Vanessa Arada de
Almeida
Mitochondrial proteome analyses for LCHAD
deficiency characterization
Vanessa Arada de
Almeida
Mitochondrial proteome analyses for LCHAD
deficiency characterization
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Bioquímica
especialização em Bioquímica Clínica, realizada sob a orientação científica da Professora Doutora Rita Ferreira e Professor Doutor Francisco Amado do Departamento de Química da Universidade de Aveiro
o júri
Presidente Prof. Doutor Pedro Domingues
professor auxiliar da Universidade de Aveiro Prof. Doutor Francisco Peixoto
professor auxiliar com agregação da Universidade de Trás-os-Montes de Alto Douro
Prof. Doutor Francisco Amado
professor associado da Universidade de Aveiro
Prof. Doutora Rita Ferreira
uma forma ou de outra comigo se cruzaram ao longo desta etapa, por todos os ensinamentos, ajuda e compreensão tenho alguns agradecimentos a fazer. À Professora Doutora Rita Ferreira tenho a agradecer a incansável orientação científica e laboratorial, todas as correções e sugestões, sem as quais este trabalho não seria possível, tenho ainda a agradecer a total disponibilidade e o exemplo profissional que é.
Ao Professor Doutor Francisco Amado agradeço por me ter aceite no grupo de espectrometria de massa.
Ao Doutor Rui Vitorino agradeço o conhecimento transmitido sobre Espectrometria de Massa, o acompanhamento laboratorial, os preciosos ensinamentos bioinformáticos e a boa disposição e simpatia.
À Ana Isabel agradeço por me ter recebido com toda a simpatia e
disponibilidade no laboratório, e por me ter aturado e esclarecido diversas dúvidas. De alguma forma tenho agradecer também ao Armando, ao Renato, à Zita, à Cláudia, ao André e à Patrícia pela companhia, simpatia e boa
disposição no laboratório.
A todo o grupo de Espectrometria de Massa agradeço toda a disponibilidade. Aos meus amigos Andreia, Sofia, Paulo, Du, Marta, Hugo, Inês, Joana, Zé e Ivo devo o incentivo, a paciência, a compreensão e o companheirismo demonstrado.
Aos meus amigos de sempre Xana, Catarina M., Catarina A., Sofia, Ricardo, Diogo, Faitan e Fernando reconheço todo o apoio incondicional, compreensão nas ausências, e agradeço por me acompanharem permanentemente.
À Marta e à Patrícia tenho de agradecer por me terem aturado todos os dias durante estes dois anos, pelo companheirismo, pelas brincadeiras, pelo apoio e incentivo.
A toda a minha família tenho de agradecer por sempre me terem apoiado e terem feito de tudo para que todos os meus sonhos sempre se realizassem. Ao meu pai e irmã a quem devo tudo o que sou e o que tenho, tenho a agradecer por estarem sempre presentes sem exceção, pelo apoio e compreensão incondicional.
A todos os que referi que de forma direta ou indireta participaram na realização deste trabalho,
palavras-chave Deficiência em LCHAD; oxidação mitocondrial de ácidos gordos; análise do proteoma mitocondrial; nanoLC-MS/MS
Resumo A deficiência em 3-hidroxiacil-CoA desidrogenase de ácidos gordos de cadeia
longa (LCHAD) é uma desordem da oxidação lipídica mitocondrial que apesar de rara está associada a um mau prognóstico devido às suas graves
consequências clínicas. Apesar de a implementação dos programas de rastreio neonatal em alguns países desenvolvidos, incluindo Portugal, ter contribuído para uma melhor compreensão das doenças metabólicas e para a prevenção das suas consequências, os mecanismos fisiopatológicos
subjacentes à LCHAD ainda são pouco compreendidos.
No sentido de contribuir para a elucidação destes mecanismos, avaliou-se a plasticidade mitocondrial em resposta à deficiência em LCHAD. Assim, foram isoladas mitocôndrias de culturas de fibroblastos obtidas a partir de biópsias de pele de doentes com deficiência em LCHAD e o seu proteoma foi
caracterizado e comparado com amostras obtidas de indivíduos saudáveis. Recorrendo a nanoLC-MS/MS 729 proteínas distintas foram identificadas, a grande maioria pertencente aos seguintes clusters funcionais “metabolismo”,
“transporte”, “transdução de sinal”, “processos de desenvolvimento e geração de percursores de metabolitos e energia”. Da análise dos resultados obtidos com marcação com iTRAQs identificaram-se 40 proteínas diferentemente expressas entre os dois doentes com défice de LCHAD e os controlos entre elas estão chaperones, protéases, proteínas associadas ao metabolismo e ainda proteínas associadas ao stress oxidativo.
Em geral, este estudo permitiu a obtenção de uma perspetiva global da plasticidade do proteoma mitocondrial perante a deficiência em LCHAD e evidenciou as vias moleculares envolvidas na sua patogénese.
keywords LCHADD; mitochondrial fatty acid oxidation; mitochondrial proteome analysis; nanoLC-MS/MS
abstract Long chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency is
a rare inborn error of the mitochondrial fatty acid β-oxidation. The
implementation of newborn screening programs in several developed countries, including Portugal, has contributed not only to a better understanding of
metabolic diseases but also to the prevention of fatal disease's consequences. Nevertheless, the pathophysiological mechanisms underlying the clinical manifestation of LCHADD (LCHAD deficiency) and its consequences are poorly understood.
In order to contribute to the elucidation of these mechanisms, mitochondria plasticity to LCHAD deficit was evaluated. In this sense cultured fibroblasts were obtained from LCHADD patients’ skin biopsies, and its proteome was characterized and compared with samples from normal controls. Using nanoLC-MS/MS 729 distinct mitochondrial proteins were identified, most of which were assigned to the functional clusters “metabolism”, “transport”, “signal transduction”, “developmental process and generation of precursor
metabolites” and “energy”. Based on iTRAQs data, 40 proteins were found differentially expressed in LCHADD patients compared with controls, among which were chaperones, proteases, metabolic proteins and oxidative stress proteins.
In overall, this study provides a global perspective of the mitochondrial proteome plasticity in LCHADD and highlights the main molecular pathways involved in its pathogenesis.
Abbreviations
ACD Acyl-CoA dehydrogenase
ACN Acetonitrile
AMP Adenosine monophosphate
ATP Adenosine triphosphate
cAMP Cyclic adenosine monophosphate
CoA Coenzyme A
CPT Carnitine palmitoyl transferase
ECH 2-enoyl-CoA hydratase
ETF Electron transfer flavoprotein
FAD Flavin adenine dinucleotide
FABP Fatty acids binding protein
FATP Fatty acids transport protein
GC Gas chromatography
GC-MS Gas chromatography couple to mass spectrometry
HACD L-3-hydroxyacyl-CoA thiolase
iTRAQ Isobaric tags for relative and absolute quantitation
LC Liquid chromatography
LC-MS/MS Liquid chromatography couple to tandem mass spectrometry
LCAD Long-chain acyl-CoA dehydrogenase
LCHAD Long-chain 3-hydroxyacyl-CoA dehydrogenase
LCHADD Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency
MCAD Medium-chain acyl-CoA dehydrogenase
MMTS S-Methyl methanethiosulfonate
mRNA Messenger ribonucleic acid
MS Mass spectrometry
MS/MS Tandem Mass spectrometry
MTP Mitochondrial Trifunctional Protein
NAD+ Nicotinamide adenine dinucleotide (reduced form of NAD+ – NADH)
OXPHOS Oxidative Phosphorylation
PMSF Phenylmethanesulfonylfluoride
PQC Protein quality control
SCAD Short-chain acyl-CoA dehydrogenase
SDS Sodium dodecyl sulfate
SOD Superoxide dismutase
TCEP Tris (2-carboxyethyl) phosphine
TEAB Triethyl ammonium bicarbonate buffer
TFA Trifluoroacetic acid
UQ Ubiquinone
INDEX
Abbreviations ____________________________________________________________ i
I. INTRODUCTION ____________________________________________________ 1
II. LITERATURE OVERVIEW ____________________________________________ 5
2.1. Fatty Acids β-oxidation ______________________________________________ 7
2.1.1. Mitochondrial β-oxidation system: enzymes involved _________________ 11 2.1.2. Mitochondrial Trifunctional Protein _______________________________ 13 2.1.2.1. Long-chain 3-hydroxyacyl-CoA dehydrogenase ___________________ 14
2.2. Disorders of mitochondrial fatty acid β-oxidation _____________________ 15
2.2.1. LCHAD deficiency ____________________________________________ 16 2.2.1.1. Clinical Aspects ____________________________________________ 16 2.2.1.2. Biochemical Diagnosis _______________________________________ 17 2.2.1.3. Molecular Characterization ____________________________________ 19
2.3. Mitochondrial proteome analyses for LCHAD deficiency characterization 21
III. AIMS ______________________________________________________________ 25
IV. MATERIALS AND METHODS ________________________________________ 29
4.1. Experimental design _____________________________________________ 31
4.2. Samples characterization _________________________________________ 32
4.3. Cell Culture ____________________________________________________ 32
4.4. Preparation of Mitochondria-enriched fraction _______________________ 32
4.5. NanoLC-MS/MS with iTRAQ labeled analysis _______________________ 33
4.6. Western Blotting ________________________________________________ 35
4.7. Zymography ____________________________________________________ 36
4.8. Statistical analysis _______________________________________________ 36
V. RESULTS __________________________________________________________ 37
5.2. Effect of LCHAD deficiency on mitochondrial proteome _______________ 41
5.3. Western blotting analysis __________________________________________ 50
5.4. Zymography analysis _____________________________________________ 54
VI. DISCUSSION _______________________________________________________ 57
VII. CONCLUSIONS _____________________________________________________ 63
VIII. REFERENCES __________________________________________________ 67
Figure Index
Figure 1 The pathway of mitochondrial β-oxidation based on Bartlett and Eaton 2004 with
alterations [12]. (ETF – Electron transfer flavoprotein; UQ – Ubiquinone; ETF:QO – Electron transfer flavoprotein-ubiquinone oxidoreductase) _________________________ 9
Figure 2 In the left side, an example of a mass spectrum from plasma acylcarnitines profile
in a LCHADD patient. (A) Acylcarnitines in plasma during an acute episode, and (B) during treatment with a low-fat and medium-chain triglycerides rich diet in the same patient [56]. In the right side, 3-OHFA results from a newborn infant with LCHADD. (C) at birth (dashed lines shows the reference intervals) and (D) showing the original profile (– ●–), as well as two samples collected after the infant was placed on formula containing medium chain triglycerides, 2 days after birth (–▲–) and 2 months after birth (–■–) (Dashed line is reference intervals) [61]. ______________________________________ 18
Figure 3 Experimental design followed in the analysis of the effect of LCHADD in the
mitochondrial proteome. ___________________________________________________ 31
Figure 4 Distribution of identified proteins by its molecular weight (kDa). ___________ 39 Figure 5 Distribution of identified proteins for their pI. __________________________ 39 Figure 6 Distribution of mitochondrial proteins identified by nanoLC-MS/MS considering
the biological function assigned by Panther. ___________________________________ 40
Figure 7 Venn diagram representing the differentially expressed proteins identified by
comparative analysis of mitochondrial fraction of each of the patients studied with control subjects. _______________________________________________________________ 41
Figure 8 Representation of up- and down regulated proteins in both patients. _________ 47 Figure 9 Distribution of the classes of differentially expressed proteins regarding their
biological function. In A is presented the profile of Patient 1 and in B the profile of Patient 2 based on GOA; In C is presented the profile of Patient 1 and in D of Patient 2 based on PANTHER. _____________________________________________________________ 49
Figure 10 Variation in expression of ATP synthase β subunit, evaluated by western blot in
the mitochondrial fractions of control subjects, Patient 1 and Patient 2. Under the graph is shown a picture of the western blot. The values (mean ± SD) are expressed in arbitrary units of optical density (OD). * p <0.05 ** p <0.01 _____________________________ 50
Figure 11 Variation in expression of MnSOD, evaluated by western blot in the
mitochondrial fractions of control subjects, Patient 1 and Patient 2. Under the graph is shown a picture of the western blot. The values (mean ± SD) are expressed in arbitrary units of optical density (OD). * p <0.05 ** p <0.01 _____________________________ 51
Figure 12 Variation in expression of paraplegin, evaluated by western blot in the
mitochondrial fractions of control subjects, Patient 1 and Patient 2. Under the graph is shown a picture of the western blot. The values (mean ± SD) are expressed in arbitrary units of optical density (OD). * p <0.05 ** p <0.01 ____________________________ 51
Figure 13 Variation in expression of ETF β subunit, evaluated by western blot in the
mitochondrial fractions of control subjects, Patient 1 and Patient 2. Under the graph is shown a representative picture of the western blot. The values (mean ± SD) are expressed in arbitrary units of optical density (OD). * p <0.05 ** p <0.01 ___________________ 52
Figure 14 Variation in expression of mtTFA, evaluated by western blot in the
mitochondrial fractions of control subjects, Patient 1 and Patient 2. Under the graph is shown a representative picture of the western blot. The values (mean ± SD) are expressed in arbitrary units of optical density (OD). * p <0.05 ** p <0.01 ___________________ 52
Figure 15 A. Representative image of the zymography gels. B. Representative image of
zymography with serine proteases inhibitor (PMSF). The respective semi-quantitative analysis of overall proteolytic activity is presented in C and D. (P1 and P2 refers to patients 1 and 2, respectively and Cont to a healthy individual) __________________________ 54
Table Index
Table 1 Enzymes of mitochondrial β-oxidation (adapted from [12, 30]). _____________ 11 Table 2 Up- and down-regulated proteins differentially expressed in patients. ________ 41 Table 3 Differentially expressed proteins identified by comparative analysis of
mitochondrial fraction of each of the patients studied with control subjects by nanoLC-MS/MS with iTRAQ labeling. The values marked with "+" represent up-regulated and "–" down-regulated proteins, comparative to control. _______________________________ 42
Newborn screening programs have been implemented in several developed countries, contributing not only to a better understanding of metabolic diseases but also to the prevention of fatal disease’s consequences. Nevertheless, there is still much debate about the pathophysiological mechanisms and the relation genotype-phenotype of these diseases [1]. With the implementation of these programs, some cases of sudden infant death syndrome [2, 3], defined as sudden unexpected and unexplained death occurring before 12 months of age, were attributed to fatty acid oxidation deficiencies. These disorders are, in general, inherited metabolic disorders and may cause as much as 5% of sudden unexpected death in infancy [4].
According to the latest annual report for early diagnosis of the National Institute Dr. Ricardo Jorge, one in 5.796 screened individuals in Portugal in 2010 had a β-oxidation associated disease. [5]. This is a concerning data and shows the crucial importance of the newborn screening program, which already allowed the characterization of some metabolic diseases regarding its incidence, underlying mechanisms and metabolites profiles [6, 7]. Mitochondrion is an essential organelle for cell metabolism [8-10]. Among the metabolic pathways that take place in mitochondria is β-oxidation [11], although this metabolic process also occurs in peroxisome. Alterations in the machinery of this metabolic pathway has serious implications for mitochondrial metabolism and consequently to human physiology. So, the clarification of the cellular mechanisms underlying LCHADD will help planning new and more specific therapeutic approaches, aiming a personalized medicine.
2.1. Fatty Acids β-oxidation
Glucose, fatty acids and amino acids are the three substrates that an organism can use to maintain metabolic homeostasis; they are required not only to generate energy, but also as building blocks for the biosynthesis of macromolecules. The relationship of fat oxidation with the utilization of carbohydrate as a source of energy is complex and depends upon tissue, nutritional state, exercise, development and a variety of other influences such as infection and other pathological states. The prime pathway for degradation of fatty acids is mitochondrial fatty acids β-oxidation [12].
β-Oxidation of fatty acids is an important metabolic process that happens in most organisms from bacteria to higher eukaryotes [13]. It was first described by George Franz Knoop in 1904, who postulated that oxidation took place on the β carbon atom. Basically, this metabolic pathway consists of successive and repetitive reactions that remove acetyl groups (as acetyl-coenzyme A) from fatty-acyl-CoA molecules. In humans, and mammals in general, two distinct β-oxidation systems exist, mitochondrial and peroxisomal [13]. Mitochondrial β-oxidation provides acetyl groups that can be degraded to CO2 and water for the production of ATP, and it is tightly coupled to mitochondrial respiratory chain [11]. In the other hand, β-oxidation in peroxisome does not necessarily go to completion, and this is a universal property of peroxisomes in most organisms. It is involved in the metabolism of a variety of fatty acids, including branched-chain fatty acids and prostaglandins [14].
In fasting conditions, when glucose supply becomes limited, fatty acids β-oxidation assumes particular importance. Under these conditions all tissues, except brain, can use fatty acids directly to generate energy [15]. In the liver, fatty acids are converted into ketone bodies that can be used as an additional energy source for all tissues including brain [16].
The primary sources of β-oxidation are dietary fatty acids and the ones mobilized from cellular stores. Fatty acids from the diet can be transported from the gut to cells where are stored in the form of triacylglycerols; these are the primary energy reserves in animals. In response to energy demands, these energetic reserves can be mobilized to be used by
tissues to generate energy. The release of metabolic energy, in the form of fatty acids, is controlled by a complex series of interrelated cascades that result in the activation of hormone-sensitive lipase [17].
Fatty acids mobilization from adipocytes starts when a hormone binds to the receptor that exists in the adipocyte membrane. After activation of the membrane receptor by glucagon, epinephrine or β-corticotropin, adenylate cyclase converts ATP into cAMP. Then, cAMP binds to a protein kinase and activates it, which in turn activates triacylglycerol lipase. Once active, triacylglycerol lipase is able to break triacylglycerols into their fatty acids components. These fatty acids are picked up by the serum albumin protein in the blood stream [18].
The uptake of fatty acids seems to be largely mediated by membrane proteins. Fatty acid transport proteins (FATP) are integral transmembrane proteins that enhance the uptake of long and very long chain fatty acids into cells. These proteins in humans comprise a family of six highly homologous proteins, FATP1–FATP6, that are found in all fatty acid utilizing tissues [19]. For example skeletal muscle express FATP1 and FATP4, whereas heart specifically expresses FATP6 and FATP1 [20], and in liver FATP5 plays an essential role in the hepatocellular uptake of fatty acids [21]. These fatty acids transport proteins present CoA synthetase activity, suggesting that fatty acids are rapidly converted to acyl-CoAs [15].
Cytoplasmic fatty acid binding proteins (FABPs) are required in order to maintain high rates of fatty acids β-oxidation in liver, heart and skeletal muscle [22-24]. There are many tissue-specific FABPs. To import acyl-CoAs into mitochondria the carnitine shuttle is used since mitochondrial membrane is impermeable to acyl-CoAs [15, 25-27], and their entry to the mitochondria is an important point of the β-oxidation flux [28].
Once in mitochondria, the acyl half can be considered as committed to complete oxidation (Figure 1). Transfer across mitochondrial membrane is accomplished by transference of the acyl group from CoA to carnitine, transfer across the inner membrane and intramitochondrial reconversion to acyl-CoA ester. This is achieved by carnitine palmitoyl transferase I (CPTI) on the outer mitochondrial membrane, carnitine acyl-carnitine translocase in the inner membrane, and carnitine palmitoyl transferase II (CPTII) on the
inner face of the inner membrane. The exchanges of acyl-carnitine for carnitine is made through the carnitine acyl-carnitine translocase and so the cytosol does not become carnitine depleted in comparison with mitochondria [12].
Figure 1 The pathway of mitochondrial β-oxidation based on Bartlett and Eaton 2004 with
alterations [12]. (ETF – Electron transfer flavoprotein; UQ – Ubiquinone; ETF:QO – Electron transfer flavoprotein-ubiquinone oxidoreductase)
Inside mitochondria, acyl-CoAs are degraded into acetyl-CoA units through the classic series of four repeated enzyme reactions (Figure 1). The first reaction is catalyzed by Acyl-CoA dehydrogenase producing trans-2,3-enoyl-Acyl-CoA; after that, there is an hydration of the double bond. In the third reaction, the resulting L-3-hydroxy-acyl-CoA is dehydrogenated
by L-3-hydroxy-acyl-CoA dehydrogenase (NAD+ linked), producing 3-keto-acyl-CoA. Finally, in the fourth reaction there is a thiolytic cleavage of the 3-keto-acyl-CoA producing a two-carbon chain-shortened acyl-CoA (saturated acyl-CoA) and acetyl-CoA [12, 15]. The first dehydrogenation step is linked to the respiratory chain via electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase, while the second dehydrogenation is linked to complex I of the respiratory chain through NADH. Therefore, the ATP production from β-oxidation comes equally from direct production of reduced cofactors, and from subsequent oxidation of acetyl-CoA [12].
In the end, the acetyl-CoA can be fully degraded to CO2 and H2O through the Krebs cycle which also involves the active participation of the mitochondrial OXPHOS system [29]. β-Oxidation of fatty acids is a long and complex pathway, which flux appears to be largely dependent on the level of acyl groups’ entry on mitochondria and on substrate supply [28].
2.1.1. Mitochondrial β-oxidation system: enzymes involved
Mitochondrial β-oxidation can be conceptually divided into two steps, the first step is the process of getting acyl groups into the mitochondrion for oxidation and the second step is the intramitochondrial chain shortening by oxidative removal of two-carbon (acetyl) [12]. Many enzymes were already implicated in mitochondrial β-oxidation and are summarized in Table 1.
Table 1 Enzymes of mitochondrial β-oxidation (adapted from [12, 30]).
Enzyme Abbreviation Structure MW
(kDa)
Acyl-CoA Synthase Unknown 78
Carnitine palmitoyl transferase I (liver) lCPT I Unknown 88 Carnitine palmitoyl transferase I (muscle) mCPT I Unknown 82 Carnitine acyl-carnitine translocase CACT Unknown 32,5
Carnitine palmitoyl transferase II CPT II Unknown 68 Acyl-CoA dehydrogenases
Very-long-chain acyl-CoA dehydrogenase VLCAD Homodimer 150 Long-chain acyl-CoA dehydrogenase LCAD Homotetramer 180 Medium-chain acyl-CoA dehydrogenase MCAD Homotetramer 180 Short-chain acyl-CoA dehydrogenase SCAD Homotetramer 168
ACAD-9 ACAD-9 Homodimer 140
Trifunctional protein MTP Heterooctamer 460
Long-chain 3-hydroxyacyl-CoA dehydrogenase LCHAD Long-chain 2-enoyl-CoA hydratase ECH Long-chain 3-oxoacyl-CoA thiolase KACT
Short-chain 2-enoyl-CoA hydratase (cronotase) SCEH Homohexamer 164 Short-chain 3-oxoacyl-CoA thiolase SCOT Homotetramer 169 Short-chain 3-hydroxyacyl-CoA dehydrogenase SCHAD Homodimer 68 General (medium-chain) 3-oxoacyl-CoA thiolase GOT Homotetramer 200
Electron transfering fravoprotein ETF Heterodimer 57
ETF-ubiquinone oxireductase ETFD Monomer 68
Carnitine acetyltransferase CAT Monomer 60
2,4-Dienoyl-CoA reductase Homotetramer 124
Short-chain Δ3,Δ2-enoyl-CoA isomerase Homodimer 70 Long-chain Δ3,Δ2-enoyl-CoA isomerase Unknown 200
Δ3,5,Δ2,4-dienoyl-CoA isomerase Homotetramer 126
There are multiple enzymes for each of the constituent steps of the pathway, which differ in their chain-length specificity. In the case of acyl-CoA dehydrogenation there are four
enzymes, each of these enzymes catalyzes the formation of 2-enoyl-CoA from the corresponding saturated ester [12, 28, 30]. These acyl-CoA dehydrogenases, as previously mentioned, are important alongside the normal performance of mitochondrial β-oxidation since they catalyze the first reaction of β-oxidation in mitochondria.
2.1.2. Mitochondrial Trifunctional Protein
The β-oxidation pathway, as stated above, involves four enzymatic activities, acyl-CoA dehydrogenase, 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT), these last three enzymes are part of mitochondrial trifunctional protein (MTP), a multienzymatic complex [29, 31].
The human MTP complex is a fatty acid β-oxidation multienzyme. MTP is a hetero-octamer that comprises of two types of subunits, four α subunits and β subunits; α, which holds the ECH and HACD activities, and β, harboring the KACT [31-33]. The two subunits of MTP are encoded by separate genes, HADHA and HADHB, respectively [34]. As judged from kinetic analysis, it is likely that mammalian MTP complex also operate by means of a channeling mechanism, in view of its similarity to the bacterial MTP [33, 35]. Mammalian MTP complex shows significant sequence insertions in both α and β subunits, however the implications of these insertions in structural and functional characteristics remain in doubt[31]. Recently, Fould et al. [31] provided the first detailed structural and functional characterization of a recombinant human MTP and opened the way to perform detailed analyses through site-directed mutagenesis.
MTP was associated with several pathologic conditions, including acute fatty liver of pregnancy (AFLP) and a defect in fatty acid oxidation [36]. There are three defects associated with fatty acid β-oxidation, described until now, linked with MTP, depending on the disturbance of a particular enzyme activity: isolated LCHADD (the most frequent of the three), complete MTP deficiency (the less common), and isolated LCKAT (Long-chain 3-ketoacyl-CoA thiolase, described recently) [37, 38].
2.1.2.1. Long-chain 3-hydroxyacyl-CoA dehydrogenase
The human LCHAD (EC 1.1.1.211) is codified by the HADHA gene, which has 54 kb, and it is localized in chromosome 2 in position 23 (2p23). LCHAD is an enzyme involved in the β-oxidation of long-chain fatty acids. LCHAD is a component of a membrane-associated MTP and displays optimal activity toward substrates with 12-16 carbons in the acyl chain [39]. LCHAD catalyze the oxidation of the hydroxyl group of 3-hydroxyacyl-CoA to a keto group with the simultaneous reduction of NAD+ to NADH, as shown is Figure 1.
There are two forms of 3-hydroxyacyl-coA dehydrogenase, LCHAD and SCHAD; these two enzymes share considerable sequence homology and display overlapping substrate specificity. SCHAD is involved in β-oxidation of fatty acids that have 4-16 carbons in the acyl chain [39-41].
2.2. Disorders of mitochondrial fatty acid β-oxidation
Fatty acids β-oxidation disorders are individually rare; however, they are collectively common because of the number of different enzymes that can be affected. These disorders are typically inherited in an autosomal recessive pattern [11]. When defects occur in fatty acids degradation, excess of acylcarnitine intermediates accumulate in tissues, including heart, liver, and skeletal muscle, potentially leading to organ dysfunction [11, 42]. These acylcarnitines that spill into blood provide a marker for diagnosis, including early detection on newborn screening [42].
A diversity of different genetic defects of mitochondrial β-oxidation has been described in humans. In addition to the uncommon primary carnitine deficiencies, other defects affect enzymes that are involved either in the entry of long-chain fatty acids in the mitochondria (deficits in CPT I, translocase or CPT II) or in the β-oxidation process itself (deficiencies in SCAD, MCAD, LCAD, VLCAD, SCHAD, LCHAD, ETF or ETF-ubiquinone oxidoreductase for example) [43].
The first documented disorder affecting the mitochondrial β-oxidation in humans was the CPT II deficiency, described in 1973 by DiMauro and coworkers [43]. Since then, more inherited β-oxidation disorders have been described, including MCAD deficiency which is the most frequent inborn error of mitochondrial fatty acid β-oxidation [43-47] and LCHADD a rare inborn FAO disorder [48].
2.2.1. LCHAD deficiency
FAO disorders have become an important group of inherited metabolic disorders characterized by an extensive range of clinical presentations. More than 20 different β-oxidation disorders have been identified [48]. LCHADD was first described in 1989 and MTP deficiency was first reported in 1992, both was described firstly by Wanders and co-workers [48]. LCHADD is a rare inborn mitochondrial error; however, since being first described isolated LCHADD is recognized as one of the more severe FAO disorders [48, 49].
A decade of experience with newborn screening for fatty acid oxidation disorders is now accessible from an increasing number of programs worldwide [1] and it allowed to reduce severe metabolic decompensations and death [50]. The main goal in treatment of LCHADD is prevention of fasting to avoid death and intellectual disability but there are some treatment recommendations, like low fat high carbohydrate diet, resulting from a consensus from the analysis of several patients [51, 52].
2.2.1.1. Clinical Aspects
In LCHADD patients, energy needs from fatty acid oxidation cannot be met during fasting periods or illness. This results in the accumulation of toxic metabolites which can lead to metabolic decompensation and severe clinical disease [53]. Current clinical therapy for mitochondrial diseases focuses more on the treatment of symptoms as an alternative of correction of the actual defective mechanism. The current management of MTP defects depends on almost entirely on dietary interventions. The main stay of long-term dietary therapy in disorders of FAO is fasting avoidance [48, 54-56].
The symptoms of LCHADD range from lethargy, cardiomyopathy, arrhythmias, hypotonia, liver dysfunction, Reye like-syndrome, retinopathy, progressive myopathy, neuropathy, to seizures or coma or even death [52, 57]. LCHADD is associated to some maternal complications including pre-eclampsia, HELLP (hemolysis, elevated liver
enzymes, low platelet count), AFLP (acute fatty liver pregnancy) and hyperemesis gravidarum (severe form of morning sickness, with excessive pregnancy-related nausea and/or vomiting that prevents adequate intake of food and fluids) [57].
In patients from northern Australia, Germany or United States the incidence of LCHADD is lowest, affecting about 1:250.000 to 1:750.000 individuals [1]. In Portugal, according to the latest report of the National Program of Early Diagnosis of the National Institute Dr. Ricardo Jorge, the prevalence of LCHADD was 1:7.866 in 2010, while in 2009 was 1:112.248 [5, 58].
2.2.1.2.
Biochemical Diagnosis
The diagnosis of LCHADD requires an integrated interpretation of multiple tests. When a suspicion of LCHADD in a patient exists, a differential diagnosis must be made because this disease has clinical signs that are common to other errors of mitochondrial fatty acid β-oxidation [59].
Changes in free fatty acid profile in plasma and urine are biochemically associated with LCHADD. As a result of enzyme block, long-chain hydroxyacyl-CoA esters accumulate in the mitochondria and are eliminated in the form of glycine conjugates in urine. Abnormalities in the acylcarnitine profile are usually very characteristic, with 3-hydroxy C16:0-, C16:1-, C18:0- and C18:1- acylcarnitines most dominant in plasma [54, 60].
The metabolites, with the exception of plasma acylcarnitines, are usually detected during periods of acute illness and very rarely during asymptomatic periods. For the diagnosis of LCHADD several analytical methods are used such as gas chromatography (GC) , gas chromatography couple to mass spectrometry (GC-MS) [61] and more recently tandem mass spectrometry (MS/MS) [51, 52, 62]. This technique, MS/MS, is currently the method of choice for the detection of metabolic errors in newborns [51]. In several countries, including Portugal, the inclusion of this technique in newborn screening programs made possible the identification of pre-symptomatic patients and a better understanding of hereditary diseases in fatty acids metabolism, such as LCHADD [1]. If a mass
spectrometry analysis evidences the increase of some specific acylcarnitines, this is probably a patient with LCHADD [56, 61]. In figure 2 is given an example of a tandem MS spectrum for a LCHAD deficient plasma sample (A) and a LCHAD deficient treated plasma sample (B).
Figure 2 In the left side, an example of a mass spectrum from plasma acylcarnitines profile in a
LCHADD patient. (A) Acylcarnitines in plasma during an acute episode, and (B) during treatment with a low-fat and medium-chain triglycerides rich diet in the same patient [56]. In the right side, 3-OHFA results from a newborn infant with LCHADD. (C) at birth (dashed lines shows the reference intervals) and (D) showing the original profile (–●–), as well as two samples collected after the infant was placed on formula containing medium chain triglycerides, 2 days after birth (– ▲–) and 2 months after birth (–■–) (Dashed line is reference intervals) [61].
In Figure 2 are shown the results obtained by Jones and Bennett [61], evidencing the 3-hydroxy fatty acids profile in an infant patient with LCHADD and the same profile in the same patient after two days of the infant placed on a medium-chain triglycerides diet and after two months after the birth. As can be depicted from Figure 2, in LCHADD patients long chain 3-hydroxy fatty acids are increased. Individuals with this disorder present persistently elevated 3-hydroxy fatty acids species of chain lengths of C16-C18 consistent with enzyme function. The confirmation of a diagnosis of LCHADD can be complemented by the determination of enzyme activity in fibroblasts, leukocytes or lymphocytes of the patient [29].
In Figure 2 are also shown the results of a treatment based on the dietary recommendations for these patients. In patients with LCHADD or even MTP deficiency, long-chain fatty acids intake should be as low as possible in order to prevent the long term neuropathic symptoms. So, in newborns a special formula with low long-chain triglycerides and high medium-chain triglycerides is also recommended. These recommendations must be followed immediately after the screening results became available and even before its confirmation, since these disorders have a high mortality rate in the first days and weeks of life [51]. These dietary treatments make possible the patient survival and avoid the devastating consequences of the disease until the definition of a personalized therapeutic approach.
During these dietary treatments the patients must be regularly monitored through the analysis of plasma free carnitine, acylcarnitines, erythrocyte fatty acid profile and creatine kinase. These parameters may be helpful in the evaluation of treatment efficiency [63]. LCHADD frequently manifests with sudden and unexpected death. If an autopsy is performed, diffuse fatty infiltration of the liver and other organs is often presented. Although collection and biochemical testing of tissues and cultured skin fibroblasts are possible, these approaches have been deemed impractical and with limited application. On the other hand, postmortem blood and bile could be routinely collected and spotted on a filter paper card of the same kind used for newborn [2]. Collection of specimens provides a better chance of detection or diagnosis confirmation, which can help a family to understand a sudden death history with they are related [4].
2.2.1.3.
Molecular Characterization
The majority (about 80 %) of patients with isolated LCHAD deficiency has a mutation G1528C (E474Q), which is the most frequent mutation in HADHA gene, located in the catalytic site of the LCHAD domain [64]. This mutation alters amino acid 474 from glutamic acid to glutamine (E747Q), replacing the acidic and negatively charged side chain with neutral, amide containing residue. This affects the NAD+ binding site of LCHAD, conducing to a loss of enzyme activity of LCHAD alone, without affecting the other two
enzymatic activities of MTP [36]. This mutation blocks the β-oxidation pathway resulting in the accumulation of 3-hydroxy fatty acid metabolites [48]. Other mutations have been described in patients with isolated LCHADD, but are very rare [49].
Although this disease was first diagnosed about 20 years ago and more information has been obtained with the newborn screening programs using mass spectrometry as a mean of diagnosis, there are still many doubts about its prevalence and on their clinical, biochemical and genetic features. Protein profiling of cells (or subcellular fractions) isolated from FAOD patients appears as an attractive approach to have an integrated perspective of disease-related alterations in cellular pathways [53, 65, 66].
2.3. Mitochondrial proteome analyses for LCHAD deficiency
characterization
The true extent of fatty acids β-oxidation disorders on mitochondrial and cellular functions is still unknown [66, 67], although it is believed that pathophysiological mechanisms leading to clinical phenotype rely mostly on the resulting energy deficiency [65]. To better characterize the degree of mitochondrial dysfunction in fatty acid oxidation disorders, and so contribute to the elucidation of the pathophysiological mechanisms underlying disease development, the characterization of mitochondrial proteome might be a powerful approach [65].
It has been estimated that there are approximately one thousand or more different polypeptides in mitochondria, which amount might vary in response to distinct pathophysiological stimuli [68]. Indeed, mitochondrial dysfunction have been implicated in numerous diseases including heart diseases, neurological diseases like Parkinson or Alzheimer, aging, cancer or disorders related with fatty acid oxidation [43, 68]. The identification and characterization of the majority or ideally all of the polypeptides that exists in mitochondria would be vital, for the reason that this organelle plays crucial and diverse roles in several cellular processes that are compromised in several diseases [8-10]. There are several methodological approaches that can be used on mitochondrial protein profiling but they mainly rely on the electrophoretic or chromatographic separation of mitochondrial proteins [68]. A critical issue in mitochondrial protein analysis is organelle isolation from tissue or cell samples. Fibroblasts are the typical samples used in the genetic or biochemistry characterization of fatty acid oxidation diseases [65, 69-72]. Skin biopsies are performed in order to isolated fibroblasts. For proteomic analysis, a considerable amount of isolated fibroblasts is needed (around 107 cells) [72]. Then mitochondria are isolated from fibroblasts by differential centrifugations using specific buffers with sucrose in their composition. The purity of the fractions obtained can be assessed by electron microscopy or by western blotting targeting specific proteins of endoplasmic reticulum, lysosomes or Golgi apparatus, the most probable contaminants in mitochondria enriched fractions [73].
Once prepared mitochondrial fractions, protein separation is performed. Two-dimensional (2D) gel electrophoresis is the preferred method to resolve and array proteins from cellular mixtures. Combined with mass spectrometry (MS) techniques, 2D gels allow the simultaneous analysis of thousands of protein species [74]. Proteins’ mass mapping using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry has become the preferred high throughput technique for initial identification of protein spots from 2D gels. Protein identifications made from peptide mass fingerprints can be confirmed using tandem mass spectrometry [74, 75]. Questions about the limitations of 2D gels with regard to the resolution of low abundance or hydrophobic proteins brought the search for methods that are unbiased in these aspects, although recent reports have shown that these perceived limitations can be overcome [76]. Methods of protein fractionation that focus on the solubilization and fractionation of hydrophobic and membrane proteins are especially valuable for proteomic studies of mitochondria. Several mitochondrial proteins have a basic pI (isoelectric point), and a large number are low molecular weight, so the fractionation methods that specifically address these properties will be most useful for the subsequent identification and characterization of mitochondrial proteins [68].
Liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) is also a powerful technique for the analysis of peptides and proteins. This methodology combines efficient separation of biological materials by liquid chromatography and sensitive identification of the individual components by mass spectrometry. LC-MS/MS can be used alone or in combination with 1D or 2D electrophoresis, immunoprecipitation, or other protein purification techniques [77]. Even though numerous methods for coupling liquid chromatography to mass spectrometry have been explored, it is electrospray ionization that has transformed LC-MS/MS into a routine laboratory method sensitive enough to analyze peptides and proteins at interesting levels in biological research [78]. Recent developments in instrument sensitivity, software control, automation, and data analysis tools provided unique high throughput capabilities for protein analysis when using LC-MS/MS [77]. Although powerful biochemical information can be obtained with these methodological approaches, few studies have characterized the mitochondrial proteome of fibroblasts [65, 69, 70, 79] and even fewer have studied fatty acid oxidation diseases-related mitochondrial proteome alterations [65, 70]. Using 2D-MS/MS, Rocha et al. [65] identified 287 distinct
mitochondrial proteins in mitochondria isolated from fibroblasts and 35 proteins were found differentially expressed in MADD patients, compared with control individuals, most of them from the functional clusters “metabolism” and “protein binding/folding”. In the study of Palmfeldt et al. [69], performed with LC-MS/MS, 38 proteins were found differentially expressed and were related to mild metabolic stress.
Although powerful, these mass-spectrometry based methodological approaches have not been explored in the study of other metabolic diseases, leaving open the opportunity of future studies. The evaluation of mitochondrial proteome plasticity in LCHADD will certainly give new insights in the pathophysiological changes that underlie disease-related phenotypic alterations and in the interplay genotype-phenotype.
The aim of the present study was to evaluate de plasticity of mitochondrial proteome in LCHADD, to better understand the pathophysiology of this disease. In order to reach this it was our purpose to:
a) Characterize the mitochondria proteome using liquid chromatography coupled to tandem mass spectrometry – nanoLC-MS/MS;
b) Identify the protein differentially expressed in LCHADD patients using iTRAQ labeling and contextualize those differentially expressed proteins in the molecular pathways;
c) Evaluate the effect of LCHADD in mitochondrial proteolytic activity; The integrative analysis of all data will allow identifying the molecular pathways modulated by LCHADD and mechanistically explain the phenotype related with this disease.
A better comprehension of the molecular mechanisms underlying LCHADD will ideally give clues for the development of more specific and effective diagnostic methods and new therapeutic approaches.
4.1. Experimental design
In order to fulfill the objectives proposed, the experimental protocol presented in Figure 4 was followed.
Figure 3 Experimental design followed in the analysis of the effect of LCHADD in the mitochondrial proteome.
The proteomic approach used in the present study was based on the characterization of the protein profile of mitochondria isolated from fibroblasts of LCHADD patients using nanoLC-MS/MS. The expression of some target proteins (paraplegin, ETF subunit β, MnSOD, mtTFA and ATP synthase subunit β) were evaluated by western blot and the mitochondria proteolytic activity was evaluated by zymography.
4.2. Samples characterization
Samples from LCHADD patients were selected by the National Institute Dr. Ricardo Jorge. Two patients with a mild LCHADD were selected. These patients are homozygous for G1528C (E474Q), the most common mutation for LCHAD deficit, and present similar clinical symptoms. Control samples (n=2) were obtained from healthy subjects.
All samples were collected after informed consent and were used anonymously.
4.3. Cell Culture
Skin fibroblasts were collected by biopsy from two healthy individuals (controls) and two LCHADD patients. These skin fibroblasts were grown in Ham F10 nutrient medium supplemented with 10 % fetal calf serum, 2 mmol/L glutamine, 1 % penicillin, streptomycin and fungizone, in 75 cm2 culture flasks, at 37 ºC. Ten culture flasks from each sample (which correspond to approximately 107 cells) were grown to confluence before mitochondria isolation.
4.4. Preparation of Mitochondria-enriched fraction
Mitochondria were isolated based on the experimental protocol proposed by Schwab et al. [72] that uses differential centrifugation. Confluent fibroblasts were harvested with trypsin and washed twice with PBS (phosphate buffer saline). The cellular pellet was suspended in isolation buffer containing 250 mM sucrose, 1 mM EGTA, 10 mM HEPES, 5 g/L BSA pH 7.5 and then was centrifuged at 500xg, at 4 ºC for 2 minutes. The supernatant was discharged and the remaining pellet was suspended in isolation buffer. The cell suspension was then homogenized in a tight-fitting Potter-Elvejhelm. After centrifugation at 1500xg, at 4 ºC for 10 minutes, the supernatant was kept on ice. The pellet was homogenized and centrifuged as described above. The two supernatants were pooled and centrifuged at
10000xg, at 4 ºC for 10 minutes. The resultant mitochondrial pellet was washed with BSA-free isolation buffer and stored at -80 ºC, for further analyses. An aliquot was used for total protein quantification using a commercial kit RC DC Protein Assay (BioRad®) which is based on Lowry et al. [80] method. It was made a calibration curve with standard solutions of bovine serum albumin (BSA), at concentrations between 10 and 0.625 mg/mL. The absorbance was read at 750 nm in a plate reader (Multiskan Go, Thermo Scientific®).
4.5. NanoLC-MS/MS with iTRAQ labeled analysis
An in-solution digestion was performed for iTRAQ labeling as previously described [81]. Briefly, 100 µg of protein was used for digestion which was performed according to the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA). Briefly, samples were mixed with triethyl ammonium bicarbonate buffer (TEAB) (1 M, pH 8.5) and RapiGest (Waters) to a final concentration of 0.5 M and 0.1 %, respectively. Samples were then reduced with 5 mM tris(2-carboxyethyl) phosphine (TCEP) for 1 h at 37 C and alkylated with 10 mM S-Methyl methanethiosulfonate (MMTS) for 10 min at RT. Two micrograms of trypsin were added to each sample and the digestion was performed for 18 h at 37 C. Samples were dried in a SpeedVac (Thermo Savant).
Digested sample peptides were subsequently labeled with the iTRAQ reagents (8-plex) following the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA). In brief, peptides were reconstituted in 70 % ethanol/ 30 % TEAB 500 mM, added to each label and carried out for 2 h at room temperature. The reaction was stopped by adding water and the labeled digests corresponding to each of the four 8-plex experiments were combined and dried using SpeedVac.
Labeled mitochondrial peptides were separated by a multidimensional LC approach based on a first dimension with high pH reverse phase (as previously described [82]) and a second dimension with the acidic reverse-phase system.
Sample loading was performed at 200 µL/min with buffers (A) 72 mM TEA, 52 mM acetic acid in H2O, pH 10 and (B) 72 mM TEA, 52 mM acetic acid in ACN, pH 10 (98 % A: 2 %
B). After 5 min of sample loading and washing, peptide fractionation was performed with linear gradient to 50 % B over 35 min followed by a 100 % B step. Sixteen fractions were collected, evaporated, and ressuspended in 2 % ACN, 0.1 % TFA.
Collected fractions were separated as previously described [83]. Briefly, peptides loaded onto a C18 pre-column (5 µm particle size, 5 mm, from Dionex) connected to an RP column PepMap100 C18 (150 mm × 75 µm I.D., 3 µm particle size). The flow rate was set at 300 nL/min. The mobile phases A and B were 2% ACN 0.1% TFA in water and 95 % ACN, 0.045 % TFA, respectively. The gradient was started at 10 min and ramped to 60 % B till 50 min and 100 % B at 55 min and retained at 100 % B till 65 min. The column was equilibrated with solvent A for 20 min before the next sample was injected. The separation was monitored at 214 nm using a UV detector (Dionex/LC Packings, Sunnyvale, CA) equipped with a 3 nL flow cell. Using the micro-collector Probot (Dionex/LC Packings) and, after a lag time of 5 min, peptides eluting from the capillary column were mixed with a continuous flow of α-CHCA matrix solution (270 nL/min, 2 mg/mL in 70 % ACN/0.3 % TFA and internal standard Glu-Fib at 15 fmol) were directly deposited onto the LC-MALDI plates at 12 seconds intervals for each spot (150 nL/fraction). For every separation run, 208 fractions in total were collected.
The spectra were processed and analysed by the ProteinPilot software (v4.0 AB Sciex, USA), which uses paragon algorithm for protein/peptide identification based on MS/MS data against the SwissProt protein database (release date 01012011, all taxonomic categories). Default search parameters were used: trypsin as the digestion enzyme, Methylthio on Cysteine residue as fixed modification, iTRAQ 8Plex, biological modification with emphasis on phosphorylation and urea denaturation as the variable modification setting. Mass tolerances for precursor and fragments were default values for ProteinPilot®. Cut-off score value for accepting protein identification for ProteinPilot® was a ProteoScore of 1.3 (95% confidence).
Data was normalized for loading error by bias correction, which is an algorithm in Protein Pilot that corrects for unequal mixing when combining the labeled samples of one experiment. It does so by calculating the median protein ratio for all proteins reported in each sample, adjusted to unity and assigning an autobias factor to it. Nevertheless, the
quantification results were reviewed manually for all proteins found to be differentially expressed (iTRAQ ratio > 1.3 or < 0.7 according to [84])
4.6. Western Blotting
For the expression analysis of target proteins, equal amounts of proteins from each sample were loaded on a 12.5 % SDS-PAGE gel prepared according to Laemmli [85]. After separation at 180 V, proteins were transferred from the gel to a nitrocellulose membrane (Millipore®, 0.45 µm porosity) by electroblotting for 2 hours at 200 mA in transfer buffer (25 mM Tris, 192 mM glycine, 20 % methanol). After blotting, non-specific binding was blocked with 5 % (w/v) nonfat dry milk in TTBS (100 mM Tris pH 8.0, 150 mM NaCl, and 0,05 % Tween 20) and the membrane was incubated with primary antibodies diluted 1:1000 in 5% (w/v) nonfat dry milk in TTBS (rabbit polyclonal anti-paraplegin, sc-135026, Santa Cruz Biotechnology; rabbit polyclonal anti-Tfam, cat. no. ab47548, abcam; mouse monoclonal ATP synthase subunit β, ab14730, abcam; mouse monoclonal anti-MnSOD, ALX-804-265, Alexis; , or anti-ETF β subunit, ab73986, abcam) for 2 hours at room temperature or overnight at 4°C. After 3 washes for 10 minutes with TTBS, membranes were incubated with secondary horseradish peroxidase-conjugated antibody (GE Healthcare®) for 1 hour, and after that 3 washes were made for 10 minutes with TTBS.
The blots were developed using the chemiluminescence ECL reagent (Amersham Pharmacia Biotech®, Buckinghamshire, UK), followed by exposure to X-ray films (Kodak Biomax Light Film, Sigma®, St. Louis, USA). The protein bands on films were visualized using a GelDoc XR (Bio-Rad®) and quantified by QuantityOne Imaging software (v4.6.3, Bio-Rad®, Hercules, CA).
4.7. Zymography
Zymography assays were performed according Caseiro et al. [86]. Briefly, 10 µg of protein was mixed with sample buffer (100 mM Tris-HCl pH 6.8, SDS 5 %, Glycerol 20 % and bromophenol blue 0.1 %) and loaded onto a 10 % SDS-PAGE gels impregnated with 0.1 % porcine gelatin. After electrophoresis, gels were washed twice for 30 min each with 2.5% Triton X-100 solution. Then, one gel was incubated overnight at 37 °C in a developing buffer (50 mM Tris–HCl pH 7.4, 10 mM CaCl2 and 10 mM ZnCl2) and other was incubated at 37 °C in a developing buffer with PMSF (50 mM), serine proteases inhibitor. After incubation, gels were stained with Colloidal Coomassie Blue (0.12 % (w/v) Coomassie blue G250, 20 % (v/v) methanol) for 4 h and then destained with (25 % (v/v) methanol) until bands resulting from proteolytic activity were observed. Gels were scanned with Gel Doc XR System (Bio-Rad).
4.8. Statistical analysis
The statistical analysis was performed using GraphPad Prism. Differences between LCHADD patients and controls were evaluated with a t-test. A p value <0.05 was considered significant. Values are presented as mean ± SD.
5.1. Mitochondrial proteome analysis by nanoLC-MS/MS
In order to evaluate the effect of LCHADD on the mitochondrial protein profile, nanoLC-MS/MS analysis of mitochondria-enriched fractions isolated from skin fibroblasts of LCHADD patients and controls was performed. From this analysis 729 proteins were identified, with a confidence degree over 95% (Table A, in appendix).
These identified proteins were grouped according to their molecular weight and the profile obtained is shown in Figure 4.
<10 10 - 20 20 - 30 30 - 40 40 - 50 50 - 60 60 - 70 70 - 80 80 -90 90 - 100 >100 0 5 0 1 0 0 1 5 0 MW (k Da)
Figure 4 Distribution of identified proteins by its molecular weight (kDa).
From the 729 identified proteins, 373 present a molecular weight in the range of 10 to 40 kDa; these proteins may be considered small proteins. Also worth of note are the 86 proteins with a molecular weight higher than 100 kDa.
The identified proteins were also distributed according to their isoelectric point (Figure 5).
< 4 4 - 5 5 - 6 6 - 7 7 - 8 8 - 9 > 9 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 pI
This distribution reveals that the proteins more frequent in fibroblasts mitochondria are the ones with pI between 5 and 6. In this range of pI, 217 proteins were included.
Identified proteins were also grouped according to the functional clusters to which they belong. Clusters assignment of these proteins was performed with the bioinformatic tool PANTHER (www.pantherdb.org) (Figure 6).
Figure 6 Distribution of mitochondrial proteins identified by nanoLC-MS/MS considering the
biological function assigned by Panther.
The most significant cluster of identified proteins was “metabolism” (47%) followed by “transport” (15%) and “signal transduction” (12%). The “metabolism” cluster includes proteins like α-enolase and mitochondrial trifunctional protein. VDAC protein is included in “transport” cluster and proteins like anexins A2 and A6 are included in “signal transduction” cluster.
Less representative clusters like “apoptosis” and “OXPHOS” include proteins like galectin-3 and ATP synthase, respectively.
47% 15% 12% 8% 5% 6% 3% 2% 1% 1% Metabolism Transport Signal Transduction Developmental Process Cell comunication
Cellular component organization OXPHOS
Generation of precursor metabolites and energy
Response to stimulus Apoptosis
5.2. Effect of LCHAD deficiency on mitochondrial proteome
Using nanoLC-MS/MS with iTRAQ (Isobaric tags for relative and absolute quantitation) labeling it was possible to obtain a relative quantification of proteins expression between patients and controls and so identify the proteins differentially expressed.
After the comparison of the mitochondria proteome between each patient and control subjects based on iTRAQs ratios, 101 differentially expressed proteins were identified. Forty of these proteins were common to Patient 1 and Patient 2, as can be depicted from the Venn diagram presented in Figure 7.
Figure 7 Venn diagram representing the differentially expressed proteins identified by
comparative analysis of mitochondrial fraction of each of the patients studied with control subjects.
In Patient 1 there were 63 differentially expressed proteins while in Patient 2 there were 78. Although 40 proteins were common, 23 were only expressed in Patient 1 and 38 were exclusively expressed in Patient 2. These differentially expressed proteins are shown in Table 3.
A deeper examination of these differentially expressed proteins allowed us to verify that in Patient 1, 36 of these proteins were over-expressed and 27 were under-expressed while in Patient 2, 52 proteins were over-expressed and 26 were under-expressed (Table 2).
Table 2 Up- and down-regulated proteins differentially expressed in patients.
Patient 1 Patient 2
Differentially expressed 63 78
up-regulated 36 52
In Table 3 are presented the differentially expressed proteins that were identified by nanoLC-MS/MS with iTRAQ labeling, through the comparative analysis of the mitochondrial fraction from each of the patients studied with control subjects.
Table 3 Differentially expressed proteins identified by comparative analysis of mitochondrial fraction of each of the patients studied with control subjects by nanoLC-MS/MS with iTRAQ labeling. The values marked with "+" represent up-regulated and "–" down-regulated proteins, comparative to control.
Protein Acession Number Funtional Cluster Patient 1 Patient 2
10 kDa heat shock protein,
mitochondrial CH10_HUMAN
Protein
binding/folding – – 1.55 14-3-3 protein zeta/delta 1433Z_HUMAN Signal Transduction – + 1.70
2-oxoglutarate dehydrogenase, mitochondrial ODO1_HUMAN Generation of precursor metabolites and energy / Metabolism / Redox – – 1.70
40S ribosomal protein S15 RS15_HUMAN DNA/RNA/Protein
Biosynthesis + 1.43 – 40S ribosomal protein S18 RS18_HUMAN DNA/RNA/Protein
Biosynthesis – + 1.21
60 kDa heat shock protein,
mitochondrial CH60_HUMAN
Apoptosis / Protein
binding/folding – 1.05 – 60S ribosomal protein L24 RL24_HUMAN DNA/RNA/Protein
Biosynthesis + 1.37 – 60S ribosomal protein L29 RL29_HUMAN Metabolism + 1.75 + 1.61
60S ribosomal protein L5 RL5_HUMAN Protein
binding/folding + 1.48 – 78 kDa glucose-regulated
protein GRP78_HUMAN
Protein
binding/folding – 1.30 + 1.11 A-kinase anchor protein 2 AKAP2_HUMAN Protein
binding/folding – + 1.71 Alpha-actinin-1 ACTN1_HUMAN Apoptosis / Protein
binding/folding + 1.26 + 1.41 Alpha-actinin-4 ACTN4_HUMAN Transport + 1.38 + 1.61
Alpha-enolase ENOA_HUMAN Metabolism + 1.43 + 1.62
Aminopeptidase N AMPN_HUMAN Proteolysis + 1.48 + 1.01 Annexin A2 ANXA2_HUMAN Signal Transduction + 2.56 + 1.91 Annexin A5 ANXA5_HUMAN Signal Transduction – 1.13 + 1.02 Annexin A6 ANXA6_HUMAN Signal Transduction + 1.58 + 1.23 ATP synthase subunit
alpha, mitochondrial ATPA_HUMAN OXPHOS – 1.35 – 1.33 ATP synthase subunit
beta, mitochondrial ATPB_HUMAN
Generation of precursor metabolites
and energy / OXPHOS
Brain acid soluble protein
1 BASP1_HUMAN
Protein
binding/folding + 1.06 + 1.61 Calmodulin CALM_HUMAN Signal Transduction + 2.32 + 1.96
Calnexin CALX_HUMAN Protein
binding/folding – 1.56 – 1.37 Calreticulin CALR_HUMAN Protein binding/folding / Signal Transduction – 1.32 –
Calumenin CALU_HUMAN Protein
binding/folding – + 1.39
Cathepsin D CATD_HUMAN Proteolysis – + 1.15
CD166 antigen CD166_HUMAN Signal Transduction – + 1.68
Citrate synthase, mitochondrial CISY_HUMAN Generation of precursor metabolites and energy / Metabolism – – 1.27
Cofilin-1 COF1_HUMAN Signal Transduction + 1.89 + 1.85 Collagen alpha-1(I) chain CO1A1_HUMAN Morphogenesis /
Signal Transduction – + 2.02 Collagen alpha-1(VI)
chain CO6A1_HUMAN Structure + 1.58 + 1.42
Collagen alpha-2(I) chain CO1A2_HUMAN Structure + 1.35 + 1.83 Collagen alpha-3(VI)
chain CO6A3_HUMAN
Morphogenesis /
Signal Transduction – + 1.01 Cytochrome b-c1 complex
subunit 1, mitochondrial QCR1_HUMAN
Generation of precursor metabolites and energy / Proteolysis / Redox – – 1.58 Cytochrome b-c1 complex subunit 7 QCR7_HUMAN Generation of precursor metabolites
and energy / Redox
– – 1.30 Cytochrome c CYC_HUMAN Apoptosis / Generation of precursor metabolites and energy / OXPHOS – 1.01 – Cytoskeleton-associated
protein 4 CKAP4_HUMAN Structure – 1.54 – 1.03
Dolichyl- diphosphooligosaccharide--protein glycosyltransferase subunit 1 RPN1_HUMAN Protein binding/folding – 1.45 – 1.23 Endoplasmin ENPL_HUMAN Response to stimulus / Protein binding/folding – 1,30 –
Ezrin EZRI_HUMAN Protein bindig/folding + 1,58 + 1,71 Fibronectin FINC_HUMAN Morphogenesis / Signal Transduction / Transport + 1.06 + 1.18 Fructose-bisphosphate
aldolase A ALDOA_HUMAN Metabolism + 1.55 + 1.52
