Browsing by Subject "Mitochondria"

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Funktionen charakteristischer Sequenzmotive endogener und toxischer mitochondrialer Proteine
(2006) Papatheodorou, Panagiotis; Rassow, Joachim
In the course of their biogenesis, mitochondria take up nuclear encoded proteins from the cytosol continuously. Protein import at the mitochondrial outer membrane is mediated by TOM proteins and by TIM proteins at the inner membrane, respectively. Now and then, toxical proteins released by pathogenic bacteria to infected tissue can also reach mitochondria. The present dissertation provides new findings on the role of characteristical sequence motifs that can be identified in endogenous and toxical mitochondrial proteins. In an extensive project the importance of sequence motifs from mitochondrial metabolite carrier proteins in their biogenesis and function was investigated in more detail. It could be shown, that the positively charged presequence of the citrate carrier from Rattus norvegicus is not involved in mitochondrial targeting but rather serves as an internal chaperone. A conserved sequence motif, PX(D/E)XX(R/K), the Carrier Signature, which can be found in all mitochondrial carrier proteins, does also not represent a mitochondrial targeting signal, as could be proven by using the dicarboxylate carrier from Saccharomyces cerevisiae as a model protein. Even the translocation across the outer membrane, the insertion into the inner membrane and the following dimerization of the dicarboxylate carrier are processes occuring independently of the Carrier Signature. Instead, it was discovered, that the Carrier Signature is primarily necessary for the function of metabolite carrier proteins in the inner membrane. In another project it could be shown for the Map toxin from enteropathogenic Escherichia coli strains (EPEC), that it is directed to the mitochondrial matrix, mediated by its typical N-terminal presequence and by the TOM and TIM complexes, respectively. The Map toxin leads then to the fragmentation of the mitochondrial network independent of the mitochondrial fission machinery and to the loss of the mitochondrial membrane potential. Moreover, it could be proven, that an internal conserved sequence motif, WXXXE, is essential for cytotoxicity of the Map toxin in the cytosol and for fission of mitochondria. A lysine residue within the WXXXE sequence serves probably as a locus of sumoylation. The investigations show, that mechanisms of intracellular protein transport are not only important for the biogenesis of mitochondria, but can also be relevant for pathological processes.
Mitochondrial haplotypes, gene expression and nuclear diversity in two strains of laying hens
(2021) Dreyling, Clara; Hasselmann, Martin
The domesticated chicken (Gallus gallus domesticus) is the most popular and widely spread domestic fowl worldwide, providing human with a stable source of protein in form of meat and eggs for centuries. The ongoing growth of human population increases the need for food and made poultry production one of the fasted growing sectors in the past decades. This need for food has resulted in several different strains which outperform their wild ancestors in terms of meat and egg production. During the past decades not only animal welfare gained importance but also ecological aspects such as global warming and the shortage of resources are becoming more important to society. One important resource for mankind which is becoming shortened is phosphorus (P), whose deposits in form of rock phosphate could be exhausted within the next 50-100 years. 90% of P supply is used in agriculture as fertilizer, whose demand will increase as well with growing population. This thesis focuses on the mitochondrial genetic background and mitochondrial related gene expression in the context of the productive life span and different diets in two contrasting high-yielding strains of laying hens, Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL). Mitochondria, which are commonly known as the powerhouse of the cell due to their role as the main producer of energy, play roles in other processes from cellular homeostasis to the process of ageing. The process of oxidative phosphorylation depends on the availability of P and thus, they become an important part of the complex framework of P utilization. In addition, mitochondrial haplotypes are known to affect physiological traits such as body weight in laying hens or important traits such as e.g. the metabolic capacity in dairy cows. It is known, that single mutations in the mitochondrial genome lead to a better adaptation to height in the Tibetan chicken or play a role in diseases from Alzheimer to obesity or lead to resistance to disease such as Marek’s disease in birds. This work provides insight into the whole mitochondrial genome of 180 laying hens of two commercial strains and links this information to physiological traits and genetic diversity. In addition, the first large-scaled gene expression analyses in the context of the productive life span and different P and Ca contents in laying hens is implemented. The analysis of mitochondrial haplotypes revealed a low level of genetic diversity with only three haplotypes within the LB strain while all LSL hens shared the same mitochondrial genome. Following from this observation, the nuclear genome was analysed based on genotyping data to reveal the whole genetic diversity of both strains. On the nuclear genetic level, both strains appeared as clearly distinct and equally diverse, while some individuals appear as strikingly close related. These individuals are mostly half-siblings sharing the same mitochondrial haplotype, underlining the need for more analyses about the genetic structure about the parental generation, especially the maternal background. Although there were no strong associations were found between the mitochondrial haplotypes and the analysed phenotypic traits (feed intake, body weight, P and Ca utilization), the differences between the strains indicate a potential involvement of the mitochondrial genetic background. The gene expression analyses revealed tissue type and point of the productive life span as the main influencers on gene expression while the influence of the strain is secondary. In addition, the expression of the gene GAPDH, which is frequently used as a reference gene for normalization in gene expression studies, was influenced by tissue and strain, leading to the decision to exclude it as a reference, that should be considered for in further studies. Further, no influence of the changes in dietary P and Ca on gene expression could be observed, suggesting that a reduction of 20% of both minerals is possible without the need to adapt gene expression. However, the results show, that a reduction of both minerals has less effect than a reduction of P alone, leading to an imbalance. In the context of the productive live span, mitochondrial and mitochondrial regulatory genes react contrary, illustrating the complexity of mitochondrial gene expression and regulation. In addition to the higher variance in the analysed phenotypic traits and mitochondrial genome in LB hens, they showed signs of increased oxidative stress compared to LSL hens. In the context of the productive life span, a potential higher demand for energy is suggested, since OXPHOS related gene expression is increasing. As a conclusion this work provides an insight into the mitochondrial genome and provides the first large scaled analysis of mitochondrial linked gene expression in two contrasting laying hen strains.
Untersuchungen zur Biogenese von Proteinen in der mitochondrialen Innenmembran
(2008) Randel, Olga; Rassow, Joachim
Mitochondria and prokaryotes show many similarities and it is a well established notion that they have common ancestors. It is therefore reasonable to expect significant similarities also in the biogenesis of their proteins. This study followed this idea in investigations on the biogenesis of protein complexes in the mitochondrial inner membrane. The yeast Saccharomyces cerevisiae served as a model organism. (1) γ-subunit of ATP synthases is highly conserved both in mitochondria and in prokaryotes. Previous studies demonstrated that deletions at the N- or C-terminus of the subunit entail only mild reductions in the enzymatic activity, and the reason of the conserved structure was enigmatic. The experiments of this study show that N- and C-terminus of the γ-subunit are essential for an efficient assembly in the ATP synthase. A deletion of 9 residues at the N-terminus or 10 residues at the C-terminus reduced the ratio of the subunit that assembled within 10 min. at 25°C by about 50%. Deletions of more than 9 N- or more than 20 C-terminal residues reduced the share of the assembled subunit by more than 90%. Yeast strains that synthesized a shortened γ- subunit did not grow on glycerole. N- and C-terminus are probably more relevant for assembly of the ATP synthase than for the transmission of energy. It is proposed that this is the case both for mitochondria and for prokaryotes. (2) The metabolite carrier proteins of the mitochondrial inner membrane were probably newly developed during the evolution of the eukaryotic cells. A common sequence motif of all carrier proteins is the carrier signature, P x (D/E) x x (K/R). The data of this study show that the carrier signature substantially facilitates the biogenesis of the dicarboxylate carrier (DIC). In particular, the translocation of this protein across the mitochondrial outer membrane is significantly accelerated. (3) The mitochondrial inner membrane protein Oxa1 is a member of a protein family that includes the bacterial protein YidC. Both Oxa1 and YidC act as mediators of protein insertion in their membranes, and both proteins participate in their own biogenesis. In this study, a series of experiments indicates that newly synthesized Oxa1 is not imported into the mitochondrial matrix, but accumulates in the inner membrane TIM23 translocase, for direct integration into the lipid bilayer. Oxa1 thereby shows a similar principle of membrane insertion as prokaryotic YidC. This was previously shown to first accumulate in the SecYEG translocase and then to directly integrate into the bacterial plasmamembrane. Oxa1 and YidC thus seem to resemble each other both in their structure and in their biogenesis. In summary, the experiments show that mitochondrial and prokaryotic proteins, after two billion years of separate evolution, have retained surprising similarities, even in molecular details of their function.
Walnut oil reduces Aβ levels and increases neurite length in a cellular model of early Alzheimer disease
(2022) Esselun, Carsten; Dieter, Fabian; Sus, Nadine; Frank, Jan; Eckert, Gunter P.
Mitochondria are the cells’ main source of energy. Mitochondrial dysfunction represents a key hallmark of aging and is linked to the development of Alzheimer’s disease (AD). Maintaining mitochondrial function might contribute to healthy aging and the prevention of AD. The Mediterranean diet, including walnuts, seems to prevent age-related neurodegeneration. Walnuts are a rich source of α-linolenic acid (ALA), an essential n3-fatty acid and the precursor for n3-long-chain polyunsaturated fatty acids (n3-PUFA), which might potentially improve mitochondrial function. (2) Methods: We tested whether a lipophilic walnut extract (WE) affects mitochondrial function and other parameters in human SH-SY5Y cells transfected with the neuronal amyloid precursor protein (APP695). Walnut lipids were extracted using a Soxhlet Extraction System and analyzed using GC/MS and HPLC/FD. Adenosine triphosphate (ATP) concentrations were quantified under basal conditions in cell culture, as well as after rotenone-induced stress. Neurite outgrowth was investigated, as well as membrane integrity, cellular reactive oxygen species, cellular peroxidase activity, and citrate synthase activity. Beta-amyloid (Aβ) was quantified using homogenous time-resolved fluorescence. (3) Results: The main constituents of WE are linoleic acid, oleic acid, α-linolenic acid, and γ- and δ-tocopherol. Basal ATP levels following rotenone treatment, as well as citrate synthase activity, were increased after WE treatment. WE significantly increased cellular reactive oxygen species but lowered peroxidase activity. Membrane integrity was not affected. Furthermore, WE treatment reduced Aβ1–40 and stimulated neurite growth. (4) Conclusions: WE might increase ATP production after induction of mitochondrial biogenesis. Decreased Aβ1–40 formation and enhanced ATP levels might enhance neurite growth, making WE a potential agent to enhance neuronal function and to prevent the development of AD. In this sense, WE could be a promising agent for the prevention of AD.