Fibrinogen chaperone activity in transthyretin amyloidosis: the effect of protein glycation

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UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

Fibrinogen chaperone activity in Transthyretin

Amyloidosis: The effect of protein glycation

Daniel Filipe Mesquita da Fonseca

Mestrado em Bioquímica

Dissertação de Tese de Mestrado orientada por

Doutor Carlos Cordeiro e Doutor Gonçalo da Costa

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FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

Fibrinogen chaperone activity in Transthyretin

Amyloidosis: The effect of protein glycation

Daniel Filipe Mesquita da Fonseca

Mestrado em Bioquímica

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ACKNOWLEDGEMENTS

Este espaço é dedicado àqueles que deram a sua contribuição para que este projecto fosse realizado. A todos eles deixo aqui o meu agradecimento sincero.

Em primeiro lugar agradeço ao Doutor Carlos Cordeiro a forma, a competência e generosidade com que orientou o meu trabalho e todos os momentos de discussão e conversa cientifica que muito prazer me deram. Ao Doutor Gonçalo da Costa agradeço a ambição, a ética e o pragmatismo com que pautou o meu trabalho. To acknowledge, ou em português reconhecer, é um gesto sobre honrar alguém por reconhecer os seus feitos, a sua contribuição e a sua influência. Nesse sentido, reconheço todo o progresso e evolução que passei é parte responsabilidade directa dos meus orientadores e por isso agradeço a oportunidade de ter trabalhado com eles durante estes três anos no grupo de Enzimologia.

No meu trabalho diário, fui abençoado com um grupo jovial e afável. Com a Cristina Silva, mais que companheira e colega de trabalho, aprendi e ainda aprendo que bondade, disponibilidade e honestidade têm lugar num laboratório. Uma palavra especial de agradecimento ao Rui Catarino: conversar contigo é como ler um bom livro. O meu agradecimento ao Prof. António Ferreira pela generosidade, paciência e o apoio com script de python. Aos restantes membros seniores do grupo Dr. Francisco Pinto pelas sábias sessões de esclarecimento estatístico e à Dra. Marta Silva por todo o auxílio oferecido no laboratório. Quero ainda agradecer aos restantes membros do laboratório, que incluem o Ricardo Gomes e a Raquel Mesquita pela companhia e paciência com que lidaram com os meus pedidos e questões espontâneas.

Pretendo ainda salientar a importância de todos aqueles não mencionados que, directa ou indirectamente, contribuíram para a minha formação como investigador, concretamente os diversos docentes do Departamento de Química e Bioquímica da Faculdade de Ciências da Universidade de Lisboa, em particular a Prof. Ana Ponces, que sempre se empenhou em transmitir da melhor forma os seus vastos conhecimentos em bioquímica.

Finalmente, quero agradecer a toda a minha família. Em especial, quero agradecer aos meus pais, pela dedicação e sacrifício que foi suportar esta jornada.

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RESUMO

A Amiloidose da Transtirretina (ATTR), também conhecida como Polineuropatia Amiloidótica Familiar (PAF), é uma doença neurodegenerativa hereditária que se caracteriza pela deposição extracelular de fibras amilóides, em particular no sistema nervoso periférico. A primeira descrição desta amiloidose foi feita em 1952 por Corino de Andrade, numa população do norte litoral de Portugal. Fenotipicamente a doença manifesta-se inicialmente com a perda de sensibilidade nos membros dos doentes, seguida de atrofia muscular, incapacidade motora e morte 10 a 20 anos após o início das primeiras manifestações.

O principal constituinte das fibras amilóides é a transtirretina (TTR), uma proteína extracelular tetramérica encontrada no plasma e fluido cerebrospinal. O tetrâmero é composto por 4 subunidades idênticas de 14 kDa, sendo a TTR maioritariamente expressa e secretada a partir do fígado. Actualmente, não existe nenhuma abordagem terapêutica que se apresente como uma solução permanente para tratar a ATTR. Porém, através da realização de um transplante hepático é possível atenuar a progressão da ATTR em doentes que manifestam os sintomas desta patologia. São conhecidas mais de 80 mutações no gene da TTR, que conferem carácter amiloidogénico à proteína e que estão associadas esta doença, sendo a maioria delas substituições pontuais na cadeia peptídica. A mutação mais comum na ATTR é a TTR V30M (em que a Valina na posição 30 é substituída por uma Metionina), a qual se encontra dispersa de forma esporádica a nível mundial, mas está principalmente associada às áreas endémicas de Portugal, Japão e Suécia.

O modelo mais aceite para descrever a patogénese desta doença, fundamentado em estudos in vitro, assenta na perda de estabilidade do tetrâmero da TTR devido à ocorrência de mutações pontuais na sua cadeia polipeptídica, que levam à sua destabilização em monómeros parcialmente desnaturados que se associam em agregados, culminando na formação de fibras amilóides compostas por TTR. No entanto, existem diversas observações fenotípicas que contradizem alguns dos pressupostos do modelo sugerindo um mecanismo in vivo mais complexo, envolvendo elementos que - juntamente com as mutações pontuais -, influenciam a manifestação e progressão da ATTR. A título

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vi de exemplo, para indivíduos que possuem a mesma mutação no gene da TTR incluindo gémeos monozigóticos, foi observado uma grande diversidade no início das manifestações clínicas da ATTR, podendo mesmo variar em décadas. Existem ainda diferenças geográficas no que toca à manifestação dos sintomas e na própria morfologia dos agregados. Ao contrário do expectável, indivíduos homozigóticos para genes de TTR mutada não apresentam uma forma mais agressiva da doença.

Por detrás destas diferenças poderão constar outros elementos, para além da desestabilização do tetrâmero da TTR que podem influenciar o fenótipo patológico associado à doença. Os mecanismos moleculares mais importantes que podem afectar directamente a estrutura e função das proteínas são interacções proteína-proteína e modificações pós-traducionais. A TTR tem dois ligandos principais no plasma, conhecidos como a T4 (tiroxina) e a RBP (do inglês, Retinol Binding Protein). Mais recentemente foram também identificados no plasma novos interactuantes da TTR. Destes, o fibrinogénio surge com particular interesse, uma vez que foi também recentemente descrito como chaperone extracelular. O fibrinogénio é uma glicoproteína de múltiplos domínios, tendo uma massa de aproximadamente 340kDa, sendo constituído pelas cadeias Aα, Bβ e γ. Esta proteína sintetizada no fígado é geralmente associada ao fenómeno de associação da fibrina e coagulação sanguínea, no entanto a sua descrição como chaperone extracelular denuncia a relevância da sua interacção com a TTR in vivo. Recentemente foram identificadas no proteoma plasmático de doentes com ATTR proteínas diferencialmente glicadas. A glicação proteica é uma modificação pós-traducional irreversível e não enzimática, onde os grupos amina das cadeias laterais de argininas e lisinas reagem com compostos com grupos carbonilo, dando origem a produtos avançados de glicação (AGEs). O metilglioxal é (MG) é o agente de glicação in vivo mais importante, formado nas células principalmente a partir dos intermediários da via glicolítica. Foi observado que chaperones podem ser alvos específicos desta modificação não-enzimática, resultando na modulação da sua função e actividade. A glicação foi anteriormente descrita como estando implicada em outras doenças conformacionais, tais como a doença de Alzheimer e de Parkinson.

A hipótese de trabalho que foi estabelecida para este estágio sugere que outros factores – para além da destabilização do tetrâmero de TTR - contribuem para o fenótipo patológico associado com a doença, de forma directa ou indirecta. Assim, propõe-se que alterações na homeostase proteica normal (proteostase) no espaço extracelular possam

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vii contribuir para a progressão da ATTR. Embora várias moléculas sejam relevantes para o estudo das doenças amilóides, uma vez que fibrinogénio é um interactuante da TTR com função de chaperone, foi a proteína de eleição para testar a hipótese de trabalho. Além disso, dada a influência do envelhecimento e/ou stress metabólico com uma proteostase em desequilíbrio, decidiu-se estudar o possível envolvimento da glicação na ATTR.

Neste trabalho realizaram-se ensaios de agregação e formação de fibras realizados na presença de fracções de fibrinogénio enriquecidas a partir de plasma de indivíduos saudáveis e doentes ATTR. Observou-se uma diferença nítida na actividade de chaperone entre as fracções de fibrinogénio de indivíduos saudáveis e ATTR. Uma vez que a glicação induzida in vitro inibe a actividade de chaperone do fibrinogénio, verificou-se que estes indivíduos apresentam perfil de glicação diferencial para esta molécula. Além disso, perfil de glicação do fibrinogénio foi caracterizado com recurso à espectrometria de massa. Encontrou-se uma distribuição espacial diferencial para a glicação em fibrinogénio de pacientes ATTR em relação a indivíduos saudáveis. Este padrão de glicação diferencial pode estar correlacionado com a diminuída actividade de chaperone do fibrinogénio em indivíduos ATTR. Estas observações apoiam fortemente o fibrinogénio como um elemento importante no mecanismo de patogénese ATTR.

Em suma, nesta dissertação é descrita a função anormal de um elemento do espaço extracelular que está envolvido na monitorização de conformação de proteínas e concentração – o fibrinogénio. A função diminuída deste chaperone é correlacionada com uma alteração metabólica – a glicação. Assim, propõe-se uma alternativa para o modelo real para a patogénese ATTR onde outros elementos, para além da desestabilização do tetrâmero de TTR, podem influenciar o fenótipo patológico associado com a doença. Estes factores adicionais incluem distúrbios mecanismos celulares, tais como alterações metabólicas - glicação, stress oxidativo, etc. - e uma homeostase proteica desequilibrada, em que a actividade de certas proteínas deficientes, tais como o fibrinogénio e outros elementos importantes (por exemplo, chaperones) podem conduzir a uma manifestação progressiva da amiloidose de transtirretina.

Palavras-chave: Amiloidose da Transtirretina, Fibrinogénio, Agregação, Glicação, Proteostase

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SUMMARY

Transthyretin Amyloidosis (ATTR), also known as Familial Amyloidotic Polyneuropathy (FAP) is an autosomal inherited neurodegenerative disorder in which the neuropathological hallmark is the extracellular deposition of amyloid fibrils on the peripheral nervous system. Transthyretin (TTR), an extracellular tetrameric protein found in serum and cerebrospinal fluid, constitutes the major component of these amyloid fibers.

TTR has several known ligands, but recently a few more were identified as interacting partners. From these, fibrinogen arises with particular interest since it has been recently described as a chaperone. Moreover, this protein was also identified as differentially glycated in ATTR patients. It is well documented that chaperones are specific targets of this non-enzymatic modification, resulting in their function and activity modulation. Also, glycation was previously described as being involved in other conformational diseases, such as Alzheimer’s and Parkinson’s diseases.

In this work aggregation and fiber formation assays were performed in the presence of fibrinogen from healthy and ATTR patients’ plasma. We could observe a clear difference in the chaperone activity between fibrinogen enriched fractions from healthy and ATTR subjects. Since these individuals show different glycation profile and in vitro glycation hampers fibrinogen chaperone activity, we further engaged fibrinogen fibrinogen’s glycation profile characterization resorting to mass spectrometry. We found a dissimilar spatial glycation pattern in fibrinogen from ATTR patients regarding healthy individuals. These observations strongly support that fibrinogen is a likely player in the mechanism of ATTR pathogenesis.

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ABBREVIATION LIST

ACN acetonitrile

AGE advanced glycation end-products AGP argpyrimidine

Apo-AI apolipoprotein-AI Apo-J apolipoprotein-AJ ATP adenosine triphosphate BSA bovine serum albumin CEL N ε -carboxiethyl-lisine

CHCA α-cyano-4-hydroxycinnamic acid CSF cerebral spinal fluid

DNA deoxyribonucleic acid DTT dithiothreitol

EDTA ethylenediamine tetraacetic acid

FAP familial amyloidotic polyneuropathy

FTICR fourier transform ion cyclotron resonance HRP horseradish peroxidase

HSA human serum albumin Hsp27 heat shock protein 27 IgG immunoglobulin G IF fluorescence intensity

L55P replacement of lysine by proline at position 55 MALDI matrix-assisted laser desorption/ionization MG methylglyoxal

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xii MS mass spectrometry

m/z mass to charge ratio

PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline

PBS-T phosphate buffered saline – tween-20 PDB protein Data Bank

PNS peripheral nervous system PMF peptide mass fingerprinting PVDF polyvinylidene difluoride

RAGE advanced glycation end-products receptor RBP retinol binding protein

SAP plasma pentraxin serum amyloid P component SDS sodium dodecyl sulphate

SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SNAP sophisticated numerical annotation procedure

SSA senile systemic amyloidosis

T119M replacement of threonine by methionine at position 119 T4 thyroxin

TBS tris buffered saline

TBS-T tris buffered saline – tween-20 TFA trifluoroacetic acid

ThT thioflavin T TTR transthyretin

V30M replacement of valine by methionine at position 30 WT wild-type

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TABLE LIST

Table II.1 – Human plasma samples. p. 23

Table III.1 – Fibrinogen peptide mass fingerprint analysis. p. 39 Table III.2 – Sequence coverage (%) of fibrinogen (α, β, γ chains) from

human ATTR patients and healthy individuals

p. 45

Table III.3 – Number of glycated peptides p. 46

Table III.4 – Number of MGO-derived modifications in fibrinogen’s p. 48 Table VII.1 – Fibrinogen glycation sites exclusively identified in ATTR

patients.

p. 79

Table VII.2 – Fibrinogen glycation sites exclusively identified in healthy individuals.

p. 81

Table VII.3 – Fibrinogen glycation sites identified in both ATTR and healthy subjects.

p.82

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FIGURE LIST

Figure I.1 – Structure of TTR p. 6

Figure I.2 – Transthyretin amyloid cascade p. 9

Figure I.3 – Model of ﬁbrinogen molecular structure p. 13

Figure I.4 – Fibrin assembly p. 14

Figure I.5 – Initial steps of the Maillard reaction of protein glycation from glucose

p. 15

Figure I.6 – Methylglyoxal-derived adducts p. 17

Figure II.1 – Peptide mass fingerprint for the identification of glycated sites by MADI-FTICR.

p. 31

Figure II.2 – Flowchart of MS data analysis using the Python script p. 33 Figure III.1 – Fibrinogen enrichment in ATTR and control individuals p. 37

Figure III.2 – Fibrinogen quantification p. 39

Figure III.3 – Fibrinogen chaperone activity is altered in ATTR patients p. 40 Figure III.4 – Fibrinogen effect on insulin aggregation p. 41 Figure III.5 – Fibrinogen from ATTR patients display an increased

glycation pattern.

p. 43

Figure III.6 – Isotopic distribution analysis p. 46

Figure III.7 – In vivo glycation sites p. 47

Figure III.8 – Mapping human fibrinogen glycation profile p. 49 Figure IV.1 – Molecular model of the fibrinogen role in ATTR p. 56

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xvi Figure VII.1 – Fibrinogen glycation sites for alfa chain. p. 83

Figure VII.2 – Fibrinogen glycation sites for beta chain. p. 84 Figure VII.3 – Fibrinogen glycation sites for gamma chain. p. 85

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I – INTRODUCTION

1 Transthyretin-related hereditary amyloidosis

Historical background and epidemiology

1.1

Transthyretin-related hereditary amyloidosis (ATTR) is a disabling disorder of autosomal dominant trait. It belongs to a class of pathologies that occur as a consequence of protein misfolding, hallmarked by protein aggregation into insoluble cross beta-fiber amyloid deposits (Buxbaum and Reixach, 2009; Fleming et al., 2009). These amyloid deposits, mainly composed by the protein transthyretin (TTR) (Costa et al., 1978), occur at different organs and tissues, affecting the heart, blood vessels and kidneys (Becker et al., 1964). However, the main pathological phenotype of this condition is the progressive dysfunction of the peripheral nervous system (PNS). As a result, a gradual suppression and ultimately loss of organ function occur, increasingly affecting life quality and causing an early death (Holt et al., 1989).

This crippling disease was first described in 1952 by Corino de Andrade in a small fisherman’s village in the north coast of Portugal, Póvoa do Varzim (Andrade, 1952), but only in 1964 was the autosomal dominant trait established (Becker et al., 1964). Although not as common as other amyloidosis, ATTR is presently considered a worldwide spread disorder, with a major incidence focus in Portugal, Sweden and Japan (Sousa and Saraiva, 2003; Hellman et al., 2008; Tawara et al., 1983). The most common ATTR mutation is the V30M variant, where valine replaces methionine in the 30th residue of TTR polypeptide chain (Saraiva et al., 1983), which is scattered sporadically worldwide but is mainly associated to the previous mentioned endemic areas (Tawara et al., 1983; Holmgren et al., 2008; Saraiva et al., 1983) .

Given the incidence of this disease and the well known historical connections, Coutinho hypothesized that a mutant allele in the Portuguese kindred could represent the origin of the ATTR mutation that was subsequently spread throughout the world, including Japan, Europe, North and South America, and Africa (Ando et al., 2005).

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4 However, some share the opinion that the Portuguese disease (V30M) was imported from Sweden, probably from Viking expeditions, and then it might have spread worldwide as a consequence of Portuguese expeditions around the world. Recent studies appear to support Coutinho’s hypothesis though additional studies of genotypes and phenotypes are needed (Ohmori et al., 2004; Zaros et al., 2008).

In endemic areas of Portugal, there are fewer individuals that carry an ATTR mutation when compared to the Swedish population. However the number of individuals that manifest the disease is higher in Portugal, which is associated with the early age at onset in the Portuguese ATTR population (Hellman et al., 2008; Sousa et al., 1993, 1995; Holmgren et al., 1994).

Symptomatology and phenotypic heterogeneity

1.2

As mentioned before, ATTR is characterized by systemic deposition of TTR amyloid fibers, with great emphasis on the peripheral nervous system (Coimbra and Andrade, 1971a; b). As a result, axonal degeneration begins, starting in the unmyelinated nerve fibers (Thomas and King, 1974). However, ATTR also manifests itself in a group of heterogeneous phenotypes that include different levels of neuropathy, cardiomyopathy (Saraiva et al., 1992; Saito et al., 2001), carpal tunnel syndrome (Izumoto et al., 1992), kidney and leptomeningeal impairment (Petersen et al., 1997; Ando et al., 1993).

ATTR symptoms appear in adult life, on average at the age 33,5 ± 9,5 (Sousa et al., 1995), usually with loss of motor sensitivity in the lower limbs. Atrophy and muscular weakness follow, as paresis progresses to the higher extremities. Death comes in the natural course of disease, 10 to 20 years after the first clinical manifestations (Sousa et al., 1995)

Individuals of Portuguese, Japanese or Swedish origin have distinct phenotypes that are mainly characterized by different ages at onset, implying diferent levels of penetrance. This is most obvious for the V30M ATTR population, since also it is the most common mutation (Mutations in hereditary amyloidosis database; http://amyloidosismutations.com/mut-attr.php). Olsson et al. proposed that these dissimilar phenotypes between Swedish and Portuguese V30M heterozygous carriers were due to differences in the concentration of the mutant variant in circulation (lower for

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5 the Nordic individuals as they typically exhibit late age onsets) (Olsson et al., 2010). However, this was not experimentally observed and it was later demonstrated that heterozygous Portuguese V30M ATTR carriers exhibit the same plasma TTR ratio (wild-type to V30M) between asymptomatic and symptomatic states, therefore the wild-(wild-type to V30M ratio does not decrease with illness progression (Ribeiro-Silva et al., 2011). On the other hand homozygous ATTR carriers should present a more severe clinical condition than patients bearing only one copy of the mutant allele. However, the firsts do not show more severe forms of this pathology (Koike et al., 2009; Tanaka et al., 1988) and some individuals (both hetero and homozygous) remain asymptomatic throughout their lives (Rudolph et al., 2008).

Although it is established that ATTR first clinical manifestations occur in adulthood, the average age at onset is not consistent among individuals carrying the same variant or between carriers with different ATTR mutations. Within the ethnically and genetically homogeneous Portuguese population, age at onset was determined to be between 17 and 78 in the 1233 patients examined to 1995 (Hund et al., 2001). Most patients present ATTR in the third or fourth decade, but onset of symptoms may be delayed until old age (Hamilton and Benson, 2001). Moreover, in monozygotic twins where a similar age at onset varies widely, against all expectations if only genetic factors were involved (Munar-Qués et al., 1999).

2 ATTR pathogenesis: transthyretin and aggregation models

Transthyretin

2.1

Transthyretin, also known as pre-albumin, is a plasmatic homotetrameric protein (Figure I.1) of 54 kDa (van Jaarsveld et al., 1973), with a monomer of 14kDa (Kanda et al., 1974). Each subunit is a polypeptide chain of 127 amino acids with two β-sheet (composed by the chains DAGH and CBEF), containing a single cysteine residue that does not participate in the formation of inter-molecular disulfide bonds. The structure assembly occurs primarily with the formation of a dimer, resulting from hydrogen bond between two β sheets in each monomer subunit (between chains HH' and FF'). Further association of two of these dimers produces the homotetrameric structure and creates

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6 two thyroxine binding sites per tetramer. This dimer-dimer interface results from hydrogen bonds and hydrophobic interactions, though these ones are much weaker than the hydrogen bonds involved in the dimer stabilization (Foss et al., 2005; Blake et al., 1978).

The regions comprising amino acids 10 to 22 and 106 to 121, strongly conserved, correspond to the sites involved in thyroid hormone, thyroxine (T4) binding, whereas the RBP (Retinol-binding-protein) binding site (residues 83-85, 99 and 100) exhibit a smaller conservation degree. The more variable regions correspond to the amino and carboxyl terminals (residues 1-10 and 123 to 127) (Schreiber and Richardson, 1997).

Figure I.1 – Structure of TTR (Protein Data Bank code: 3TCT). Ribbon diagram depiction of TTR with the crystallographic 2-fold axis (z-axis) bisecting the T4 binding channel comprising the weaker of TTR's two dimer–dimer interfaces.

Although mainly plasmatic (170-420 mg/L), TTR can also be found in the cerebrospinal fluid (Vatassery et al., 1991) and saliva (da Costa et al., 2010). Moreover, salivary TTR has its origins in the plasma (da Costa et al., 2010). In humans, transthyretin (TTR) is a protein coded by a gene of 7kb located in chromosome 18 (18q11.2-q12.1) (Whitehead et al., 1984). TTR synthesis occurs mainly at the liver (Dickson et al., 1985), but it is also expressed in the choroid plexus and retina in lower amounts (Soprano et al., 1985; Herbert et al., 1986; Getz et al., 1999). TTR Has a short turnover, with a half-life of aproximatelly 2 days, being mainly degraded in the liver (Makover et al., 1988).

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7 TTR main role in the blood stream is the transport of the thyroid hormone T4 and retinol, the last one through formation of a complex TTR:RPB (retinol binding protein) (Buxbaum and Reixach, 2009; Fleming et al., 2009). 15% of T4 transport is carried by TTR in plasma, but in the cerebrospinal fluid TTR is the main transporter of this hormone (Bartalena and Robbins, 1993). As for retinol, TTR binds to the complex RBP-retinol still in the endoplasmic reticulum (Raz et al., 1970) preventing its removal through filtration in the kidney (Noy et al., 1992). Though it was speculated the possibility of TTR to play as a retinol reservoir, it was later confirmed that TTR is not involved in its absorption (Sundaram, 1998).

2.1.1 Transthyretin mutations

More than one hundred TTR mutations with amyloid character are known (Connors et al., 2003) and all, except for the deletion of one amino acid at position 122, result of point mutations in TTR polypeptide chain. Most ATTR patients are heterozygous, producing two different TTR copies: the wild-type and the mutant form, being V30M variant the most common amyloid mutation in TTR gene (Saraiva et al., 1984). In Portugal, besides the V30M variant, other mutations of amyloid character were identified as well as a non-amyloidogenic one - the T119M variant. The latter is more stable than wild-type TTR and reduces amyloidogenesis in heterozygous individuals (Hammarström et al., 2003; Coelho, 1996; Sekijima et al., 2003). TTR L55P is one of the most aggressive mutants and results from the replacement of a leucine residue by a proline in position 55 (TTR L55P) (Jacobson et al., 1992). This variant is very amyloidogenic, which makes it more prone to aggregate and to form amyloid fibers than other common TTR variants (Bonifácio et al., 1996). For instance, most TTR amyloid mutants have a three-dimensional structure quite similar to the wild-type protein. For the V30M variant, the X-ray crystallography studies showed only an increase between the distances of the β sheets that cause a distortion in the cavity responsible for T4 binding (Hamilton et al., 1993), while in the case of the L55P mutation occurs a disruption of the hydrogen bonds between the chains A and D (Sebastião et al., 1998).

Some ATTR associated mutations are clinically indistinguishable (Booth et al., 1998; Misrahi et al., 1998; de Carvalho et al., 2000) while others exhibit variable and heterogeneous phenotypes. It is also interesting to note that there are also mutations that preferentially affect a particular tissue or organ, despite being the same protein that is

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8 mutated (Connors et al., 2003). Thus, assessing the phenotypic implications of a given mutation is not trivial, due to extensive clinical heterogeneity and also the intrinsic difficulty in symptom evaluation in Amyloidosis.

Transthyretin aggregation models

2.2

One of the first models for TTR aggregation was proposed by Kelly and Colon and described TTR tetramer dissociation into monomer as a process dependent on pH and protein concentration occurring at the lysosomes (Colon and Kelly, 1992; Lai et al., 1996). The low pH conditions in these organelles would induce tetramer rearrangement and dissociation, resulting in the formation of a partially denatured amyloidogenic intermediate and eventually in the deposition of amyloid fibers (Colon and Kelly, 1992). Supporting this hypothesis was the reduced stability observed for different TTR variants at low pH, correlated with the aggressiveness of each mutation (McCutchen et al., 1993). However, the mechanism suggested by Kelly implies that the formation of TTR amyloid fibrils occur inside the cell, which is not consistent with the observations of TTR deposits in the extracellular space (Adams and Said, 1996). Moreover, TTR tetramer dissociates in non-native monomeric species under physiological conditions (including pH and ionic strength), contradicting the hypothesis that lysosomes would be essential for TTR aggregates formation (Quintas et al., 1997).

Currently, the most widely accepted ATTR model is based on in vitro studies, and relates transthyretin tetramer stability with point mutations that promote the dissociation of the protein tetramer, followed by misfolding of the monomers into an aggregation-prone conformation (Hurshman et al., 2004; Bonifácio et al., 1996). Aggregation occurs in a downhill polymerization process where oligomers, soluble and insoluble aggregates and finally amyloid fiber are formed (Figure I.2) (Hurshman et al., 2004; Quintas et al., 2001).

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9 Figure I.2 – Transthyretin amyloid cascade. TTR Mutations destabilize the native tetramer leading to dissociation into monomers that undergo partial denaturation and are able to misassemble into a variety of aggregate morphologies including oligomers and amyloid fibers.

The current model was proposed by Quintas (Quintas et al., 1999) based in the dissociation of the tetrameric protein in physiologic conditions, where exposure of the monomer’s hydrophobic surface to the solvent is a pivotal factor. It was later demonstrated that tetramer dissociation, the first and most important step, followed by partial monomer denaturation precedes amyloid fiber formation (Quintas et al., 2001). The presence of a mutation shifts the equilibrium between tetrameric and monomeric species towards the accumulation of the misfolded monomer. The more amyloidogenic the mutation, the greater the tendency to TTR assume the non-native monomer conformation (Hurshman et al., 2004; Schneider et al., 2001).

Although several kinetic and thermodynamic studies have been performed on different recombinant TTR proteins, the mechanism underlying the formation of TTR amyloid fibers is not yet completely understood (Hurshman et al., 2004; Schneider et al., 2001). Most assays to study the process of fiber formation are performed in vitro, not regarding the physiological conditions. Also, the models do not predict and explain the phenotypic variability that is associated with the ATTR, such as different age onset, penetrance levels between individuals carrying the same mutation, homo-and heterozygous, previously discussed in greater detail. Therefore, mutations in TTR cannot be the only factor behind ATTR pathogenesis responsible for destabilizing the structure of tetrameric TTR, promote their aggregation and amyloid deposition. There must be other conditions which directly or indirectly influence the progression of Transthyretin amyloidosis, particularly the stability and conformation of TTR. The two most important molecular mechanisms that may directly affect protein structure and function are protein-protein interactions and post-translational modifications.

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3 Non-genetic factors in ATTR

One key element for protein stabilization is the interaction with ligands or other proteins. For instance, although TTR is a very abundant protein in the cerebrospinal fluid its interaction with T4 prevents TTR amyloid fiber formation in the brain (Miroy et al., 1996).

Molecular chaperones also play an important role in protein stabilization (Hartl et al., 2011). They are the main elements that interact with proteins and endorse structure rearrangement or lead to the selective clearance of polypeptides with incorrect conformations. Chaperones help to minimize alternative folding pathways by shielding the hydrophobic regions of unstructured intermediates or with a non-native conformation (Mannini et al., 2012; Hartl et al., 2011). This process avoids inappropriate intra or intermolecular interactions that would lead to protein aggregation. Alternatively, proteins with incorrect fold can be signaled to degradation in the proteasome by polyubiquitination in the endoplasmic reticulum or in cytosol (Powers et al., 2009). Chaperones and stress-inducible responses constitute two prominent modulators of protein homeostasis. Proteostasis (protein homeostasis) refers to all those processes that act together to maintain the repertoire of proteins in an environment at steady-state levels of abundance and function (Balch et al., 2008).

Loss of protein stability is a pivotal step preceding the formation and accumulation of amyloid fibers. These processes are common to many disorders, including neurodenegerative diseases, referred to as “conformational disorders”. Changes in the normal proteostasis might contribute to the pathogenesis of these disorders, since the decrease ability of the proteostasis network to cope with inherited misfolded-prone proteins, aging and/or metabolic/environmental stresses appear to trigger or exacerbate proteostasis diseases (Balch et al., 2008; Powers et al., 2009). This could be particularly relevant in ATTR since the loss of TTR tetrameric structure is the first step towards amyloid formation which is why the role of natural stabilizing agents (as chaperones) should be studied in conformational-like pathologies.

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Extracellular chaperones, proteostasis and amyloidosis

3.1

In the extracellular space, proteins are exposed to a more oxidizing environment that inside cells and the mechanical stress resulting from the continuous pumping of plasma around the body can induce protein unfolding and aggregation (Bekard et al., 2011; Di Stasio and De Cristofaro, 2010). Clusterin was the ﬁrst extracellular chaperone (EC) to be identiﬁed (Wilson and Easterbrook-Smith, 2000) but the number of known ECs now amounts to seven: α2-macroglobulin, haptoglobin, apolipoprotein E, serum amyloid P component (SAP), caseins and fibrinogen (Wyatt et al., 2012). In vivo formation of extracellular amyloid fibers deposits is associated with several human diseases and many newly identified extracellular chaperones (ECs) are found associated with these deposits. For instance, clusterin, haptoglobin, α2-macroglobulin are found co-localized in amyloid deposits of Alzheimer’s disease, Systemic amyloidosis and type II diabetes (Calero et al., 2000; Fabrizi et al., 2001; Greene et al., 2011; Narayan et al., 2012). As for ATTR, so far clusterin was the only chaperone identified co-localized with TTR in amyloid fiber deposits (Brambilla et al., 2012; Greene et al., 2011; Magalhães and Saraiva, 2011). Interestingly, a recent study using a transgenic mice model for SSA related systemic (cardiac) TTR deposition with a strong decrease in the chaperoning capacity of the liver (Buxbaum et al., 2012).

Destabilized or non-native structures can initiate the aggregation process at concentrations as low as 1% (Chiti and Dobson, 2006). Therefore, such species are present at concentrations low enough to account for the potent sub-stoichiometric effects of the ECs that act as scavengers and most likely reduce the availability of these species to participate in nucleation events that precede fibril formation (Mannini et al., 2012). More also, ECs may also be involved in the physical clearance of protein aggregates guiding ‘‘damaged’’ protein substrates to specific receptors for lysosomal degradation (Yerbury et al., 2005), affecting in vivo amyloid toxicity. It should be noted that all described ECs have the ability to influence amyloid formation in vitro (Yerbury et al., 2007, 2009) and are also found co-localized with clinical amyloid deposits in vivo (Calero et al., 2000; Fabrizi et al., 2001; Greene et al., 2011; Narayan et al., 2012).

Though many studies have highlighted the ability of ECs to suppress amorphous aggregation, their effects on amyloid formation are, apart from clusterin, less well-documented.

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4 Fibrinogen

Fibrinogen structure and function

4.1

Fibrinogen is an acute phase 340-kDa glycoprotein circulating in plasma at a concentration of 160–400 g L−1, with a half-life of about 4 days. It is mainly expressed in hepatocytes, although extra-hepatic synthesis has been demonstrated (Doolittle 1984; Mosesson et al. 2006; Asselta et al. 2007). Fibrinogen is secreted into the bloodstream as a disulphide-linked hexamer composed of two identical heterotrimers, each consisting of one Aα, one Bβ, and one γ chain, with molecular masses of 67 (610 residues), 57 (461 residues) and 47 kDa (411 residues), respectively (Doolittle, 1984; Redman and Xia, 2001). The three chains are encoded by paralogous genes (FGA, FGB, and FGG, coding for Aα, Bβ, and γ chains, respectively), clustered in a 50-kb region on chromosome 4 (4q31.3) (Asselta et al., 2007). The coordinated transcription of the three fibrinogen genes is up-regulated in response to various stress stimuli, such as inflammation and tissue injury (Gabay and Kushner, 1999).

Circulating fibrinogen shows extensive heterogeneity, mainly arising from alternative splicing of both Aα and γ mRNAs and from C-terminal degradation of the Aα-chain (Henschen, 1993; Michelsen et al., 2000). Concerning the Aα Aα-chain, a minor extended αE chain with a 236-residue carboxyl extension homologous to the C termini of the other two constituent chains has been identified. The αE chain, which originates from the inclusion of a sixth exon in the mature transcript, is preferentially assembled in a homodimeric αE/αE 420-kDa fibrinogen variant (Fu and Grieninger, 1994). The quaternary structure of fibrinogen is achieved in a pairwise fashion forming the hexameric circulating molecule composed of two symmetrical distal globular D domains (constituted by the C termini of the Bβ and γ chains) joined to a central E domain (consisting of the N termini of the six chains) by coiled-coil regions (Spraggon et al., 1997; Kollman et al., 2009). The hexameric molecule is stabilized by 29 inter- and intra-chain disulphide bonds (Redman and Xia, 2001).

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13 Figure I.3 – Model of fibrinogen molecular structure. The central E region containing the N-termini of the six fibrinogen chains. The N-terminal regions of the a and b chains are cleaved during fibrinopeptide release. Coiled coil regions extend to the outer D domains which include the C-termini of the c and b chains. The C-terminal region of the a chains extends back towards the central E domain (retrieved form Mosesson 2005).

Fibrinogen plays a central role in the hemostatic balance by originating fibrin clot formation and for the support for platelet aggregation (Doolittle, 1984; Mosesson, 2005). Fibrinogen participation in cell–matrix interactions – through its binding to heparin, fibronectin, and cell adhesion molecules – mediates additional roles in angiogenesis, cell proliferation, wound healing and tumor progression, being also involved in inflammatory responses (Mosesson et al., 2001; Asselta et al., 2007). Elevated fibrinogen levels are associated with age, atherosclerotic disease, acute myocardial infarction, and stroke (Asselta et al., 2007; Gabay and Kushner, 1999). However, the roles of fibrinogen in many of these physiological and pathological conditions are still not clear. Conversion of fibrinogen to fibrin occurs after removal, by thrombin, of fibrinopeptides A (FPA) and B (FPB) from the N-termini of the Aα and Bβ chains at the Arg16-Gly17 and the Arg14-Gly15 bonds, respectively. The removal of FPA exposes a polymerization site (named EA) that initiates polymerization by binding to a constitutive complementary-binding pocket (Da) located in the D domain of a neighbouring molecule (Mosesson, 2005). Binding of the D domain of one molecule to the central E domain of an adjacent fibrin monomer gives rise to a protofibril. Fibrils also undergo lateral associations to create multi-stranded fibers, which constitute the physical meshwork of the coagulum (Mosesson, 2005; Blombäck et al., 1978).

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14 Figure I.4 – Fibrin assembly. Fibrin molecules are represented in two color schemes for ease of recognition (adapted form Mosesson 2005).

Fibrinogen as an extracellular chaperone

4.2

Recent evidence from our group showed that fibrinogen is overexpressed in the plasma of ATTR patients and it was also found to interact with TTR (da Costa et al., 2011). Interestingly, fibrinogen is also described in other amyloid-like pathologies (e.g.: Alzheimer’s and Parkinson’s Diseases) as an important element (Ahn et al., 2010; Yano et al., 2001; Wong et al., 2010). Fibrinogen specifically interacts with and suppresses aggregation of several proteins, including yeast prion protein Sup35. Moreover, fibrinogen is rescues thermally-induced protein aggregation in the plasma of fibrinogen-deficient mice (Tang et al., 2009a; b). It is likely that increased levels of fibrinogen in patients with different conformational pathologies that share common molecular mechanisms may be a response to the increased need of extracellular chaperone activity under such pathological conditions (Wyatt et al., 2012).

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5 Protein Glycation

Protein glycation

5.1

Glycation is a non-enzymatic reaction whereby amino groups are modified by highly reactive dicarbonyl compounds (usually reducing sugars), including protein amino groups in the N-terminal, the lysine and arginine side chains (Baynes et al., 1989; Rabbani and Thornalley, 2012; Walton and Shilton, 1991). This modification contrasts with enzymatic glycosylation, which is highly specific and regulated and one of the main mechanisms of post-translational modifications (Kikuchi et al., 2003). Glycation can affect protein structure, and thereby its stability and function in a similar way as a point mutation does. Though this chapter will be focused on protein glycation, other biomolecules can also be glycated, including lipids and nucleic acids (Thornalley, 2008; Fu et al., 1996; Thorpe and Baynes, 1996).

Glycation involves a series of reactions called collectively the Maillard reaction (Figure I.7) (O’Brien, 1997). Essentially, the protein’s amino groups react with a carbonyl from the glycation agent forming a reversible Schiff base. Subsequently, adducts undergo a spontaneous rearrangement - classified as early glycation - (Baynes et al., 1989; Westwood and Thornalley, 1997) to form an Amadori product. These products undergo a series of complex reactions, such as intramolecular rearrangements and non-oxidative hydrolysis, to form stable end-stage adducts termed advanced glycation endproducts – AGEs (Figure I.7) (Bucala and Cerami, 1992; Vlassara, 1994; Westwood and Thornalley, 1997).

Figure I.5 – Initial steps of the Maillard reaction of protein glycation from glucose. Adapted (O’Brien, 1997; Westwood and Thornalley, 1997).

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16 As a non-enzymatic process glycation is not regulated following a first order kinetics. Thus concentration and reactivity of the glycation agent (McPherson et al., 1988), pH, temperature and the half-life of proteins are parameters that affect this process. Moreover, glycation in a target protein residue can also be influenced by its tertiary structure, since neighbor amino acids can affect the side chains’ pKa and thus the thermodynamic of the reaction (Ahmed et al., 2005b; Westwood and Thornalley, 1997).

Glycation is likely to act at three levels. First, glycation, either itself or synergistically with point-mutations, might cause the initial unfolding, increased beta-sheet content and the formation of soluble aggregates. Secondly, it may alter the turnover and the refolding and recovery pathways involving chaperones proteins (Morgan et al., 2002). Thirdly, glycation could contribute to the transition from soluble protein aggregates to amyloid fibers, stabilizing the latest, increasing its resistance to proteolysis and its receptor activation capacity (Fleming et al., 2011). Arginine and lysine residues are most often found in enzymes active sites, therefore AGEs formation is associated with enzymes inactivation. Upon glycation, some enzymes involved in metabolism show reduced activity – as lactate dehydrogenase, glutathione reductase and enolase – (Morgan et al., 2002; Yan and Harding, 1997; Gomes et al., 2006), while others, as the α-crystallin, Hsp27 chaperone, increase its basal activity and expression (Nagaraj et al., 2003; Sakamoto et al., 2002). Molecular chaperones, such as α-crystallin and Hsp27 , are themselves major targets of methylglyoxal-induced modification and aggregation (Satish Kumar et al., 2004; Schalkwijk et al., 2006).

Advanced glycation end-products

5.2

Several carbonyl-containing compounds have been shown to modify protein molecules by the Maillard reaction, including sugars such as glucose and fructose. Glucose was intensively studied as a glycation agent due to the association between blood glucose levels in diabetes and extensive protein glycation in these patients. However, the glucose-induced damage is not confined to diabetic patients: glycation occurs even at normal concentrations of glucose, accumulating with age (Thornalley et al. 1999).

Another metabolite, an α-oxalaldehyde termed methylglyoxal (MG) has been described as the most potent glycation agent in vivo (Thornalley et al., 1999; McPherson et al., 1988; Thornalley, 2008). This highly reactive dicarbonyl compound is an

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17 unavoidable product of cellular metabolism and therefore is present in all cells, either in normal or pathological conditions (Ahmed, 2005; Thornalley, 2003). In fact, the most important pathway for its formation in eukaryotic cells is a by-pass to glycolysis, through non-enzymatic phosphate β-elimination of glycolysis metabolites: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (Richard, 1993; Fleming et al., 2011).

Figure I.6 – Methylglyoxal-derived adducts. Adapted from Ahmed, 2003.

Methylglyoxal irreversibly modifies arginine and lysine side chains, resulting in a chemically heterogeneous group of advanced glycation end-products (Rabbani and Thornalley, 2012; Westwood and Thornalley, 1997; Walton and Shilton, 1991) (Figure I.8), named methylglyoxal-derived advanced glycation end products (MAGE) (Gomes et al., 2006). Argpyrimidine and hydroimidazolones are speciﬁc markers of protein glycation by methylglyoxal in arginine residues, while Nε-(carboxyethyl)lysine (CEL) is derived of the speciﬁc reaction between methylglyoxal and lysine residues (Rabbani and Thornalley, 2012; Capote et al., 2009). Arginine-derived AGEs appear to be more relevant considering the existence of specific receptors for hydroimidazolones - sRAGE (Fleming et al., 2011; Westwood et al., 1997).

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Glycation: aging and disease

5.3

AGEs are thought to be also implicated in the aging process, acting as universal hallmarks of aging. This is supported by multiple observation of aged tissue presenting accumulation of several types of AGEs adducts, including skin, lungs, muscles and blood vessels (Thornalley, 2008; Grillo and Colombatto, 2008). The actual role of AGEs in the aging process is under great speculation: there is no consensus whether they act as cause or as biomarkers. However, the evidence collected so far points for a more intriguing contribution, as it seems they can be implicated as an important to the progression the aging process.

Macromolecular damage and biochemical changes that occur in aging and age-related disorders point to the process of glycation as a common event in all of them (Monnier and Cerami, 1981; Bucala and Cerami, 1992). Recent studies have suggested links between protein aggregation and glycation (Bouma et al., 2003). Advanced glycation end products (AGEs) have been implicated as mediators of various complications of age-related disease, such as diabetes, Parkinson’s and AD (Auburger and Kurz, 2011; Ahmed, 2005; Lüth et al., 2005; Ahmed et al., 2005a). Regarding diabetes, several plasma proteins exhibit high glycation levels including immunoglobulins, transferrin and fibrin (Jaleel et al., 2005). In diabetic patients, immunoglobulins and transferrin were shown to loose function upon glycation (Dolhofer et al., 1985), while the fibrin clot shows an abnormal structure and is more resistant to plasmin proteolysis (Pieters et al., 2008).

In Alzheimer’s disease, there is an elevated AGE content in Aβ plaques(Vitek et al., 1994) and glycation of Tau and Aβ protein increases its propensity to form fibrils (Necula and Kuret, 2004; Chen et al., 2006). Some data suggest that the formation of these glycation products arise as a consequence of the pathological condition. However, though AGEs levels naturally increase with age, Alzheimer patients show early accumulation of glycation products and a marked increase in the formation of these over time (Lüth et al. 2005). Also, in patients with Parkinson's disease it was demonstrated the presence of higher amounts of glycated end-products adjacent to Lewi bodies when compared to other brain areas (Castellani et al. 1996).

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Protein glycation in ATTR

5.4

Also in ATTR there is evidence for glycation involvement. One of the main observations was made by the detection and quantification of AGP in the deposits of ATTR patients (Gomes et al., 2005) and, more recently, da Costa et al found that plasma proteins are differentially glycated by methylglyoxal in ATTR patients (da Costa et al., 2011). Moreover, the same study shows increasing glycation levels over time in plasma proteins in individuals after being subjected to a domino liver transplantation, i.e. that received an ATTR liver. Since these individuals did not bear any know TTR mutation, an amyloidogenic TTR is introduced in the blood stream with the domino transplant, allowing a closer follow-up of ATTR progression. In the same study, one of the proteins pointed as differentially glycated in the plasma of ATTR patients was fibrinogen. Based on the observation that fibrinogen shows chaperone activity (Tang et al., 2009a) the authors proposed that fibrinogen loss of chaperone function due to glycation as a pivotal element in the destabilization of TTR tetramer (da Costa et al., 2011).

Regardless the AGEs formation timing, it is known that its accumulation is related to sustained inflammatory responses and with oxidative stress, both recurrent in neurodegenerative disorders. Glycation may contribute in a dynamic fashion to these multifactorial diseases (e.g. ATTR), promoting, stabilizing or accelerating the aggregation of pathologic-like proteins and inducing responses that lead to cellular damage and death.

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6 Work hypothesis and objectives

Previous unpublished work performed by members of our group correlated a progressive impairment of fibrinogen’s chaperone activity with increase of in vitro induced glycation. Moreover, it was developed a method, resorting to mass spectrometry, to map fibrinogen’s glycation sites upon in vitro incubation with methylglioxal. These observations and the whole methodology developed during that work were valuable tools to continuing study fibrinogen in ATTR context.

Our working hypothesis for ATTR pathogenesis states that other factors, beyond the destabilization of the TTR tetramer can contribute to accelerate the pathological phenotype associated with the disease (da Costa et al., 2011). We propose that changes in the normal protein homeostasis (proteostasis) in the extracellular space contribute to ATTR progression. Though several molecules are relevant to study amyloid disorders, fibrinogen was found to be of particular relevancy in ATTR studies since it is described as a molecular chaperone and a TTR partner and is overexpressed in the plasma of patients with this disease. Also, we believe that aging and/or metabolic stress – with an unbalanced proteostasis network – can influence ATTR progression and manifestation. An example of such kind of stresses is protein glycation.

Once a hypothesis was established, three main objectives were pursued:

i) Investigate the chaperone activity of an element in the extracellular space involved in monitoring protein conformation and concentration – fibrinogen – in ATTR patients regarding control subjects.

ii) Relate the differential chaperone function with a molecular mechanism, namely with a metabolic alteration – glycation.

iii) Characterize the extent of this modification in fibrinogen from ATTR patients. To achieve these objectives four main tasks were defined:

1 – To purify human plasma fibrinogen from ATTR individuals and healthy controls. 2 – To quantify chaperone activity for fibrinogen from different subjects.

3 – To compare fibrinogen’s glycation profile in the two study groups 4 – To map fibrinogen glycation sites

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II – METHODS

1 Human samples

Blood samples from control individuals and symptomatic Transthyretin amyloidosis (ATTR) Portuguese type TTR V30M patients (three subjects each, age ranging from 23 to 38 years – TABLE I) were collected to citrate containing tubes (Sarstedt). All subjects were characterized by gene typing for the V30M mutation and are heterozygous carriers except the controls that do not bear any known TTR mutation. This was later verified in our lab, by peptide mass fingerprinting using MALDI-FTICR (Ribeiro-Silva et al., 2011; da Costa et al., 2009). At the time of transplantation, ATTR patients showed peripheral or autonomic polyneuropathy (Ribeiro-Silva et al., 2011). All individuals gave informed written consent and the protocol was approved according to EEC (Executive Ethics Commission) ethic rules at Hospital de Curry Cabral, Lisboa and in full compliance with the Helsinki protocol.

The blood was centrifuged at 1800 g for 5 minutes at 4°C. The supernatant plasma was stored in 200 μL aliquots and kept frozen at −80°C until further analysis.

Table II.1 – Human plasma samples. Three ATTR patients and three healthy control subjects, age and gender matched were used.

Sample Name Gender Age ATTR 1 F 36 ATTR 2 M 31 ATTR 3 F 33 Healthy 1 F 38 Healthy 2 F 34 Healthy 3 M 23

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2 Fibrinogen enriched fraction

Fibrinogen enrichment

2.1

To characterize and perform functional studies with fibrinogen from human plasma of ATTR and control individuals a methodology was adapted from Doolitle et. al to obtain a fibrinogen enriched solution (Doolittle et al., 1967). Briefly, human plasma from ATTR and healthy control subjects was diluted (1/10) in Mili-Q water (Millipore) and precipitated by the cautious addition of 0.22 volumes of cold 50% ethanol, lowering the temperature to 4ºC instead of the -3ºC used by Doolitle et al. (Doolittle et al., 1967). After centrifugation (10 minutes at 10000 rpm), the precipitate was washed with 0.5 original volumes of 7% ethanol at 4ºC and the solution was again centrifuged in the previous conditions. The precipitate was collected and dissolved in 0.25 original volume 55 µM trisodium citrate buffer, pH 6.5, at 30ºC for 30 min. The solution was cooled to 4ºC followed by the addition of cold 20% ethanol to a final concentration of 2%. After centrifugation (20 seconds at 6000 rpm) a mucous-like precipitate was removed. Addition of more 20% ethanol to a final concentration of 8%, followed by another centrifugation (10 minutes at 10000 rpm), resulted in the precipitation of fibrinogen. All steps of this procedure were conducted in a cold room at 4ºC. Ethanol solutions were freshly prepared before use, and kept also at 4ºC. The enrichment process was evaluated by western blot against fibrinogen.

2.1.1 Fibrinogen Quantification

To evaluate the amount of fibrinogen in the enriched fraction, samples were first separated by Sodium Dodecil Sulphate Poliacrilamide Gel Electrophoresis (SDS-PAGE), followed by gel densitometric analysis using TotalLab™ Quant v12 software (TotalLab). This analysis is achieved by determining the volume of each band in the gel’s different lanes based on its pixel intensity. The volume of the band is proportional to the protein concentration (Vincent et al., 1997), allowing a relative quantification. The signal for each band is normalized using the total protein lane signal.

First, total protein concentration was determined using the Bradford method (Bradford, 1976). Secondly, fibrinogen percentage in the enriched fraction was

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25 determined by gel densitometry and consequently calculated the approximate molar concentration of fibrinogen.

Insulin aggregation studies

2.2

To investigate the chaperone activity of a target molecule we used the insulin aggregation assay, in acidic conditions (Colon and Kelly, 1992; Pasquato et al., 2007). Protein aggregation was evaluated by light scattering. To moniter amyloid fiber formation, the fluorescence thioflavin T assay was used (Naiki et al., 1989).

Aggregation conditions

2.3

A stock solution of human insulin (SAFC Biosciences) 0,1 mM was prepared in sodium acetate buffer 0,1 M, pH 4,6 (with 137 mM NaCl and 2,7 mM KCl). To monitor fibrinogen’s chaperone activity from ATTR and healthy subjects, a fibrinogen:insulin ratio of 40:1 was chosen, since it was previously observed by our group as sufficient to reduce in more than two and three fold the formation of aggregates and fibril structures, respectively three independent biological replicates were selected per study group (Healthy vs ATTR). However, it was necessary to prepare a sample pool, assembling the independent biological samples of the same biological condition to achieve the required fibrinogen concentration for the aggregation assays. These assays were performed in a volume of 150 μL with 20 μM to insulin (previously optimized by our group) and 0,5 μM fibrinogen (enriched fraction) in sodium acetate buffer 0,1 M, pH 4,6 (137 mM NaCl and 2,7 mM KCl). 0.002% of sterile, filtered sodium azide (Merck) was added to prevent microbial growth. For the aggregation studies in the presence of anti-fibrinogen policlonal antibody the same reaction mixture was prepared using a molar ratio fibrinogen:antibody of 4:1. Samples were incubated for 48 hours at 37ºC with constant stirring (900rpm) in an Analog Dry Block Heater (VWR).

Monitoring aggregation and amyloid fiber formation

2.4

To detect insoluble aggregates formation, light scattering measurements at 600 nm were carried in a Sunrise microplate reader (Tecan) with a 96 wells pureGrade TM BRANDplates® (BRAND). Amyloid fibril formation was monitored by adding 20 μL of

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26 our aggregation mixtures to a 975 μL solution of Glycine-NaOH 50mM pH 8,5 buffer with 0,5μM of thioflavin T. Amyloid formation was revealed by the appearance of new excitation and emission maxima of the thioflavin T fluorophore, at 450 nm and 482 nm, respectively, corresponding to the described maxima in its fluorescence spectra after binding to amyloid fibrils of different nature (Naiki et al., 1989). The fluorescence measures were carried out on a spectrofluorimeter Fluorolog-3 (Horiba Jobin Yvon) with a quartz cuvette with 1 cm optical path. Fluorescence intensity was measured at 482 nm (excitation at 450 nm). Both excitation and emission bandwidths were defined at 2.5 nm and 10 readings were made for each fluorescence measurement. Data were correct using insulin without incubation.

Statistical analysis

2.5

For aggregation and amyloid fiber formation studies - without antibody - 3 independent biological replicates per study group were selected to exclude inherent variability. As previously mentioned these samples were pooled, each pool containing three independent biological samples of the same study group (healthy pool vs ATTR pool). These pools were prepared three times and three experimental replicates were performed for each of them, making a total of nine replicates per study group. To compare the significance of the chaperone activity of the fibrinogen enriched fraction from different study groups, we applied the Student’s t-test with a confidence interval of 95%. P-values ≤ 0.05 were considered statistically significant.

For the aggregation assay in the presence of anti-fibrinogen antibody only one pool was considered, from the control group, being subjected to three experimental replicates. To compare the significance of the different conditions (with or without antibody), we applied a Two-Way ANOVA test with a confidence interval 95%. P-values ≤ 0.05 were considered statistically significant.

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3 Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Gels castings

3.1

Fibrinogen chains were separated by SDS-PAGE in mini-gel format (Mini-PROTEAN® 3 Cell - 8,3 x 7,3 cm from Bio-Rad), using 4% and 10% of acrylamide for concentration and resolution gels, respectively, to obtain better resolution for proteins with a molecular weight ranging from 30 to 70 kDa.

Sample preparation

3.2

Fibrinogen enriched fraction was dissolved in 30 μL of 0,1M sodium acetate buffer pH 4,6 (137 mM NaCl and 2,7 mM KCl) and protein concentration was determined using the Bradford method (Bradford, 1976). For blue stained coomassie gels 5μg of total protein was added to 5μL of loading buffer (6.25 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 0.01% (v/v) bromophenol blue). The samples were boiled for 2 mins at 100 ° C in an Analog Dry Block Heater (VWR) and then applied on the gel. For western blot, 2μg were used per lane for enriched fractions and 0,5μg for Calbiochem fibrinogen – glycated and non glycated. For peptide mass fingerprint and glycation site mapping, 5μg were used per lane. Glycerol and bromophenol blue was purchased from Merck, and β-mercaptoethanol from Sigma.

Electrophoretic analysis

3.3

Electrophoresis was performed with denaturing running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS; pH ≈ 9), using PowerPac Basic Power Supply TM (Bio-Rad) at 60V. The electrophoretic running was stopped when the 25kDa molecular weight marker reached the end of resolution gel. We used molecular weight markers ranging 250 to 10kDa (5μL, Precision ProteinTM All Blue Standards Plus, Bio-Rad).

Gel staining

3.4

Protein bands were stained with coomassie blue G-250 (50% water, 40% methanol, 10% glacial acetic acid, 2% (w/v) coomassie G-250) overnight with constant

Fibrinogen chaperone activity in transthyretin amyloidosis: the effect of protein glycation

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

Fibrinogen chaperone activity in Transthyretin

Amyloidosis: The effect of protein glycation

Daniel Filipe Mesquita da Fonseca

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

Fibrinogen chaperone activity in Transthyretin

Amyloidosis: The effect of protein glycation

Daniel Filipe Mesquita da Fonseca

Mestrado em Bioquímica

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

RESUMO

SUMMARY

ABBREVIATION LIST

TABLE LIST

FIGURE LIST

I – INTRODUCTION

1 Transthyretin-related hereditary amyloidosis

Historical background and epidemiology

1.1

Symptomatology and phenotypic heterogeneity

1.2

2 ATTR pathogenesis: transthyretin and aggregation models

Transthyretin

2.1

Transthyretin aggregation models

2.2

3 Non-genetic factors in ATTR

Extracellular chaperones, proteostasis and amyloidosis

3.1

4 Fibrinogen

Fibrinogen structure and function

4.1

Fibrinogen as an extracellular chaperone

4.2

5 Protein Glycation

Protein glycation

5.1

Advanced glycation end-products

5.2

Glycation: aging and disease

5.3

Protein glycation in ATTR

5.4

6 Work hypothesis and objectives

II – METHODS

1 Human samples

2 Fibrinogen enriched fraction

Fibrinogen enrichment

2.1

Insulin aggregation studies

2.2

Aggregation conditions

2.3

Monitoring aggregation and amyloid fiber formation

2.4

Statistical analysis

2.5

3 Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Gels castings

3.1

Sample preparation

3.2

Electrophoretic analysis

3.3

Gel staining