible reason are the conditions in which the peptide
structure is simulated. As shown in Fig. 3, the peptide
conformation and dynamics is highly determined by the
surrounding environment, which is influenced by the proce-
dures used for the peptide purification. For example, residual
ions in the purified sample could be the key factor for inducing
the differences between folded and unfolded helix, as shown in
Fig. 3d being the increase in helical character due to an increase
in chloride ions concentration.
The simulations of the monomeric form of the peptide, in a
totalsamplingof about 0.1 msinthis study, suggesta rathersimple
explanation for the discrepancy so far existing in the literature
about the peptide structure and its biochemical properties in
solution. As shown here the conformation of melittin can be
highly influenced by physical–chemical conditions (Fig. 3),
inducing the peptide to adopt a diversity of forms under different
media. On the other hand, both crystallographic and NMR
structuresare usuallyobtained underartificialaqueousconditions
(i.e. different from plasma composition), as has been the case for
melittin. As a consequence, the helical structure observed in
crystallographic and NMR data for this peptide should be
interpreted as a consequence of both high salt and melittin
concentrations. In this context, our calculations suggest that in
physiological medium such as human plasma, melittin should
assume a monomeric form, mostly unfolded.
3.3. Implications to current understanding of melittin
biological properties
The melittin conformation responsible for its pore formation
capabilities has been a matter of controversy [3,30–32],
especially because some authors assume that melittin
approaches and binds to membranes in a defined conformation
and orientation. In fact, the orientation of the peptide over lipid
bilayers has been proposed as a function of the peptide
concentration, e.g. at low concentration melittin binds parallel
to the membrane surface and as the peptide concentration
increases in the medium the binding turns to a perpendicular
orientation [32]. The pore model has also been assumed as a
toroidal model by neutron diffraction [32], and a previous
molecular dynamics simulation study has proposed that even
when inserted into lipid bilayers the position of the melittin
monomers is not stabilized as a perpendicular tetramer [14].
Besides, the orientation of melittin chains in a pore is
enormously different [32] to that proposed by X-ray crystal-
lography [10,11]. Thus, considering the whole of experimental
and theoretical data available for melittin structure and
dynamics, the peptide appears to fold into a helix conformation
only when interacting with highly negative biological surfaces,
as membranes of activated platelets or to membrane proteins, as
phospholipases. Such charged surfaces would perform a role
equivalent to high salt conditions in the peptide folding.
Actually it has been experimentally shown [33] that melittin
binds to a lipid vesicle as a disordered monomeric peptide form,
inserts itself into the lipid membrane, and its concerted folding
as a helix is promoted upon the peptide penetration. The
characterization of this process at the atomic level was recently
shown, with the observation that DMPC bilayers are capable to
stabilize the secondary structure of melittin related to the
unfolded peptide in aqueous solution [34].
4. Conclusion
In this work we performed molecular dynamics simulation of
melittin in three different oligomeric states, i.e. monomeric,
dimeric, and tetrameric forms, in order to have a complete
overview of the peptide structure, conformation and dynamics
according to environmental conditions. Our results show that the
tetrameric organization of melittin is not possible in plasma, but
only in extreme physical–chemical conditions. We have seen that
the crystallographically observed inner channel does not remain
stable in solution under physiological conditions and cannot be
responsible for the biological properties of melittin.
Additionally, we observed that the peptide is not a perfect
helix, but a random structure at pH 7.0 in physiological ionic
strength. Our findings are important not only for the under-
standing of the melittin mechanism of action concerning its
lytic activity, but are also relevant for the study of its ability to
modulate phospholipases and others physiologically important
proteins.
Acknowledgements
We thank Coordenac¸a
˜
o de Aperfeic¸oamento de Pessoal de
Nı
´
vel Superior (CAPES-MEC) and Conselho Nacional de
Desenvolvimento Cientı
´
fico e Tecnolo
´
gico (CNPq-MCT),
Brazil, for financial support.
Appendix A. Supplem entary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.jmgm.2006.06.006.
References
[1] E. Haberman, Bee wasp venoms, Science 177 (1972) 314–322.
[2] J. Gauldie, J.M. Hanson, F.D. Rumjanek, R.A. Shipolini, C.A. Vernon,
Peptide components of bee venom, Eur. J. Biochem. 61 (1976) 369–376.
R.M.S. Terra et al. / Journal of Molecular Graphics and Modelling xxx (2006) xxx–xxx 5
+ Models
JMG-5593; No of Pages 6
Please cite this article as: Renata M.S. Terra et al., Structural and functional behavior of biologically active monomeric melittin, Journal of
Molecular Graphics and Modelling (2006), doi:10.1016/j.jmgm.2006.06.006.
[3] C.E. Dempsey, The actions of melittin on membranes, Biochim. Biophys.
Acta 1031 (1990) 143–161.
[4] B. Bechinger, Structure and functions of channel-forming peptides:
magainins, cecropins, melittin and alamethicin, J. Membr. Biol. 156
(1997) 197–211.
[5] W.T. Shier, Activation of high levels of endogenous phospholipases A2 in
cultured cells, PNAS 76 (1979) 195–199.
[6] S.S. Saini, J.W. Peterson, A.K. Chopra, Melittin binds to secretory
phospholipase A(2) and inhibits its enzymatic activity, Biochem. Biophys.
Res. Commun. 238 (1997) 436–442.
[7] A. Scaloni, N. Miraglia, S. Orru, P. Amodeo, A. Motta, G. Marino, P.
Pucci, Topology of the calmodulin–melittin complex, J. Mol. Biol. 277
(1998) 945–958.
[8] J. Bello,H.R. Bello,E. Granados, Conformation and aggregationof melittin-
dependence on pH and concentration, Biochemistry 21 (1982) 461–465.
[9] R. Bazzo, M.J. Tappin, A. Pastore, T.S. Harvey, J.A. Carver, I.D. Camp-
bell, The structure of melittin—A H-1-NMR study in methanol, Eur. J.
Biochem. 173 (1998) 139–146.
[10] T.C. Terwillinger, D. Eisenberg, The structure of melittin. 1. Structure
determination and partial refinement, J. Biol. Chem. 257 (1982) 6010–6015.
[11] T.C. Terwillinger, D. Eisenberg, The structure of melittin. 2. Interpretation
of the structure, J. Biol. Chem. 257 (1982) 6016–6022.
[12] M. Iwadate, T. Asakura, M.P. Williansom, The structure of melittin
tetramer at different temperatures—an NOE-based calculation with che-
mical shift refinement, Eur. J. Biochem. 257 (1998) 479–487.
[13] F. Wang, P.L. Polavarapu, Conformational analysis of melittin in solution
phase:vibrationalcirculardichroismstudy, Biopolymers70 (2003)614–619.
[14] J.H. Lin, A. Baumgaertner, Stability of a melittin pore in a lipid bilayer: a
molecular dynamics study, Biophys. J. 78 (2000) 1714–1724.
[15] H.L. Liu, C.M. Hsu, The effects of solvent and temperature on the
structural integrity of monomeric melittin by molecular dynamics simula-
tions, Chem. Phys. Lett. 375 (2003) 119–125.
[16] J.C. Talbot, J. Dufourcq, J.D. Bony, J.F. Faucon, C. Lussan, Conforma-
tional change and self association of monomeric melittin, FEBS Lett. 102
(1979) 191–193.
[17] P. Liu, X.H. Huang, R.H. Zhou, B.J. Berne, Observation of a dewetting
transitionin the collapseof the melittintetramer, Nature 437(2005)159–162.
[18] W.F. van Gunsteren, H.J.C. Berendsen, Computer simulation of molecular
dynamics: methodology, application, and perspectives in chemistry,
Angew. Chem. Int. Ed. Engl. 29 (1990) 992–1023.
[19] M. Karplus, G.A. Petsko, Molecular dynamics simulations in biology,
Nature 347 (1990) 631–639.
[20] E. Lindahl, B. Hess, D. van der Spoel, Gromacs 3.0: a package for
molecular simulation and trajectory analysis, J. Mol. Model. 7 (2001)
306–317.
[21] N. Guex, M.C. Peitsch, SWISS-MODEL and Swiss-PDB viewer: an
environment for comparative protein modeling, Electrophoresis 18
(1997) 2714–2723.
[22] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PRO-
CHECK: a program to check the stereochemical quality of protein
structures, J. Appl. Crystallogr. 26 (2003) 283–291.
[23] H.J.C. Berendsen, J.R. Grigera, T.P. Straatsma, The missing term in
effective pair potentials, J. Phys. Chem. 91 (1987) 6269–6271.
[24] W.F. van Gunsteren, S.R. Billeter, A.A. Eising, P.H. Hu
¨
nenberger, P.
Kru
¨
ger, A.E. Mark, W.R.P. Scott, I.G. Tironi, Biomolecular Simulation:
The Gromos96 Manual and User Guide, vdf Hochschulverlag, ETH
Zu
¨
rich, Switzerland, 1996.
[25] B. Hess, H. Bekker, H.J.C. Berendsen, J.G.E.M. Fraaije, LINCS: a linear
constraint solver for molecular simulations, J. Comput. Chem. 18 (1997)
1463–1472.
[26] S. Miyamoto, P.A. Kollman, SETTLE: an analytical version of the
SHAKE and RATTLE algorithm for rigid water models, J. Comput.
Chem. 13 (1992) 952–962.
[27] I.G. Tironi, R. Sperb, P.E. Smith, W.F. van Gunsteren, A generalized
reaction-field method for molecular dynamics simulations, J. Chem. Phys.
102 (1995) 5451–5459.
[28] J.D. Hirst, C.L. Brooks III, Helicity, circular dichroism and molecular
dynamics of proteins, J. Mol. Biol. 243 (1994) 173–178.
[29] W.H. Qiu, L.Y. Zhang, Y.T. Kao, W.Y. Lu, T.P. Li, J. Kim, G.M.
Sollemberger, L.J. Wang, D.P. Zhong, Ultrafast hydration dynamics in
melittin folding and aggregation: helix formation and tetramer self-
assembly, J. Phys. Chem. B 109 (2005) 16901–16910.
[30] A. Okada, K. Wakamatsu, T. Miyazawa, T. Higashijima, Vesicle-bound
conformation of melittin: transferred nuclear Overhauser enhancement
analysis in the presence of perdeuterated phosphatidylcholine vesicles,
Biochemistry 33 (1994) 9438–9446.
[31] A. Naito, T. Nagao, K. Norisada, T. Mizuno, S. Tuzi, H. Saito, Con-
formation and dynamics of melittin bound to magnetically oriented lipid
bilayers by solid state
31
P and
13
C NMR spectroscopy, Biophys. J. 78
(2000) 2405–2417.
[32] L. Yang, T.A. Harroun, T.M. Weiss, L. Ding, H.W. Huang, Barrel-stave
model or toroidal model? A case study on melittin pores, Biophys. J. 81
(2001) 1475–1485.
[33] I. Constantinescu, M. Lafleur, Influence of lipid composition on the
kinetics of the concerted insertion and folding of the melittin in bilayers,
Biochim. Biophys. Acta 1667 (2004) 26–37.
[34] A. Gla
¨
ttli, I. Chandrasekhar, W.F. van Gunsteren, A molecular
dynamics study of the bee venom melittin in aqueous solution, in
methanol, and inserted in a phospholipid bilayer, Eur. Biophys. J. 35
(2006) 255–267.
R.M.S. Terra et al. / Journal of Molecular Graphics and Modelling xxx (2006) xxx–xxx6
+ Models
JMG-5593; No of Pages 6
Please cite this article as: Renata M.S. Terra et al., Structural and functional behavior of biologically active monomeric melittin, Journal of
Molecular Graphics and Modelling (2006), doi:10.1016/j.jmgm.2006.06.006.
33
2. Caracterização dos efeitos da melitina na função
plaquetária
2.1 Função e avaliação de função plaquetária
As plaquetas têm um papel fundamental na hemostasia. Durante um dano
vascular o subendotélio exposto provoca uma reação rápida de adesão plaquetária
ao tecido, seguida da ativação das plaquetas, promovendo a formação de um
tampão e impedindo a hemorragia (Rand et al., 2003; Willoughby et al., 2002). A
adesão das plaquetas às proteínas dos sítios de dano vascular, principalmente fator
de von Willebrand, é mediada pelo receptor glicoprotéico (GP)Ib-V-IX,
exclusivamente expresso em plaquetas e megacariócitos (Nieswandt et al., 2005).
Este processo ainda é auxiliado pela presença de receptores específicos para outras
proteínas de membrana basal, como a integrina α
2
β
1
ligadora de colágenos
(Nieswandt et al., 2005). O processo de adesão é seguido da ativação das plaquetas
e conseqüente recrutamento de outras plaquetas circulantes.
As plaquetas circulantes são discóides e possuem uma meia-vida aproximada
de 8-10 dias (Willoughby et al., 2002). O processo de ativação ocorre na presença
34
de agonistas sendo mediado por receptores na superfície das plaquetas. A ativação
desencadeia quatro distintos processos: (a) alteração morfológica (shape change),
(b) agregação, (c) secreção e (d) produção de ácido araquidônico (Chow e Kini,
2001; Smith e Brinkhous, 1991). A indução deste processo pode ser realizada por
uma série de substâncias endógenas - como adenosina difosfato (ADP), colágeno,
tromboxana e trombina – ou substâncias exógenas – como ionóforos e análogos de
endoperóxidos cíclicos (Willoughby et al., 2002). Existe ainda uma grande
quantidade de toxinas de venenos animais que também possuem a capacidade de
interagir e alterar a função plaquetária (Chow e Kini, 2001; Smith e Brinkhous, 1991).
A maioria dos agonistas utiliza receptores acoplados a proteína G, sendo a proteína
α
IIb
β
3
essencial na ativação plaquetária. Quando ativada, a α
IIb
β
3
provoca a ativação
de fosfolipases, aumento do cálcio intracelular, supressão da produção de AMPc e
reorganização do citoesqueleto (Nieswandt et al., 2005).
Uma vez ativada, as plaquetas sofrem uma mudança de forma (shape
change); a mobilização de actina, miosina, tropomiosina e outras proteínas do
citoesqueleto resulta na emissão de pseudópodos, a exposição de diversos
receptores na superfície da célula e a sua mudança para uma forma globular (Zucker
e Nachmias, 1985). Na Figura 1 pode ser observada a diferença morfológica das
plaquetas por microscopia eletrônica. Após o processo de shape change, as células
passam então a formar agregados. A secreção dos conteúdos de grânulos
intracelulares, principalmente ADP, e a liberação de ácido araquidônico provoca um
aumento no sinal de ativação e o recrutamento de outras plaquetas, promovendo a
formação de grandes agregados, tampão vascular, impedindo a hemorragia
(Willoughby et al., 2002).
35
FIGURA 1 – Ativação e agregação plaquetária. Diferenças morfológicas evidenciadas por
microscopia eletrônica de varredura. Em (a) plaquetas circulantes e em (b) plaquetas
ativadas. Retirado de Willoughby, S. et al (2002)
Diversas técnicas são utilizadas para avaliar a função plaquetária. O primeiro
ensaio descrito para esta finalidade foi o tempo de sangramento, porém sua falta de
sensisibilidade e especificidade provocaram o desenvolvimento de novas técnicas
(Rand et al., 2003). O ensaio mais utilizado atualmente para avaliar a função
plaquetária é a medida de agregação plaquetária em plasma rico em plaquetas
(PRP) (Rand et al., 2003). A técnica espectrofotométrica baseia-se no princípio de
que, na presença de um agonista, as plaquetas sofrem ativação provocando sua
alteração morfológica (shape change) com conseqüente e transitória diminuição na
transmitância do meio medida a 650 nm. Em seguida, as plaquetas agregam
resultando em um aumento na transmitância do meio. A quantificação da resposta
de agregação é realizada através da intensidade com que ocorre esta diferença na
passagem da luz (Rand et al., 2003).
36
2.2 Artigo 2: Terra, RMS; Pinto AFM; Berger, M; de Oliveira, SK;
Juliano, MA; Juliano, L; Guimarães, JA. Melittin-induced
platelet signaling and aggregation. Manuscrito em preparação.
O presente trabalho é uma avaliação da influência do peptídeo melitina na
função plaquetária. Para tal, foi realizada a análise da sua atividade pró-agregante
assim como de sua capacidade secretora. Durante o desenvolvimento deste estudo
foi possível ainda identificar a seqüência de aminoácidos relacionada a esta
atividade biológica através da avaliação de peptídeos sintéticos derivados de
melitina. Além disso, uma demonstração clara da interação direta peptídeo-plaqueta
está aqui apresentada. O trabalho ainda indica uma ativação pouco específica,
envolvendo várias possíveis rotas de sinalização.
Os resultados apresentados aqui estão escritos na forma de manuscrito que
será submetido para publicação no periódico Biochemical and Biophysics Research
Communications.
37
Melittin-Induced Platelet Signaling and Aggregation
Renata M.S. Terra
1
, Antonio F.M. Pinto
1
, Markus Berger
1
, Simone K. de Oliveira
1
,
Maria Aparecida Juliano
2
, Luiz Juliano
2
, Jorge A. Guimaraes
1*
1
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto
Alegre, Brazil
2
Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil
* Corresponding Author. Tel.: +55 51 3316 6068; Fax: +55 51 3316 7309
e-mail address:
[email protected] (J.A. Guimarães)
38
ABSTRACT
Envenomation involving africanized bees results in many biochemical alterations,
including coagulation disorders. The main toxic substance of Apis mellifera venom is
melittin, a peptide with the ability to interact with cell membranes. In this work we
evaluate melittin and five melittin-derived peptides (Mel-1 to Mel-5) in their ability to
affect platelet function. Here we describe the capability of melittin to promote human
platelet aggregation in dose-dependent manner and to secrete ATP from dense
granules. The peptides Mel-1 and Mel-2, which correspond to the amino-terminal
portion of melittin, are related to the pro-aggregating activity as well as the adhesion
of the peptide to platelet surfaces. Additionally, we demonstrate that melittin induced
platelet aggregation is inhibited by indomethacin, esculetin, verapamil, naloxone and
cilostazol. Altogether these findings contribute to the actual knowledge of the
biological activities of melittin and give new insights to the better understanding of
the haemostatic abnormalities during bee envenomations.
Keywords: Melittin, platelet aggregation, platelet adhesion, bee venom, Apis
mellifera, indomethacin, esculetin, cilostazol, naloxone, verapamil
39
INTRODUCTION
Accidents involving africanized bees are frequently reported and massive
envenomations results in nausea, hemolytic and other haemostatic abnormalities,
shock, disseminated intravascular coagulation, kidney failure and coma and, in some
cases, a delayed multi-organ-failure [1, 2]. Altogether these clinical symptoms points
to profound blood coagulation disorder appearing in patients after bee sting. For a
long time, practically all biological effects of bee venoms were ascribed to its
phospholipase activity, specially regarding the haemostatic abnormalities [3-5]. In
fact, the effect of bee venom phospholipase A2 upon platelet reactivity has been
already described [6-8]. However bee venom is a complex mixture of
pharmacologically and toxicologically active proteins and peptides that could
interfere in platelet function. Recently, the publication of the bee genome gave rise to
the interest to investigate others platelet active proteins such as desintegrins [9].
Melittin, a 2.8 kDa peptide that accounts for about 50% of the venom dry weight [3],
is the most important toxic compound present in Apis mellifera venom with respect to
weight and activity. The cationic 26 amino acid long peptide
(GIGAVLKVLTTGLPALISWIKRKRQQ) is composed by a hydrophobic amino-
terminal portion and a carboxy-terminal end rich in hydrophilic amino acids. As we
have previously demonstrated [10], in solution melittin is found in a random
conformation, a fact that could explain its wide range of biological activities [11, 12].
The hemolytic action of melittin as well as its ability to interact and disrupt natural
and artificial membranes is well described [3, 13, 14]. The peptide is also able to
modulate phospholipases [15-18] and calmodulin being then an important inhibitor of
cell growth [19]. The stimulation of arachidonic acid-derived lipoxygenase
40
metabolites induced by melittin in leukocytes and platelets [20] denotes its
interesting potential as a platelet aggregating agent [6, 8, 21]. However, such aspect
has been neglected or poorly explored so far.
Considering the variability of actions of melittin upon many target proteins and cells,
the importance of a better understanding of both the pharmacological and
toxicological effects produced by bee venoms and furthermore, its impact on the
clinical management of bee envenomation, this work aimed to investigate the effects
of melittin upon platelet morphology and function.
MATERIALS AND METHODS
Materials
Melittin was either obtained from commercial source (Sigma Chemical Co., St. Louis,
MO, USA) or synthesized. Luciferin-luciferase (Chrono-lume) was obtained from
Chrono-log (Havertown, PA, USA); glutaraldehyde 25% EM grade was purchased
from Electron Microscopy Sciences (Hatfield, PA, USA) and von Willebrand factor
(vWF) was a commercial preparation (vWF/fVIII) from Octapharma (Langfeld,
Germany). Verapamil (Sandoz pharmaceuticals, Brazil) and cilostazol (Libbs
pharmaceuticals, Brazil) were obtained as commercial drugs. Indomethacin,
esculetin, naloxone and apyrase grade VII as well as all other chemicals used were
purchased from Sigma (St. Louis, MO, USA).
41
Peptides synthesis and purification
Peptides were synthesized by the Fmoc [N-(9-fluorenyl)methoxycarbonyl]
methodology as previously described [22] using an automated benchtop
simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from
Shimadzu, Tokyo, Japan) and were purified to homogeneity by high-pressure liquid
chromatography on a Vydac C18 analytical column. The molecular mass and
sequence were checked by amino acid analysis, MALDI-TOF (TOFSpec E
instrument from Micromass, Manchester, U.K.), and Edman degradation (PPSQ 23
system from Shimadzu).
Blood collection and platelet preparation
Human venous forearm blood was collected from five healthy volunteers in
anticoagulant ACD (2:10 v/v) or 3.2 % sodium citrate (1:10 v/v). Platelet rich plasma
(PRP) was prepared from blood anticoagulated with sodium citrate, centrifuged at
200 X g thrice for 5 minutes. Washed platelets were obtained from blood collected
on ACD, centrifuged as above and re-centrifuged at 620 X g for 10 min. The platelet
pellet was re-suspended in 1 mL ACD and washed through a modified protocol from
Timmons and Hawinger [23] by gel filtration in a Sepharose 2B column equilibrated
and eluted with Albumin-Tyrode’s buffer, pH 7.4. The final platelet suspension O.D.
(650nm) was adjusted to 0.15 O.D.
42
Platelet aggregation and ATP secretion
Platelet aggregation (PRP and washed platelets) was measured turbidimetrically as
described [24] using either a Lumi-aggregometer (Chrono-log, USA) and/or a
SpectraMax microplate reader (Molecular Devices, USA). Briefly, platelets were
incubated for 2 minutes at 37 °C under stirring and aggregation was induced by
melittin and melittin-derived peptides. ADP (10 µM) and collagen (2 µg/mL) were
used as control inducers.
Adenosine triphosphate (ATP) secretion was determined as described [24], using a
Luciferin-luciferase reagent (Chrono-lume). Luminescence gain was monitored at 37
°C for 5 minutes in the dark.
Scanning electron microscopy (SEM)
SEM sample preparation procedure was modified from Gear [25]. Briefly, PRP
samples were pre-warmed at 37 °C and exposed to ADP, saline or melittin under low
stirring. Platelets were fixed overnight in 2.5% glutaraldehyde in 0.1 M cacodilyc
buffer, pH 7.2. The samples were washed twice for 30 minutes in 0.1 M cacodilyc
buffer and filtered in a 0.4 µm polycarbonate membranes (Millipore, USA). The fixed
cells were sequentially dehydrated for 5 minutes in 30, 50, 70 and 90% (v/v) acetone
and finally treated twice for 10 minutes in 100% acetone. Critical-point drying and
gold coating treatments were performed at the University’s Center of Electron
Microscopy (CEM-UFRGS, Brazil). Specimens were visualized in a JEOL-JSM 6060
scanning electron microscope with automated image digitization and archiving.
43
Platelet aggregation inhibition
Platelet aggregation inhibition assays were performed in PRP as already described
[26]. Indomethacin (100 µM), esculetin (10 µM), verapamil (50 µM), naloxone (0.5
mM), cilostazol (50 µM) and apyrase grade VII (5 units/mL) were used as inhibitors
of platelet aggregation. ADP (5 µM) was used as experiments control. The inhibitors
were incubated with pre-warmed (37 °C) PRP for 5 minutes under low stirring.
Platelet aggregation was induced upon the addition of melittin or ADP.
Platelet Adhesion
A modified platelet adhesion procedure described by Bellavite et al. [27] was used.
Melittin was assayed either as an adhesion protein (Direct Adhesion) or as a
competitive agonist when other proteins were added as an adhesive substrate
(Competitive Adhesion). In the direct adhesion experiment, 96 wells microtiter plates
(Flacon, USA) were coated overnight at 4 °C and then for 1 hour at 37 °C with 10 µg
of melittin or melittin-derived peptides (100 µL aliquots). Immediately before use,
plates were manually washed twice with 0.9 % NaCl solution. Aliquots (100 µL each)
of washed platelets preparation (2.5 X 10
6
cells) were added to the wells and
incubated at 37 °C for 30 minutes. At the end of incubation the wells were washed
twice with 0.9 % NaCl and each one immediately supplemented with 100 µL of 0.1 M
citrated buffer, pH 5.4, containing 5 mM ρ-nitrophenyl phosphate and 0.1% Triton X-
100 in order to measure platelets acid phosphatase activity. The plate was incubated
for one hour at room temperature and the reaction was stopped with 50 µL of 2M
NaOH. The amount of ρ-nitrophenol produced by the reaction was measured at 405
44
nm against a platelet-free blank by using a SpectraMax microplate reader (Molecular
Devices, USA). The number of adherent platelets was calculated on the basis of a
standard curve obtained with known number of lysed platelets. For the competitive
adhesion experiments the plates were coated with 100 µL of diluted human plasma
(1:2 in saline), bovine fibrinogen (2mg/mL), collagen types I and III (2 mg/mL) and
human von Willebrand Factor (20 µg/mL). The experiment then followed the protocol
described above. In these experiments melittin was assayed as a competitive
agonist of known pro-aggregating inducers. In these cases, platelets were treated
with 10 µg of melittin for 10 minutes or immediately before their addition to the
plates.
Statistical analysis
Results were analyzed by ANOVA followed by the Bonferroni test using Instat Graph
Pad software in order to estimate difference between groups. Values of *p<0.05,
**p<0.01 and ***p<0.001 were considered statistically significant.
RESULTS
Melittin capability to induce platelet aggregation was tested in both platelet rich
plasma and washed human platelets. As shown in Figure 1A, the aggregation effect
in PRP was produced in a dose-dependent manner where the platelets shape
change response could be clearly visualized as an initial decrease in the medium
transmittance. Melittin was also able to induce aggregation in washed platelets (not
shown) indicating that its pro-aggregating activity is not dependent or mediated by
45
plasma proteins. ATP secretion was demonstrated by luminescence gain in platelet
rich plasma. The secretion of ATP is related to dense granules content release and
was comparable to the secretion obtained by known platelet-aggregation agonists,
such as ADP and collagen type I (Figure 1B). Electron microscopy techniques
showed (Figure 1C) that the platelet aggregates produced by melittin presents a
morphological pattern similar to the one induced by ADP. It also confirms that at
these concentrations, melittin does not produce lyses of human platelets.
With the aim of identify a possible amino acid sequence responsible for melittin
effects regarding both platelet activation and aggregation, five peptides (Mel-1 to
Mel-5) containing from 5 to 9 amino acid residues and covering the entire melittin
sequence were designed and synthesized (Table I). Platelet pro-aggregating activity
was found only in peptides Mel-1 and Mel-2 (Figure 2A). Even presenting such
activity at a much higher concentration (ca 0.8 mM or about 40-fold) than the full-
length peptide, these results are indicative of a possible role of peptides in melittin-
induced platelet aggregation.
Inhibitory studies of platelet aggregation induced by melittin were performed upon
pre-incubation of PRP with indomethacin, esculetin, verapamil, naloxone, cilostazol
and apyrase. As shown in Figure 3, indomethacin (100 µM) and esculetin (10 µM)
produced a discrete inhibitory effect of melittin-induced platelet aggregation (ca 18
and 15% inhibition, respectively) while a more expressive effect (ca 70% inhibition)
was produced by verapamil (50 µM). Naloxone was able to produce a 30% inhibition
which was comparable to the effect of the phosphodiesterase inhibitor (cilostazol, ca
25%). On the other hand, apyrase (5 units/mL) was unable to block the pro-
aggregating effect of melittin (Figure 3) although, in this concentration, the enzyme
was able to reduce 75% of 5 µM ADP induced aggregation.
46
In order to evaluate the interaction of melittin and its platelet active derived-peptides
Mel-1 and Mel-2 the direct adhesion experiment was conducted. Peptides to be
tested were immobilized in a 96-well plate (100 µg per well) and then incubated with
washed platelets for 30 minutes at 37 °C. The adherent platelets were lysed and acid
phosphatase activity measured in a colorimetric assay. Figure 2B shows that melittin
as well as its sequence related peptides Mel-1 and Mel-2 were able to induce direct
platelet adhesion, being however, the full-length peptide more effective to adhere to
the platelets surface. When testing melittin interference (competitive adhesion
experiment) in platelet adhesion to known adhesive proteins, such as collagen,
fibrinogen and von Willebrand factor, we found that the peptide inhibited platelet
interaction with all these adhesion proteins (Figure 4). There were no statistical
differences between pre-incubation (10 minutes, 37 °C) and no incubation of melittin
with washed platelets before their addition to coated microplates.
DISCUSSION
Accidents involving bee stings can cause several hematological manifestations that
are still not fully understood [1, 2, 5]. In this work we have investigated the platelet-
aggregating activity of melittin, the main toxic component of honey bee venom. Using
a series of different though complementary methodologies we were able to
demonstrate that melittin interacts with human platelets leading to its activation and
subsequent aggregation. This pro-aggregating effect is coherent with the platelet-
aggregating activity of the whole venom, which is also a platelet-aggregating agent
(data not shown), even thought the bee phospholipase posses an inhibitory activity
in the tested concentration range [7]. Thus, it should be concluded that during
47
envenomation the predominant platelet effect of bee venom components can be
attributed to melittin rather than to phospholipase A
2
.
The platelet-aggregating activity of bee venom components has been poorly studied.
In the case of its component melittin a single and preliminary report [21] just mention
such activity but this action of melittin has never been included in any inventory of
venoms components that cause platelet aggregation [6, 8]. Here we demonstrated
that the aggregation induced by melittin is dose-dependent and is followed by the
platelets shape change as observed by the slight decrease in light transmission seen
in Figure 1A and also demonstrated by electron microscopy (Figure 1B). Moreover,
the activation of blood platelets by the peptide was followed by dense granules
release with ATP secretion (Figure 1B) and possibly other granules components
such as ADP, an important platelet-aggregating agonist.
The known amino acid composition of melittin allowed us to design and synthesize
partial-sequence melittin peptides in order to evaluate the possible involvement of
structural motifs of melittin molecule responsible for its unique platelet pro-
aggregating activity. Five peptides were tested and among then only Mel-1
(GIGAVLKV) and Mel-2 (AVLKVLTTG) promoted platelet aggregation (Table I),
although much less potent than the full peptide (Figure 2A). The two designed
peptides with an overlap in the sequence AVLKV, represent the N-terminal portion of
melittin and furthermore, they constitute the first structural helix before the proline
kink in the folded conformation of the entire peptide [10]. Interestingly, this region is
also responsible for the known hemolytic activity of melittin [28]. However, such lytic
effect of melittin upon human platelets was not detected neither using electron
microscopy (Figure 1C) nor in the release of lactic acid dehydrogenase experiments
(data not shown). Moreover, large platelets aggregates could be easily visualized in
48
a naked eye inspection when platelets were stimulated with melittin, thus confirming
the absence of a lytic action of the peptide in this case.
Considering the pro-aggregating effect and the release of dense granules content,
an inhibitory assay was designed in order to elucidate the mechanism by which
melittin activates blood platelets. It was already reported that melittin has a
modulatory effect over phospholipases and lipoxygenases that are essential to
arachidonic acid pathway, a key signaling route in platelets [20]. Intracellular
phospholipases A
2
and C catalyze the production of arachidonic acid from platelet
membrane phospholipids [29]. Arachidonic acid is a substrate for lipoxygenases and
cyclooxygenases and their activity, together with other intracellular enzymes,
produces thromboxane A
2
(TXA
2
) [29]. This pathway enhances the aggregating
signal since TXA
2
is also an agonist that can liberate intracellular calcium thus
amplifying platelet aggregation [29]. It seems that melittin is able to modulate more
than one enzyme in this cascade. This let us to use several platelet inhibitors in
order to investigate melittin’s pro-aggregating effects: indomethacin (a
cyclooxygenase inhibitor), esculetin (a lipoxygenase inhibitor), verapamil (a voltage-
dependent calcium-gated channel blocker) and naloxone (an intracellular calcium
mobilization inhibitor) to investigate the role of the peptide in this signaling pathway.
All four platelet-aggregation inhibitors were effective in blocking melittin signaling
although calcium intracellular mobilization and influx seems to be pivotal in this
process (Figure 3). The role of phosphodiesterase in melittin-induced aggregation
was also evaluated by the incubation of platelets with cilostazol and it seems that
melittin could also activate this enzyme during the signaling route. However, despite
melittin’s ability to induce cellular secretion of dense granules contents, apyrase (an
ADP hydrolase), by itself was ineffective in inhibiting platelet aggregation induced by
49
the peptide (Figure 3). These data could be understood as ATP and ADP release
being not the most essential event in platelet activation produced by melittin.
Moreover, melittin as well as mastoparan – a toxic peptide from wasp venom - was
already described as an important G-protein modulator [30, 31]. This remarkable
activity is of special concern once many platelet receptors are G-coupled proteins.
ATP P2Y
1
and ADP P2Y
12
receptors are both G-coupled-proteins [32]. Then is
plausible to speculate that melittin could interact directly with platelets receptors
generating a pro-aggregating effect or even mimic the G-coupled receptors
promoting the cell signaling response like mastoparan seems to do [33].
The direct interaction of melittin with human platelets was demonstrated in an
adhesion assay. The adhesion occurs directly to melittin as well as to the platelet-
active melittin-fragments Mel-1 and Mel-2, reflecting the affinity of the peptides for
the platelets surface (Figure 2B). Our data also reveled that both melittin (Figure 4)
and Mel-1 and Mel-2 (data not shown) could inhibit the adhesion of platelets to
surfaces coated with plasma, fibrinogen, vWF and collagens I and III. Altogether,
these findings indicate a direct interaction of melittin to platelet surface, an action
that corroborate to our hypothesis of an unspecific association of melittin to platelet
surface proteins and receptors.
In conclusion, our results show that melittin is able to aggregate human platelets in a
dose-dependent manner, releasing dense granules contents. Moreover, we were
able to identify the amino terminal portion of the peptide as partially responsible for
the observed activity. In addition we demonstrated a direct adhesion of the peptide to
the cell surfaces. Here we could also explore some of the platelet signaling pathways
involved in melittin platelet activation such as the arachidonic acid activating
cascade. This study contributes to the actual knowledge of the biological activities of
50
melittin and could give insights to the better understanding of the haemostatic
abnormalities during bee envenomations and its clinical management.
ACKNOWLEDGEMENTS
The authors would like to thank Lucélia Santi and Walter O. B. da Silva from the
Center of Biotechnology - UFRGS for the valuable help with SEM technique. We
also thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES-MEC), Brazil and Conselho Nacional de Desenvolvimento Científico and
Tecnológico (CNPq-MCT), Brazil for fellowships and financial support.
51
FIGURE LEGENDS
Table I – Peptides based on the melittin sequence. The full sequence of melittin is
shown together with the designed peptides (Mel-1 to Mel-5) derived from its full
length sequence.
Figure 1 – Melittin induced platelet aggregation and ATP secretion. A. Dose-
response curve of melittin induced aggregation of human platelets. Results (mean ±
SD) are shown as percentage of maximal aggregation (considering ADP 10 µM as
100%) for four replicates measured in a microplate reader. Shape change and profile
of the aggregation registered in an aggregometer are shown in the inset. B. Melittin
induced ATP secretion was detected by an increase in luminescence. PRP (270 µL)
was incubated with 30 µL of luciferin-luciferase reagent (Chrono-log) for 2 minutes at
37 °C in the dark. Platelet aggregation was induced by 23 µM melittin or ADP (10
µM) and collagen type I (2 µg/mL) as control. Luminescence was monitored for 5
minutes. C. Surface morphology of human platelets by scanning electron microscopy
(SEM). Control (saline), 10 µM ADP and 23 µM melittin stimulated platelets are
shown in panels, white bar represents 2 µm.
Figure 2 – Effects of melittin and melittin-derived peptides upon platelet aggregation
and adhesion. A. Platelet aggregation induced by melittin, Mel-1 (GIGAVLKV) and
Mel-2 (AVLKVLTTG) peptides was recorded in a microplate reader. Results (mean ±
SD) are expressed in percentage of aggregation, considering 10 µM ADP as 100%
of aggregation, for four replicates. B. Direct adhesion was measure as number of
platelets adhered to immobilized 10 µg melittin, 10 µg Mel-1 or 10 µg Mel-2 by an
acid phosphatase colorimetric assay. Control is represented as the number of
52
platelet adhered to 20 µg bovine albumin. Results are shown as mean ± SD for
triplicates, values of *p<0.05 and **p<0.01 were considered statistically significant.
Figure 3 – Effect of platelet aggregation inhibitors. PRP was incubated with
inhibitors for 5 minutes under low stirring and platelet aggregation was stimulated
with 23 µM melittin (dark grey) or 5 µM ADP (light grey - as a standard platelet-
aggregating agent). Results (mean ± SD) are expressed as percentage of
aggregation (5 µM ADP and 23 µM melittin as 100% of aggregation) for four
replicates. Values of *p<0.05, **p<0.01 and ***p<0.001 were considered statistically
significant.
Figure 4 - Competitive adhesion assay. Interference of melittin in platelet adhesion
to immobilized adhesive proteins. Microplates were coated with 100 µL of human
plasma (1:2 in saline), collagens I and III (20 mg/mL), bovine fibrinogen (2 mg/mL)
and von Willebrand factor (vWF) (20 µg/mL). Immediately before the addition of
human washed platelets to the microplates, 10 µg of melittin was added. After 30
minutes at 37 °C, platelets were lysed and the adhesion was measure by a
colorimetric assay of acid phosphatase activity. Results (mean± SD) are expressed
in number of platelets for triplicates, values of *p<0.05, **p<0.01 and ***p<0.001
were considered statistically significant. Control (ADP 10 µM) is represented in light
grey bars and melittin treatment is represented in dark grey bars.
53
Table I
54
Figure 1
55
Figure 2
56
Figure 3
57
Figure 4
58
BIBLIOGRAPHY
[1] P. Kolecki, Delayed toxic reaction following massive bee envenomation, Ann.
Emerg. Med. 33 (1999) 114-116.
[2] R. Gawlik, B. Rymarczyk, B. Rogala, A rare case of intravascular coagulation
after honey bee sting, J. Investig. Allergol. Clin. Immunol. 14 (2004) 250-252.
[3] E. Habermann, Bee and wasp venoms, Science 177 (1972) 314-322.
[4] G. Petroianu, J. Liu, U. Helfrich, W. Maleck, R. Rufer, Phospholipase A2-induced
coagulation abnormalities after bee sting, Am. J. Emerg. Med. 18 (2000) 22-27.
[5] F.O. Franca, L.A. Benvenuti, H.W. Fan, D.R. Dos Santos, S.H. Hain, F.R. Picchi-
Martins, J.L. Cardoso, A.S. Kamiguti, R.D. Theakston, D.A. Warrell, Severe and fatal
mass attacks by 'killer' bees (Africanized honey bees--Apis mellifera scutellata) in
Brazil: clinicopathological studies with measurement of serum venom concentrations,
Q. J. Med. 87 (1994) 269-282.
[6] G. Chow, R.M. Kini, Exogenous factors from animal sources that induce platelet
aggregation, Thromb. Haemost. 85 (2001) 177-178.
[7] C. Ouyang, T.F. Huang, Effect of the purified phospholipases A2 from snake and
bee venoms on rabbit platelet function, Toxicon 22 (1984) 705-718.
[8] S.V. Smith, K.M. Brinkhous, Inventory of exogenous platelet-aggregating agent
derived from venoms, Thromb. Haemost. 66 (1991) 259-263.
[9] H.G.S. Consortium, Insights into social insects from the genome of the honeybee
Apis mellifera, Nature 443 (2006) 931-949.
[10] R.M.S. Terra, J.A. Guimaraes, H. Verli, Structural and functional behavior of
biologically active monomeric melittin, J. Mol. Graph. Model. 25 (2007) 767-772.
59
[11] F. Wang, P.L. Polavarapu, Conformational analysis of melittin in solution phase:
vibrational circular dichroism study, Biopolymers 70 (2003) 614-619.
[12] I. Constantinescu, M. Lafleur, Influence of the lipid composition on the kinetics of
concerted insertion and folding of melittin in bilayers, Biochim. Biophys. Acta 1667
(2004) 26-37.
[13] C.E. Dempsey, The actions of melittin on membranes, Biochim. Biophys. Acta
1031 (1990) 143-161.
[14] M.T. Tosteson, S.J. Holmes, M. Razin, D.C. Tosteson, Melittin lysis of red cells,
J. Membr. Biol. 87 (1985) 35-44.
[15] I. Mingarro, E. Perez-Paya, C. Pinilla, J.R. Appel, R.A. Houghten, S.E.
Blondelle, Activation of bee venom phospholipase A2 through a peptide-enzyme
complex, FEBS Lett. 372 (1995) 131-134.
[16] S.S. Saini, J.W. Peterson, A.K. Chopra, Melittin binds to secretory
phospholipase A2 and inhibits its enzymatic activity, Biochem. Biophys. Res.
Commun. 238 (1997) 436-442.
[17] S.S. Saini, A.K. Chopra, J.W. Peterson, Melittin activates endogenous
phospholipase D during cytolysis of human monocytic leukemia cells, Toxicon 37
(1999) 1605-1619.
[18] K. Koumanov, A. Momchilova, C. Wolf, Bimodal regulatory effect of melittin and
phospholipase A2-activating protein on human type II secretory phospholipase A2,
Cell Biol. Int. 27 (2003) 871-877.
[19] W.N. Hait, L. Grais, C. Benz, E.C. Cadman, Inhibition of growth of leukemic cells
by inhibitors of calmodulin: phenothiazines and melittin, Cancer Chemother.
Pharmacol. 14 (1985) 202-205.
60
[20] H. Salari, P. Braquet, P. Borgeat, Stimulation of lipoxygenase product synthesis
in human leukocytes and platelets by melittin, Mol. Pharmacol. 28 (1985) 546-548.
[21] W.J. Shi, H.J. Xu, J.A. Cheng, C.X. Zhang, Expression of the melittin gene of
Apis cerana cerana in Escherichia coli, Protein Expr. Purif. 37 (2004) 213-219.
[22] I.Y. Hirata, M.H.S. Cezari, C.R. Nakaie, P. Boschov, A.S. Ito, M.A. Juliano, L.
Juliano, Internally quenched fluorogenic protease substrates: Solid-phase synthesis
and fluorescence spectroscopy of peptides containing ortho-
aminobenzoyl/dinitrophenyl groups as donor-acceptor Lett. Pept. Sci. 1 (1995) 299-
308.
[23] S. Timmons, J. Hawiger, Isolation of human platelets by albumin gradient and
gel filtration, Methods Enzymol. 169 (1989) 11-21.
[24] A.L. Fuly, A.M. Soares, S. Marcussi, J.R. Giglio, J.A. Guimaraes, Signal
transduction pathways involved in the platelet aggregation induced by a D-49
phospholipase A2 isolated from Bothrops jararacussu snake venom, Biochimie 86
(2004) 731-739.
[25] A.R. Gear, Rapid platelet morphological changes visualized by scanning-
electron microscopy: kinetics derived from a quenched-flow approach, Br. J.
Haematol. 56 (1984) 387-398.
[26] D. Olivera-Severo, G.E. Wassermann, C.R. Carlini, Bacillus pasteurii urease
shares with plant ureases the ability to induce aggregation of blood platelets, Arch.
Biochem. Biophys. 452 (2006) 149-155.
[27] P. Bellavite, G. Andrioli, P. Guzzo, P. Arigliano, S. Chirumbolo, F. Manzato, C.
Santonastaso, A colorimetric method for the measurement of platelet adhesion in
microtiter plates, Anal. Biochem. 216 (1994) 444-450.
61
[28] C. Subbalakshmi, R. Nagaraj, N. Sitaram, Biological activities of C-terminal 15-
residue synthetic fragment of melittin: design of an analog with improved
antibacterial activity, FEBS Lett. 448 (1999) 62-66.
[29] P. Clutton, J.D. Folts, J.E. Freedman, Pharmacological control of platelet
function, Pharmacol. Res. 44 (2001) 255-264.
[30] N. Fukushima, M. Kohno, T. Kato, S. Kawamoto, K. Okuda, Y. Misu, H. Ueda,
Melittin, a metabostatic peptide inhibiting G(s) activity, Peptides 19 (1998) 811-819.
[31] T. Higashijima, J. Burnier, E.M. Ross, Regulation of Gi and Go by mastoparan,
related amphiphilic peptides, and hydrophobic amines. Mechanism and structural
determinants of activity, J. Biol. Chem. 265 (1990) 14176-14186.
[32] C. Oury, E. Toth-Zsamboki, J. Vermylen, M.F. Hoylaerts, The platelet ATP and
ADP receptors, Curr. Pharm. Des. 12 (2006) 859-875.
[33] T. Higashijima, S. Uzu, T. Nakajima, E.M. Ross, Mastoparan, a peptide toxin
from wasp venom, mimics receptors by activating GTP-binding regulatory proteins
(G proteins), J. Biol. Chem. 263 (1988) 6491-6494.
62
Conclusões
Os resultados obtidos no desenvolvimento deste trabalho e aqui
apresentados demonstram a complexidade conformacional e funcional do peptídeo
melitina do veneno da abelha Apis mellifera. Os dados aqui descritos evidenciam
que:
1. A estrutura terciária do peptídeo é altamente influenciada pelo meio;
2. A melitina em condições fisiológicas de pH e força iônica apresenta-se
monomérica uma vez que as estruturas oligoméricas só são mantidas em
elevado pH;
3. A presença de solvente orgânico (metanol) é capaz de desestabilizar a
estrutura em hélice da melitina;
4. A conformação cristalográfica, em hélice, pode ser mantida apenas em
condições elevadas de pH e força iônica, não sendo influenciada pelo caráter
do íon;
5. A estrutura cristalográfica (PDB 2MLT), antes proposta como biologicamente
ativa, não tem relevância biológica, uma vez demonstrado aqui que o
peptídeo é randômico em condições fisiológicas de pH e íons;
6. O enovelamento da proteína é dependente de condições ambientais
específicas;
63
7. A melitina é capaz de provocar a agregação plaquetária sem lise celular;
8. O peptídeo provoca secreção de grânulos densos das plaquetas, com
liberação de ATP;
9. A porção amino-terminal da melitina (GIGAVLKVLTTG) é a responsável pela
atividade biológica aqui estudada;
10. A melitina é capaz de interagir diretamente com a superfície das plaquetas
assim como seus peptídeos relacionados (Mel-1 e Mel-2) aqui avaliados;
11. Ocorre inibição da adesão das plaquetas a algumas proteínas, indicando
uma interação inespecífica com a superfície plaquetária;
12. A modulação de diversas proteínas intracelulares, assim como a interação
inespecífica parecem ser responsáveis pela ativação plaquetária.
Os resultados aqui apresentados mostram o quão importante é o estudo conjunto
de estrutura e função.
A falta de estrutura definida da melitina em condições fisiológicas e seu
enovelamento sob diferentes condições ambientais ajudam-nos a entender a
diversidade de atividades biológicas. Este entendimento é importante na avaliação e
conduta clínica em casos de envenenamento. Os acidentes causados por ataques
de abelhas hoje são considerados um problema de saúde pública a ser manejado e
a urbanização das abelhas africanizadas tem aumentado o número de acidentes. A
melitina, sendo a principal toxina do veneno, tem um papel essencial em diversas
manifestações bioquímicas e patológicas. Neste trabalho foi possível descrever seus
efeitos sobre a função plaquetária e contribuir para o conhecimento das
coagulopatias causadas pelo envenenamento. Porém, há ainda muito a ser
entendido a respeito da fisiopatologia do envenenamento e do impacto de cada um
de seus componentes na evolução clínica.
64
Referências Bibliográficas
Asthana, N., Yadav, S.P. e Ghosh, J.K. (2004) Dissection of antibacterial and toxic
activity of melittin: a leucine zipper motif plays a crucial role in determining its
hemolytic activity but not antibacterial activity. J Biol Chem, 279, 55042-55050.
Baker, K.J., East, J.M. e Lee, A.G. (1995) Mechanism of inhibition of the Ca(2+)-
ATPase by melittin. Biochemistry, 34, 3596-3604.
Bello, J., Bello, H.R. e Granados, E. (1982) Conformation and aggregation of melittin:
dependence on pH and concentration. Biochemistry, 21, 461-465.
Blostein, M.D., Rigby, A.C., Furie, B.C., Furie, B. e Gilbert, G.E. (2000) Amphipathic
helices support function of blood coagulation factor IXa. Biochemistry, 39,
12000-12006.
Buku, A. (1999) Mast cell degranulating (MCD) peptide: a prototypic peptide in
allergy and inflammation. Peptides, 20, 415-420.
Chow, G. e Kini, R.M. (2001) Exogenous factors from animal sources that induce
platelet aggregation. Thromb Haemost, 85, 177-178.
Dempsey, C.E. (1990) The actions of melittin on membranes. Biochim Biophys Acta,
1031, 143-161.
Ewan, P.W. (1998) Venom allergy. Bmj, 316, 1365-1368.
Franca, F.O., Benvenuti, L.A., Fan, H.W., Dos Santos, D.R., Hain, S.H., Picchi-
Martins, F.R., Cardoso, J.L., Kamiguti, A.S., Theakston, R.D. e Warrell, D.A.
(1994) Severe and fatal mass attacks by 'killer' bees (Africanized honey bees--
65
Apis mellifera scutellata) in Brazil: clinicopathological studies with
measurement of serum venom concentrations. Q J Med, 87, 269-282.
Fukushima, N., Kohno, M., Kato, T., Kawamoto, S., Okuda, K., Misu, Y. e Ueda, H.
(1998) Melittin, a metabostatic peptide inhibiting G(s) activity. Peptides, 19,
811-819.
Garcia, A. (2006) Proteome analysis of signaling cascades in human platelets. Blood
Cells Mol Dis, 36, 152-156.
Gawlik, R., Rymarczyk, B. e Rogala, B. (2004) A rare case of intravascular
coagulation after honey bee sting. J Investig Allergol Clin Immunol, 14, 250-
252.
Glattli, A., Chandrasekhar, I. e Gunsteren, W.F. (2006) A molecular dynamics study
of the bee venom melittin in aqueous solution, in methanol, and inserted in a
phospholipid bilayer. Eur Biophys J, 35, 255-267.
Grisotto, L.S., Mendes, G.E., Castro, I., Baptista, M.A., Alves, V.A., Yu, L. e
Burdmann, E.A. (2006) Mechanisms of bee venom-induced acute renal failure.
Toxicon, 48, 44-54.
Habermann, E. (1972) Bee and wasp venoms. Science, 177, 314-322.
Hait, W.N., Grais, L., Benz, C. e Cadman, E.C. (1985) Inhibition of growth of
leukemic cells by inhibitors of calmodulin: phenothiazines and melittin. Cancer
Chemother Pharmacol, 14, 202-205.
Higashijima, T., Burnier, J. e Ross, E.M. (1990) Regulation of Gi and Go by
mastoparan, related amphiphilic peptides, and hydrophobic amines.
Mechanism and structural determinants of activity. J Biol Chem, 265, 14176-
14186.
Higashijima, T., Uzu, S., Nakajima, T. e Ross, E.M. (1988) Mastoparan, a peptide
toxin from wasp venom, mimics receptors by activating GTP-binding
regulatory proteins (G proteins). J Biol Chem, 263, 6491-6494.
Jones, R.G., Corteling, R.L., Bhogal, G. e Landon, J. (1999) A novel Fab-based
antivenom for the treatment of mass bee attacks. Am J Trop Med Hyg, 61,
361-366.
66
Kaetzel, M.A. e Dedman, J.R. (1987) Affinity-purified melittin antibody recognizes the
calmodulin-binding domain on calmodulin target proteins. J Biol Chem, 262,
3726-3729.
Karplus, M. (2002) Molecular dynamics simulation of biomolecules. Accounts of
Chemical Research, 35, 321-323.
Karplus, M. e McCammon, J.A. (2002) Molecular dynamics simulations of
biomolecules. Natrure Structural Biology, 9, 649-652.
Kataoka, M., Head, J.F., Seaton, B.A. e Engelman, D.M. (1989) Melittin binding
causes a large calcium-dependent conformational change in calmodulin. Proc
Natl Acad Sci U S A, 86, 6944-6948.
Kolecki, P. (1999) Delayed toxic reaction following massive bee envenomation. Ann
Emerg Med, 33, 114-116.
Koumanov, K., Momchilova, A. e Wolf, C. (2003) Bimodal regulatory effect of melittin
and phospholipase A2-activating protein on human type II secretory
phospholipase A2. Cell Biol Int, 27, 871-877.
Lam, Y.H., Wassall, S.R., Morton, C.J., Smith, R. e Separovic, F. (2001) Solid-state
NMR structure determination of melittin in a lipid environment. Biophys J, 81,
2752-2761.
Lin, J.H. e Baumgaertner, A. (2000) Stability of a melittin pore in a lipid bilayer: a
molecular dynamics study. Biophys J, 78, 1714-1724.
Markovic-Housley, Z., Miglierini, G., Soldatova, L., Rizkallah, P.J., Muller, U. e
Schirmer, T. (2000) Crystal structure of hyaluronidase, a major allergen of bee
venom. Structure, 8, 1025-1035.
McCammon, J.A., Gelin, B.R. e Karplus, M. (1977) Dynamics of folded proteins.
Nature, 267, 585-590.
Metz, M., Piliponsky, A.M., Chen, C.C., Lammel, V., Abrink, M., Pejler, G., Tsai, M. e
Galli, S.J. (2006) Mast cells can enhance resistance to snake and honeybee
venoms. Science, 313, 526-530.
Mingarro, I., Perez-Paya, E., Pinilla, C., Appel, J.R., Houghten, R.A. e Blondelle, S.E.
(1995) Activation of bee venom phospholipase A2 through a peptide-enzyme
complex. FEBS Lett, 372, 131-134.
67
Nicolas, J.P., Lin, Y., Lambeau, G., Ghomashchi, F., Lazdunski, M. e Gelb, M.H.
(1997) Localization of structural elements of bee venom phospholipase A2
involved in N-type receptor binding and neurotoxicity. J Biol Chem, 272, 7173-
7181.
Nieswandt, B., Aktas, B., Moers, A. e Sachs, U.J. (2005) Platelets in
atherothrombosis: lessons from mouse models. J Thromb Haemost, 3, 1725-
1736.
Okamoto, T., Isoda, H., Kubota, N., Takahata, K., Takahashi, T., Kishi, T., Nakamura,
T.Y., Muromachi, Y., Matsui, Y. e Goshima, K. (1995) Melittin cardiotoxicity in
cultured mouse cardiac myocytes and its correlation with calcium overload.
Toxicol Appl Pharmacol, 133, 150-163.
Ouyang, C. e Huang, T.F. (1984) Effect of the purified phospholipases A2 from snake
and bee venoms on rabbit platelet function. Toxicon, 22, 705-718.
Ownby, C.L., Powell, J.R., Jiang, M.S. e Fletcher, J.E. (1997) Melittin and
phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine
skeletal muscle in vivo. Toxicon, 35, 67-80.
Peiren, N., Vanrobaeys, F., de Graaf, D.C., Devreese, B., Van Beeumen, J. e
Jacobs, F.J. (2005) The protein composition of honeybee venom reconsidered
by a proteomic approach. Biochim Biophys Acta, 1752, 1-5.
Petroianu, G., Liu, J., Helfrich, U., Maleck, W. e Rufer, R. (2000) Phospholipase A2-
induced coagulation abnormalities after bee sting. Am J Emerg Med, 18, 22-
27.
Qiu, W., Zhang, L., Kao, Y.T., Lu, W., Li, T., Kim, J., Sollenberger, G.M., Wang, L. e
Zhong, D. (2005) Ultrafast hydration dynamics in melittin folding and
aggregation: helix formation and tetramer self-assembly. J Phys Chem B
Condens Matter Mater Surf Interfaces Biophys, 109, 16901-16910.
Raghuraman, H. e Chattopadhyay, A. (2005) Cholesterol inhibits the lytic activity of
melittin in erythrocytes. Chem Phys Lipids, 134, 183-189.
Rand, M.L., Leung, R. e Packham, M.A. (2003) Platelet function assays. Transfusion
and Apheresis Science, 28, 307-317.
Rivera, J. (2006) Snake bites and bee stings: the mast cell strikes back. Nat Med, 12,
999-1000.
68
Saini, S.S., Chopra, A.K. e Peterson, J.W. (1999) Melittin activates endogenous
phospholipase D during cytolysis of human monocytic leukemia cells. Toxicon,
37, 1605-1619.
Salari, H., Braquet, P. e Borgeat, P. (1985) Stimulation of lipoxygenase product
synthesis in human leukocytes and platelets by melittin. Mol Pharmacol, 28,
546-548.
Scaloni, A., Miraglia, N., Orru, S., Amodeo, P., Motta, A., Marino, G. e Pucci, P.
(1998) Topology of the calmodulin-melittin complex. J Mol Biol, 277, 945-958.
Sengupta, D., Meinhold, L., Langosch, D., Ullmann, G.M. e Smith, J.C. (2005)
Understanding the energetics of helical peptide orientation in membranes.
Proteins, 58, 913-922.
Sherman, R.A. (1995) What physicians should know about Africanized honeybees.
West J Med, 163, 541-546.
Shorina, E.A., Dolgova, N.V., Rubtsov, A.M., Storey, K.B. e Lopina, O.D. (2004)
Melittin induces both time-dependent aggregation and inhibition of Na,K-
ATPase from duck salt glands however these two processes appear to occur
independently. Biochimica Et Biophysica Acta-Biomembranes, 1661, 188-195.
Smith, S.V. e Brinkhous, K.M. (1991) Inventory of exogenous platelet-aggregating
agent derived from venoms. Thromb Haemost, 66, 259-263.
Steen, C.J., Janniger, C.K., Schutzer, S.E. e Schwartz, R.A. (2005) Insect sting
reactions to bees, wasps, and ants. Int J Dermatol, 44, 91-94.
Terwilliger, T.C. e Eisenberg, D. (1982) The structure of melittin. I. Structure
determination and partial refinement. J Biol Chem, 257, 6010-6015.
Tosteson, M.T., Holmes, S.J., Razin, M. e Tosteson, D.C. (1985) Melittin lysis of red
cells. J Membr Biol, 87, 35-44.
van der Staay, F.J., Fanelli, R.J., Blokland, A. e Schmidt, B.H. (1999) Behavioral
effects of apamin, a selective inhibitor of the SK(Ca)-channel, in mice and rats.
Neurosci Biobehav Rev, 23, 1087-1110.
Vetter, R.S., Visscher, P.K. e Camazine, S. (1999) Mass envenomations by honey
bees and wasps. West J Med, 170, 223-227.
69
Voss, J.C., Mahaney, J.E. e Thomas, D.D. (1995) Mechanism of Ca-ATPase
inhibition by melittin in skeletal sarcoplasmic reticulum. Biochemistry, 34, 930-
939.
Wang, F. e Polavarapu, P.L. (2003) Conformational analysis of melittin in solution
phase: vibrational circular dichroism study. Biopolymers, 70, 614-619.
Willoughby, S., Holmes, A. e Loscalzo, J. (2002) Platelets and cardiovascular
disease. Eur J Cardiovasc Nurs, 1, 273-288.
Winningham, K.M., Fitch, C.D., Schmidt, M. e Hoffman, D.R. (2004) Hymenoptera
venom protease allergens. J Allergy Clin Immunol, 114, 928-933.
Yang, L., Harroun, T.A., Weiss, T.M., Ding, L. e Huang, H.W. (2001) Barrel-stave
model or toroidal model? A case study on melittin pores. Biophys J, 81, 1475-
1485.
Zucker, M.B. e Nachmias, V.T. (1985) Platelet activation. Arteriosclerosis, 5, 2-18.
70
Curriculum vitae – Renata Maria Soares Terra
71
CURRICULUM VITAE
RESUMIDO
Outubro, 2006
DADOS PESSOAIS
Nome: Renata Maria Soares Terra
Filiação: Lafaiete Oliveira Terra e Nadir Soares Terra
Nascimento: 23/10/1981, Porto Alegre/RS - Brasil
Endereço profissional: Universidade Federal do Rio Grande do Sul, Centro de
Biotecnologia, Laboratório de
Bioquímica Farmacológica.
Av. Bento Gonçalves, 9500 - Prédio 43431- Laboratório 214 -
Campos do Vale
Agronomia
91501970 Porto Alegre, RS - Brasil
Telefone: (51) 33166062
Endereço residencial: Avenida Protásio Alves, 7157 apto 401 bl 4
Alto Petrópolis
91310003 Porto Alegre, RS - Brasil
Telefone: (51) 33813710
TÍTULOS ACADÊMICOS
2006 Doutorado em Biologia Celular e Molecular.
Universidade Federal do Rio Grande do Sul, UFRGS, Rio Grande do
Sul,Brasil.
Título: CARACTERIZAÇÃO DE ENZIMAS ENVOLVIDAS NO
METABOLISMO DE NUCLEOTÍDEOS E SEU PAPEL NO
ENVENENAMENTO OFÍDICO
Orientador: Jorge Almeida Guimarães.
Bolsista do(a): Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior, CAPES, Brasil
2005 Mestrado em Biologia Celular e Molecular.
Universidade Federal do Rio Grande do Sul, UFRGS, Rio Grande do Sul,
Brasil.
Título: ANÁLISE CONFORMACIONAL DA MELITINA POR DINÂMICA
MOLECULAR E CARACTERIZAÇÃO DOS EFEITOS DO PEPTÍDEO NA
FUNÇÃO PLAQUETÁRIA
Orientador: Jorge Almeida Guimarães.
Bolsista do(a): Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior, CAPES, Brasil.
2000 - 2004 Graduação em Farmácia / Farmácia Industrial.
Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS, Rio Grande
do Sul, Brasil.
Título: UTILIZAÇÃO DA TÉCNICA DE ESPECTROSCOPIA DE
INFRAVERMELHO (FTIR-ATR) NO ESTUDO DE MECANISMOS DE AÇÃO
DE FÁRMACOS ANTIMICROBIANOS.
72
Orientador: Mercedes Passos Geimba.
Bolsista do(a): Conselho Nacional de Desenvolvimento Científico e
Tecnológico, CNPQ, Brasil.
ATIVIDADES CIENTÍFICAS E TÉCNICAS
Publicações em Periódicos
1 TERRA, Renata Maria Soares; GUIMARÃES, Jorge Almeida; VERLI, Hugo. Structural
and functional behavior of biologically active monomeric melittin. Journal of molecular
graphics & modelling, in press, 2006
2 PINTO, Antônio Frederico Michel;TERRA, Renata Maria Soares; GUIMARÃES, Jorge
Almeida; FOX, Jay William. Mapping von Willebrand factor A domain binding sites on a
snake venom metalloproteinase cystein-rich domain. Archives of biochemistry and
biophysics, aceito para publicação, 2006.
3 PINTO, Antônio Frederico Michel; TERRA, Renata Maria Soares; GUIMARÃES, Jorge
Almeida; KASHIWAGI, Masahide; NAGASE, Hideaki; SERRANO, Solange Maria de
Toledo; FOX, Jay William. Structural features of the reprolysin atrolysin C and tissue
inhibitors of metalloproteinases (TIMPs) interaction. Biochemical and Biophysical
Research Communications, v. 347, p. 641-648, 2006.
4 BIZZANI, Delmar; MOTTA, Amanda S; MORRISSY, Juliana Alonso Campos; TERRA,
Renata Maria Soares; SOUTO, André Arigony; BRANDELLI, Adriano. Antibacterial
activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus. International
microbiology, v. 8, p. 125-131, 2005.
Trabalhos apresentados em Congressos e Simpósios
1 RECK JR., José; BERGER, Markus; TERRA, Renata Maria Soares Terra; MARKS,
Fernanda; DA SILVA VAZ JR., Itabajara; GUIMARÃES, Jorge Almeida; TERMIGNONI,
Carlos.Avaliação de parâmetros Hemostáticos de bovinos infestados pelo carrapato
Rhipicephalus (Boophilus) Microplus. In: CONGRESSO BRASILEIRO DE
PARASITOLOGIA VETERINÁRIA, 2006, Ribeirão Preto. 2006
2 TERRA, Renata Maria Soares; KOBE, Simone de Oliveira; GUIMARÃES, Jorge Almeida.
Evaluation of melittin effect over hemostasis. In: REUNIÃO ANUAL DA SOCIEDADE
BRASILEIRA DE BIOQUÍMICA, 2006, Águas de Lindóia. 2006.
3 PINTO, Antônio Frederico Michel; TERRA, Renata Maria Soares; NAGASE, Hideaki;
SERRANO, Solange Maria de Toledo; GUIMARÃES, Jorge Almeida; FOX, Jay William.
Insight into the molecular interaction of snake venom metalloproteinase, atrolysin C, and
tissue inhibitors of metalloproteinases. In: REUNIÃO ANUAL DA SOCIEDADE
BRASILEIRA DE BIOQUÍMICA,2006, Águas de Lindóia. 2006.
4 TERRA, Renata Maria Soares; VERLI, Hugo; GUIMARÃES, Jorge Almeida. Evaluation of
Melittin Structure, Conformation and Oligomerization in Solution Using Molecular Dynamics
Simulation. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE BIOQUÍMICA, 2005,
Águas de Lindóia. 2005.
5 JAGER, Alessandro; TERRA, Renata Maria Soares; SOUTO, André Arigony. Altas
concentrações de trans-resveratrol em sucos de uva ecológicos. In: 26ª REUNIÃO ANUAL
DA SOCIEDADE BRASILEIRA DE QUÍMICA, 2003, Poços de Caldas. 2003.
73
Participação em Congressos e Reuniões
1 Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq).
2006. (Participação em eventos/Congresso).
2 Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq).
2005. (Participação em eventos/Congresso).
3 III Jornada de Terapia Nutricional do HCPA. 2004. (Participação em eventos/Outra).
4 The 2nd Brazilian Symposium on Medicinal Chemistry: Current Trends in Drug Discovery
and Development. 2004. (Participação em eventos/Simpósio).
5 X Escola de Verão em Química Farmacêutica e Química Medicinal. 2004. (Participação
em eventos/Outra).
6 2ª Semana de Antibióticos do Hospital de Clínicas de Porto Alegre. 2003. (Participação
em eventos/Seminário).
7 26ª Reunião Anual da Sociedade Brasileira de Química. 2003. (Participação em
eventos/Congresso).
8 Encontro de Farmácia Hospitalar AFARGS/SBRAFH-RS. 2003. (Participação em
eventos/Encontro).
9 6º Congresso de Produtos Farmacêuticos e Cosméticos do Rio Grande do Sul. 2002.
(Participação em eventos/Congresso).
Participação em outros eventos
Cursos de extensão
2004 - 2004 História da Descoberta de Fármacos. (Carga horária: 12h)
Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil.
2004 - 2004 Relações Quantitativas 3D Estrutura-Atividade. (Carga horária: 12h)
Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil.
2004 - 2004 Síntese em Fase Sólida Aplicada ao Desenho de Fármacos. (Carga horária:
12h) Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil.
2004 - 2004 Gestão em Projetos de Pesquisa, Desenv. e Inovação. (Carga horária: 12h)
Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil.
2004 - 2004 Extensão universitária em Bioinformática e Modelagem Molecular. (Carga
horária: 40h)
Associação Brasileira de Química, ABQ-SUL, Rio Grande do Sul, Brasil.
2004 - 2004 Extensão universitária em Escola de Inverno Em Química Orgânica. (Carga
horária: 30h) Universidade Federal do Rio Grande do Sul, UFRGS, Rio
Grande do Sul, Brasil.
2004 - 2004 Extensão universitária em Delineamentos Experimentais em Biologia. Carga
horária: 24h) Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS,
Rio Grande do Sul, Brasil.
74
2003 - 2003 Atuação do Farmacêutico na Pesquisa Clínica. (Carga horária: 4h)
Associação dos Farmacêuticos do Rs, AFARGS, Rio Grande do Sul, Brasil.
2003 - 2003 Química Medicinal. (Carga horária: 6h)
Sociedade Brasileira de Química, SBQ, São Paulo, Brasil.
2002 - 2002 Estabilidade de Produtos Farmacêuticos/Cosméticos. (Carga horária: 7h)
Associação dos Farmacêuticos do Rs, AFARGS, Rio Grande do Sul, Brasil.
2002 - 2002 Radicais Livres e Nutracêuticos. (Carga horária: 7h)
Associação dos Farmacêuticos do Rs, AFARGS, Rio Grande do Sul, Brasil.
2001 - 2001 As Bases da Oncologia. (Carga horária: 8h)
Hospital de Clínicas de Porto Alegre, HCPA, Rio Grande do Sul, Brasil.
Aprovação em Língua Estrangeira
1 Proficiência em Lingua Inglesa Test of English as a Foreign Language (TOEFL) - Score
250. 2002.
2 Proficiência em leitura de lingua inglesa – UFRGS. 2005
3 Proficiência em leitura de lingua francesa – UFRGS. 2006
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