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1.  Introduction

1.1. Introduction to mitochondria

1.1.1. Mitochondrial morphology, biogenesis and composition

Mitochondria are essential cell organelles in the cytoplasm which have a double-membrane. They are thought to have arisen about 1.5 billion years ago and to originate from a symbiotic association between oxidative bacteria and glycolytic proto-eukaryotic cells [Margulis, 1974]. “Modern” mitochondria retain a number of features that reflect their endosymbiotic origin. These include the double membrane structure and a bacteria-like circular mitochondrial genome with mitochondria-specific transcription, translation, and protein assembly systems [Margulis, 1974; Gray et al., 1999; Lopez et al., 2002].

Mitochondria are made up of two highly specialized membrane systems. These are the inner and the outer membranes. In the center of the mitochondrion and between the membranes there are two aqueous compartments: the matrix and the inter-membrane space [Frey et al., 2000]. The two membrane systems contain carrier proteins and channels that regulate the exchange of substrates between the compartments. The inner membrane is especially rich in proteins, e.g. the high molecular weight multi-protein-complexes of the respiratory chain are located at the inner mitochondrial membrane. The total number of different proteins or polypeptides making up a mitochondrion is estimated to be around 1000 [Lopez et al., 2002].

1.1.2. Functions of the mitochondria

Mitochondria serve many important functions for the cell. These are the oxidative ATP-production, the degradation of fatty acids, the modulation of intracellular calcium homeostasis and a major role in cell signaling and apoptosis, as well as biosynthesis (e.g. heme-groups, nucleotides, and amino acids) and degradation (e.g. urea cycle) of metabolites [Lopez et al., 2002]. Below I describe the functions of the mitochondria shortly:

1.1.2.1. Oxidative phosphorylation

The oxidative phosphorylation takes place in the mitochondrion and is the main pathway of oxidative ATP-production in animals, plants and many forms of microbial life (e.g. yeast). One mole ATP hydrolyzes into one mole ADP and inorganic phosphate with concomitant release of 3054 Joules. This free energy can be made available to all cellular compartments that take up ATP. Most mammalian cells rely on the ATP produced this way for survival and anabolism [Grossman et al., 1996]. The respiratory chain-oxidative phosphorylation system consists of five multi-subunit enzyme complexes [Smeitink et al., 2001]. Mitochondrial complexes I, III and IV function as proton pumps to generate an electro-chemical gradient across the inner membrane. This proton gradient is then utilized by the ATP-synthase (complex V) to generate ATP from ADP and inorganic phosphate.

1.1.2.2. β-Oxidation

The carnitine-dependent transport of fatty acids and their βoxidation is another important metabolic pathway located in the mitochondrion. Most of the fatty acids to be oxidized for energy production by intra-mitochondrial β-oxidation have to be transported from the cytosol into the mitochondrion. For transport, the fatty acids are first esterified with Coenzyme A (CoA) for “activation”, and are then coupled to carnitine to transverse the mitochondrial dou[page 2↓]ble membrane. All enzymes of the β-oxidation are mitochondrial enzymes [Stryer, 1995; Kerner et al., 2000]. Acetyl-CoA, NADH, and FADH2, which are generated in each round of fatty acid oxidation, will later be channeled either into the citric acid cycle or directly into the respiratory chain to produce ATP.

1.1.2.3. Citric acid cycle

The citric acid cycle, also named the “Krebs’ cycle” or “tricarboxylic acid cycle”, is located in the mitochondrion too. This is the final common pathway for different metabolites such as carbohydrates, fatty acids and amino acids. The details of this cycle are shown in Fig. 1-1. The compounds with a high redox-potential [reduced nicotinamide-adenine-dinucleotide (NADH) and reduced flavin-adenine-dinucleotide (FADH2)], which are generated in this cycle, are later delivered to the respiratory chain of the mitochondrion in order to generate ATP.

Fig. 1-1: The citric acid cycle is the final common pathway for the oxidation of fuel molecules. Most fuel molecules enter the cycle as acetyl-CoA. The cycle starts with the fusion of oxaloacetate and acetyl-CoA to citrate. Citrate then undergoes a series of isomerisation-, oxidation-, and decarboxylation-steps that finally regenerate oxaloacetate. The free energy of these intermittent steps is used for the reduction of three molecules NAD+ and one molecule FAD+ . The NADH- and FADH2 -molecules thus generated, are subsequently delivered to the respiratory chain of the mitochondrion to generate ATP.

1.1.2.4. Urea cycle

The urea cycle has a role in the degradation of amino acids. It is partially located in the mitochondria of liver cells. In this pathway ammonia is detoxified, which is a by-product of amino acid catabolism. The cycle comprises four reactions and enzyme systems. The first reaction, the formation of citrulline from ammonia and ornithine, takes place in the matrix of the mitochondrion. Citrulline is then exported from the mitochondrion to the cytosol, where the other steps of the urea cycle take place [Krebs et al., 1932; Katunuma et al., 1966]. The details of this cycle are shown in Fig. 1-2.


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1.1.2.5.  Heme biosynthesis

Heme, which is needed as a prosthetic group in several important proteins such as hemoglobin, myoglobin and cytochrome C, is partly synthesized in the mitochondrion. The condensation of succinyl-CoA and glycine to δ-aminolevulic acid is the key-step of the heme-synthesis and takes place in the mitochondrion. δ-Aminolevulic acid is then delivered into the cytosol where coproporphyrinogen III is formed after a series of reactions. This molecule later returns into the mitochondrion to be converted into heme. The details of this process are depicted in Fig. 1-3.

Fig. 1-2: The urea cycle is part of the degradation pathway of amino acids. It converts the NH4 + generated by amino acid degradation into urea. The first reaction of the urea cycle ― the condensation of ornithine and carbamaylphosphate - takes place in the mitochondrial matrix. Citrulline is then exported into the cytosol.

Fig. 1-3: The heme biosynthesis occurs partly in the mitochondrion and partly in the cytoplasm. The first step (the condensation of succinyl-CoA and glycine to δ- aminolevulic acid) and the final two steps (production of heme) take place in the mitochondrion. Most of the intermediate steps take place in the cytoplasm.


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1.1.2.6.  Apoptosis

In recent years mitochondria have been discovered to be able to initiate apoptosis by the release of several mediators like cytochrome c and apoptosis-inducing factor. These mediators activate the caspase family proteases which result in apoptosis [Osiewacz, 1997; Green et al., 1998].

Beyond that there are still other biochemical pathways located in the mitochondrion such as pathways for iron metabolism and for calcium signaling. Recent findings also indicate that mitochondria appear to be responsible for functional age-related impairments of human tissues and organs [Osiewacz, 2002] and may influence cellular mechanisms and pathways located in the cytosol such as insulin secretion [Green et al., 1998].

1.1.3. Mitochondrial genetics

Each mitochondrion contains up to 10 copies of mitochondrial DNA (mtDNA). The mtDNA, which was completely sequenced in 1981 [Anderson et al., 1981], is a 16.56 kbp circular and double-stranded molecule. It encodes 13 polypeptides, 12S and 16S rRNA and 22 transfer-RNAs. All of these products are essential for the formation of a functional mitochondrion. All 13 polypeptides encoded by the mtDNA are components of the respiratory chain complexes. However, the total number of polypeptide subunits of all five mitochondrial respiratory complexes exceeds 88 [Lestienne, 1992; “Neuromuscular Disease Center” (see list of internet sites)]. Four of five enzyme complexes of the respiratory chain-oxidative phosphorylation system are encoded by both the nuclear DNAand the mtDNA. Only complex II (succinate: ubiquinone oxidoreductase; SDH) is made up exclusively of four nuclear encoded polypeptides. Seven of the 43 subunits of complex I (NADH: ubiquinone oxidoreductase), one of the eleven subunits of complex III (ubiquinol: cytochrome c oxidoreductase), three subunits of 13 subunits of complex IV (cytochrome c oxidase; COX), and two membrane components of complex V (adenosine triphosphate (ATP) synthase) are encoded by the mtDNA [Pesole et al., 2000].

The genetics of vertebrate mtDNA is characterized by these unique features:

Therefore, in inherited mitochondrial diseases the genetic defect might reside in the mitochondrial DNA or in the nuclear DNA. For example, in the former case the inheritance pattern [page 5↓]is maternal, while it might be autosomal or X-chromosomal recessive or autosomal dominant in the later case.

1.1.4. Mitochondrial disorders

1.1.4.1. Definition of mitochondrial disorders

Traditionally, the term “mitochondrial disorders” describes defects in the energy-generating apparatus of the mitochondrion, i.e. the respiratory chain coupled to the oxidative phosphorylation [Bauer et al., 1999]. Mitochondrial disorders comprise a heterogeneous group of clinical phenotypes, which can result from mutations in the mtDNA, the nuclear DNA or both. Abnormalities of the electron transport and the oxidative phosphorylation system are probably the most common causes of mitochondrial disorders [Schapira et al., 1999]. However, mitochondrial diseases can also result from defects in metabolic pathways located only partially in the mitochondria (e.g. the pyruvate-dehydrogenase-complex deficiency). Mitochondrial disorders may manifest themselves at any time of life, from infancy to late adulthood. They may affect virtually any tissue either alone or in combination. Tissues with high energy-requirements such as heart, muscles, brain, kidney and endocrine organs are most commonly affected [Lopez, 2002].

1.1.4.2. Classification of mitochondrial disorders

The first mitochondrial disease that was understood at the molecular level was Leber’s hereditary optic neuropathy (LHON) with a mutation in a mtDNA encoded subunit of complex I [Wallace et al., 1988] and the Kearns-Sayre syndrome with a large deletion in the mtDNA [Holt et al., 1988]. The current classification of mitochondrial disorders is based on the kind and the location of the genetic defect (mtDNA versus nuclear DNA).

Mutations in the mtDNA

Mutations in the nuclear DNA

The second group of mitochondrial disorders is due to mutations in nuclear genes. These mutations may affect structural subunits of the respiratory chain, their assembly, the replication of the mtDNA and the transport of polypeptides through the mitochondrial double membrane [Zeviani et al., 1999; Leonard et al., 2000b; Sue et al., 2000; Orth et al., 2001]. These gene defects can be grouped as follows:

1.1.4.3. Diagnosis of mitochondrial disorders

The diagnosis of mitochondrial disorders has to rely on the sum of clinical, morphological, biochemical, and molecular genetic investigations since there is no explicit relation between genotype and phenotype. Atypical clinical pictures can be observed quite frequently in mitochondrial disorders. With the exception of typical syndromes like MELAS or MERRF, histological studies of muscle biopsy specimens are usually recommended in suspected cases. Characteristic changes include the presence of paracrystalline mitochondrial inclusions, mitochondria with abnormal size and shape, ragged-red-fibres (RRFs) in muscle, fat deposits and histochemically focal enzyme deficiencies (e.g. patchy COX-deficiency or SDH-deficiency) [Zeviani et al., 1998; Parker, 2000]. In most cases, however, biochemical analysis have to be performed in order to formulate a diagnosis [Letellier et al., 2000]. Using enzymatic tests, the activities of pyruvate dehydrogenase complex (PDHc), carnitine-palmitoyl-transferase and all complexes of the respiratory chain-oxidative phosphorylation system can be determined in muscle homogenate. Single enzyme activities can also be measured in cultured fibroblasts and in blood cells (lymphocytes and platelets). But only the molecular genetic analysis can verify the diagnosis of a mitochondrial disorder. In the case of a maternal inheritance pattern the investigations will focus on the analysis of the mtDNA. Otherwise, the biochemical results may narrow possible candidate genes to screen for mutations. For example, in the case of an isolated complex I deficiency, one would at first sequence the structural subunits of complex I in which mutations have been described before.

1.1.5. Characteristics of mitochondrial proteins and preproteins

It is estimated that the mitochondrial proteome consists of approximately 1000 distinct proteins [Lopez et al., 2002]. With the exception of 13 proteins, which are encoded by the mtDNA, most mitochondrial proteins are encoded by nuclear genes, including most of the mitochondrial OXPHOS proteins, the metabolic enzymes, the DNA and RNA polymerases, the ribosomal proteins, and the mtDNA regulatory factors [Grivell et al., 1988; Wallace, 1999]. These proteins are synthesized at the encoplasmatic reticulum and are later imported into the mitochondrion. Fig. 1-4depicts this principle of the transportation of the preproteins through the double membrane. Before being transported into the mitochondrion, proteins are synthesized as preproteins, i.e. precursors that contain transit sequences either as amino-terminal targeting pre-sequences, or as targeting and sorting information sequences within the mature proteins. The cytosolic preproteins are imported through the translocases of the outer membrane (TOM) when their targeting information is recognized by the receptors of TOM. They are then sorted either directly to the outer membrane, the inter-membrane space or to the [page 8↓]translocases of the inner-membrane (TIM). Preproteins with a typical amino-terminal targeting sequence engage the TIM17/TIM23 complex that guides preproteins into the matrix. In the matrix the targeting sequencesare removed by the matrix-processing-protease, and the remaining polypeptide chains are folded by chaperones into mature proteins. Preproteins, which lack a targeting sequence, engage with the TIM22 complex to be inserted into the inner membrane [Millar et al., 1994; Shore et al ., 1995; Hanson et al., 1996; Koehler, 2000].

1.2. Proteome analysis

1.2.1. Definition of proteome analysis

The term “proteome” was first advocated by Marc Wilkins in 1996 as a linguistic equivalent to “genome” which indicates all chromosomes and their genes of any cell type of a given organism. The proteome was defined as the entire protein complement expressed by a celltype, tissue or an organism [Wilkins et al., 1996]. Genome research usually refers to sequencing the total genomic DNA of an organism and mapping all genes within these sequences. In contrast, the aim of proteome research focuses on the structural and functional analysis of the proteome and the interaction of proteins with one another. This includes the isolation, identification and characterization of all proteins encoded by the genome of an organism.

Fig. 1-4: Preprotein import pathways into the mitochondrion . Before being transported into the mitochondrion, proteins are synthesized as preproteins in the cytosol. They are then imported through the translocases of the outer membrane (TOM). Preproteins with a typical amino-terminal targeting sequence engage with the inner-membrane (TIM) complex 17+23 (pathway A) to be imported into the matrix. In the matrix the targeting sequences are removed by the matrix-processing protease to form the mature proteins. Preproteins, which lack a targeting sequence, engage with the TIM22 complex (pathway B) to be inserted into the inner membrane.

Proteome analysis could lead the way to explain the function of an organism dynamically rather than statically. This is important since the protein compositions and concentrations change from cell type to cell type, even within sub-cellular compartments. Moreover, they [page 9↓]also differ between various stages of development [Abbott, 1999]. Proteome analysis can also offer the opportunity to examine entire pathways, or multiple enzymatic pathways simultaneously [Lopez et al., 2000]. High throughput two-dimensional protein electrophoresis [Klose, 1975; O’Farrell, 1975] coupled with peptide mass fingerprinting analysis by MALDI-TOF mass spectrometry [Karas et al., 1988] have become the most powerful techniques for proteome analysis.

1.2.2. Previous work on the proteome

Using two-dimensional protein electrophoresis in order to establish a whole proteome map was first done by Boucherie et al. (1995) in yeast. The work was extended in 1999 [Perrot et al., 1999]. They identified more than 400 proteins on their reference proteome map. Similar results have been reported by Garrels et al. (1997), Shevchenko et al. (1996b) and Maillet et al. (1996). Lopez et al.(2000) established a 2D-electrophoresis map of the mitochondrial proteome of rat liver that included ca. 70 proteins by using high–throughput automated equipment in combination with mini-spin affinity columns. Analysis of the human mitochondrial proteome has first been done by Rabilloud et al. (1998). They investigated human mitochondrial proteins from placenta using 2D-electrophoresis and MALDI-TOF mass spectrometry, complemented by protein sequencing and immunodetection. They detected ca. 1500 spots on a silver-stained reference gel and finally identified 46 proteins [Rabilloud et al., 1998]. Most recently, Fountoulakis et al.(2003) identified approximately 185 different proteins in mitochondria isolated from a cultured neuroblastoma cell line (IMR-32) using similar methods.

1.3. The aim of my study

Despite the advent of high throughput sequencing, with the exception of typical syndromes like MELAS, MERRF and LHON, most cases of mitochondrial diseases are difficult to diagnose on the molecular level.

The difficulties in making a molecular diagnosis are:

Until now less than 20% of mitochondrial diseases can be diagnosed on the molecular level. Therefore, new tools should be established in order to increase the identification rate of mitochondrial diseases. Since the proteome bridges the genotype with the phenotype, we hypothesize that mutations in mitochondrial genes encoded by the mtDNA or nuclear DNA cause changes on the proteome level. These changes might be primary (e.g. a mutated protein is absent or has different running characteristics) or secondary (e.g. other proteins that are up- or down-regulated to compensate for a mutated protein).


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However, to lay a basis for these proteome analyses we first have to establish what is “normal”. Furthermore we have to choose a model system that guaranties purification of a sufficient amount of mitochondria from patients.

Since lymphocytes generally express the functional defects of mitochondrial enzymes, we chose to work on immortalized lymphoblastoid cell lines since they can be cultivated permanently in order get sufficient material for analysis.

The aim of the present study is to establish a method to purify mitochondria from as little as possible patient material (cultured lymphoblastoid cells) and to establish a reference map for the mitochondrial proteome of lymphoblastoid cells.

The reference map and database can be later used to compare deviating protein patterns between healthy and diseased individuals. This might direct the attention to disease-specific proteins and genes and open new ways to diagnose mitochondrial diseases on proteome level or with a combined genetic-proteomic approach.


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