Protein phosphorylation in yeast mitochondria: enzymes, substrates and function

Protein phosphorylation is one of the major post-translational modifications to allow for signal transmission and fine tuning of metabolism on the cellular proteomic level. As such it is “one of the last instances” to modulate the activity of enzymes and hence to impact the cellular life irrespective of the basic conditions provided by the genome – and epigenome– controlled gene expression. The evolutionary increase in cellular complexity is reflected by highly sophisticated regulatory networks in multicellular eukaryotes based on the transfer of phosphate mostly onto the side chains of serine, threonine and tyrosine residues. Nature has chosen phosphate for inter- and intracellular communication, which is also an integral component of nucleic acids and can be regarded as the molecule of choice for life. Currently, life science is interested to unravel the network of reversible protein phosphorylation that is catalyzed by two antagonistic enzyme classes: the protein kinases and protein phosphatases. We are currently in the era of proteomics and enormously benefit from the progress of mass-spectrometry methods. This is documented by a huge number of “proteomic studies” that mostly provide a simple inventory of the existence of proteins – and/or their phosphorylated forms – under more or less defined conditions. So far, the physiological correlations could be established only in a few cases, e.g. by comparing two physiological conditions. Another strategy, which was addressed in this work, is the systematic screening of mutants defective in genes encoding either protein kinases or protein phosphatases. This approach benefits from the ease to predict these enzymes due to the presence of characteristic protein motifs. In combination with the major goal of this work – to shed light on the impact of protein phosphorylation in the mitochondrial (mt) compartment – the yeast Saccharomyces cerevisiae was chosen as a model system because of its respiro-fermentative metabolism, that allows for the maintenance of respiratory defective mutants. Indeed, this reverse genetic approach successfully revealed two kinases (Pkp1p, Pkp2p) and two phosphatases (Ppp1p, Ppp2p) as the key components regulating the pyruvate dehydrogenase complex by phosphorylation of serine 313 of its α- subunit Pda1p. In addition, evidence is provided that Pkp1p has an additional role in the assembly process of the PDH complex. Also, the effect of the deletion of the COQ8 gene (gene engaged in coenzyme Q synthesis; originally named ABC1) leading to respiratory deficiency, could be correlated with the phosphorylation of subunit Coq3p of the mitochondrial ubiquinone biosynthesis complex. Finally, in the case of the kinase Sat4p (protein involved in salt tolerance), overexpression of the enzyme was used as an alternative approach to unravel the molecular basis of the originally observed salt sensitivity of sat4 mutants. The data suggest that Sat4p has a direct or indirect role in the late steps of iron-sulfur (Fe/S) cluster assembly of the so-called “aconitase-type” enzymes in mitochondria, accompanied by a strongly reduced steady state concentration of the Fe/S-cluster protein aconitase. Interestingly, a secondary phenotype became apparent upon overexpression of Sat4p: the abundance of the lipoic acid containing mitochondrial proteome was markedly reduced. Most likely this phenotype is due to the fact that the synthesis and/or attachment of lipoic acid depend on a Fe/S-cluster bearing enzyme. In the course of the work it became clear that the regulatory (mt) protein phosphorylation network of yeast evolved to meet the criteria of a life adapted to the ecological niche on temporarily available sugar rich sources. Clearly, the transfer of the respective data to higher eukaryotes is limited. However, it shows that yeast is primarily an excellent model system for the principal molecular reactions shared with higher eukaryotes.:1 SUMMARY 1 ZUSAMMENFASSUNG 3 2 INTRODUCTION 5 2.1 Why phosphate? 5 2.2 Protein phosphorylation in prokaryotes 6 2.3 Protein phosphorylation in mitochondria 7 2.4 Regulation of mammalian pyruvate dehydrogenase complex (PDH) by phosphorylation 9 2.5 Mammalian cytochrome c oxidase (COX) 10 2.6 Protein phosphorylation in yeast mitochondria 11 3 AIM OF THIS WORK 13 4 PROLOG 14 4.1 Critical evaluation of tools for phosphoproteomics 14 4.2 Introducing a new method for in gel profiling of phospho-proteins 17 4.3 In-gel screening of phosphorus of yeast mitochondrial proteins by LA-ICP-MS 20 4.4 Detection of phosphorylated subunits of ATPase 22 5 RESULTS 23 5.1 YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex 28 5.2 Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases 29 5.3 Proteomic analysis reveals a novel function of the kinase Sat4p in yeast mitochondria 30 5.4 Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O –methylase Coq3p depends on Abc1p/Coq8p 53 6 DISCUSSION AND PERSPECTIVES 54 6.1 Mitochondrial phosphorylation in yeast 54 6.1.1 An evolutionary view 54 6.1.2 The yeast ABC1-kinase family 55 6.1.3 Regulation of yeast pyruvate dehydrogenase (PDH) complex 57 6.1.4 Regulation of iron sulfur cluster biogenesis 60 6.2 Challenges to investigate the network of (mt) protein phosphorylation 62 6.2.1 When is a kinase a mitochondrial kinase? 64 6.2.2 Epilog 66 7 LITERATURE 69 8 APPENDIX 78 8.1 Related publications 78 8.2 List of publications 80 8.3 ERKLÄRUNGEN 82Phosphorylierungen von Aminosäuren ist eine der verbreitetsten post-translationalen Modifikationen für zelluläre Signalübertragungswege und zur Regulation des Metabolismus auf Proteom-Ebene. Mit der reversiblen Protein-Phosphorylierung eng verbunden ist die unabhängige Modulation der Aktivität von Enzymen ungeachtet der Genom- und Epigenom-basierten Genexpression. Die evolutionäre Zunahme der zellularen Komplexität äußert sich in zunehmend komplexeren Regulations-Netzwerken in mehrzelligen eukaryotischen Organismen basierend auf dem Transfer von Phosphatgruppen vorzugsweise auf die Aminosäuren Serin, Threonin und Tyrosin. Die Natur hat evolutionär als Baustein der inter- und intrazellulären Kommunikation Phosphat gewählt, welches auch ein integraler Bestandteil der Nukleinsäuren ist und somit als das „Molekül der Wahl“ für das Leben bezeichnet werden darf. Die Lebenswissenschaften sind gegenwärtig daran interessiert das Netzwerk der Proteinphosphorylierung aufzuklären, welches durch zwei antagonistisch wirkende Enzymklassen, die Proteinkinasen und Proteinphosphatasen charakterisiert ist. Dabei profitieren wir gegenwärtig von den Fortschritten der „Proteomics-Ära“ auf dem Gebiet der massenspektrometrischen Proteinidentifizierung. Ausdruck dessen ist eine Vielzahl von Proteom-Studien, die jedoch meist nur eine einfache Inventarisierung der unter mehr oder weniger gut definierten zellulären Bedingungen existierenden Proteine in ihrer Phosphat-modifizierten oder unphosphorylierten Form darstellen. Die beteiligten Enzyme werden dabei kaum berücksichtigt. Insbesondere gilt dies für extra-cytoplasmatische Ereignisse. Bisher gelang es nur in wenigen Fällen eine Korrelation der physiologischen Rolle dieser Proteinmodifikation, z.B. durch den Vergleich der Phospho-Proteome unter zwei unterschiedlichen physiologischen Bedingungen, herzustellen. Eine andere Strategie, die auch Gegenstand dieser Arbeit ist, sieht ein Screening von Mutanten vor, die durch Deletionen von Genen, die für Proteinkinasen bzw. –phosphatasen kodieren, gekennzeichnet sind. Dieser Ansatz profitiert von der Existenz und leichten bioinformatischen Vorhersagbarkeit charakteristischer Kinase- bzw. Phosphatase- Sequenzmotive. In Kombination mit dem Hauptziel der Arbeit – Licht ins Dunkel der Proteinphosphorylierung im mitochondrialen Kompartiment zu bringen – wurde die Hefe Saccharomyces cerevisiae als Modellsystem gewählt, insbesondere vor dem Hintergrund ihres fermentativen Metabolismus. Als Beleg der prinzipiellen Funktionalität des vorgeschlagenen Ansatzes konnten zwei Kinasen (Pkp1p, Pkp2p) und zwei Phosphatasen (Ppp1p, Ppp2p) als Schlüsselkomponenten der Regulation des Pyruvatdehydrogenase (PDH) Komplexes identifiziert und charakterisiert werden. Darüber hinaus konnte sowohl das Zielprotein der Phosphorylierung, Pda1p, die α-Untereinheit des Komplexes, als auch die modifizierte Aminosäure (Serin 313) experimentell bestätigt werden. Ferner konnte der Atmungsdefekt von Stämmen mit einer nicht-funktionellen Abc1p-Kinase mit dem Phosphorylierungszustand der Untereinheit Coq3p des Ubiquinon-Biosynthese Komplexes und dem Ausfall der Ubiquinonsynthese korreliert werden. Eine alternative Herangehensweise, die Überexpression einer Kinase, führte zur Identifizierung möglicher Zielproteine von Sat4p. Vergleichende Analysen des 2D-gelelektrophoretisch separierten mitochondrialen Genoms mit dem des Wildtyps legen die Vermutung nahe, dass Sat4p eine direkte oder indirekte Rolle bei der Regulation der „Aconitase-Typ“ Eisen-Schwefel (Fe/S) Proteine besitzt. Der darüber hinaus beobachtete Effekt einer Abnahme von Liponsäure-tragenden mitochondrialen Enzymen, ist wahrscheinlich sekundärer Natur und kann durch die Zugehörigkeit der Liponsäure-Synthase zur oben erwähnten Gruppe der „Aconitase-Typ“ -Fe/S-Proteine erklärt werden. Im Verlauf der Arbeit wurde deutlich, dass das regulatorische Netzwerk der Proteinphosphorylierung der Hefe eher den Kriterien einer evolutionären Adaptation an eine spezifische ökologische Nische – der temporären Verfügbarkeit zuckerreicher Substanzen – entsprechen. Das schränkt die Übertragbarkeit der gewonnen Einsichten in die Regulation des mitochondrialen Metabolismus auf höhere Eukaryonten ein. Es zeigt jedoch, dass Hefe in erster Linie ein exzellentes Modellsystem für die prinzipiellen molekulare Mechanismen ist, die sie mit den höheren Eukaryonten teilt.:1 SUMMARY 1 ZUSAMMENFASSUNG 3 2 INTRODUCTION 5 2.1 Why phosphate? 5 2.2 Protein phosphorylation in prokaryotes 6 2.3 Protein phosphorylation in mitochondria 7 2.4 Regulation of mammalian pyruvate dehydrogenase complex (PDH) by phosphorylation 9 2.5 Mammalian cytochrome c oxidase (COX) 10 2.6 Protein phosphorylation in yeast mitochondria 11 3 AIM OF THIS WORK 13 4 PROLOG 14 4.1 Critical evaluation of tools for phosphoproteomics 14 4.2 Introducing a new method for in gel profiling of phospho-proteins 17 4.3 In-gel screening of phosphorus of yeast mitochondrial proteins by LA-ICP-MS 20 4.4 Detection of phosphorylated subunits of ATPase 22 5 RESULTS 23 5.1 YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex 28 5.2 Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases 29 5.3 Proteomic analysis reveals a novel function of the kinase Sat4p in yeast mitochondria 30 5.4 Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O –methylase Coq3p depends on Abc1p/Coq8p 53 6 DISCUSSION AND PERSPECTIVES 54 6.1 Mitochondrial phosphorylation in yeast 54 6.1.1 An evolutionary view 54 6.1.2 The yeast ABC1-kinase family 55 6.1.3 Regulation of yeast pyruvate dehydrogenase (PDH) complex 57 6.1.4 Regulation of iron sulfur cluster biogenesis 60 6.2 Challenges to investigate the network of (mt) protein phosphorylation 62 6.2.1 When is a kinase a mitochondrial kinase? 64 6.2.2 Epilog 66 7 LITERATURE 69 8 APPENDIX 78 8.1 Related publications 78 8.2 List of publications 80 8.3 ERKLÄRUNGEN 8

Idealkristalle als Abelsche Varietäten

Es wird gezeigt, daß sich Idealkristalle in natürlicher Weise als hauptpolarisierte Abelsche Varietäten darstellen lassen. Es existiert demzufolge auf Λ × Λ eine ganzzahlige schiefsymmetrische Matrix E: Λ × Λ → ℤ, deren Elementarteiler sämtlich gleich 1 sind, wobei Λ das Translationsgitter des Idealkristalls im Phasenraum V ist. Bezüglich derartiger Gitter kann die Gitterdarstellung der Heisenberggruppe definiert werden oder mit anderen Worten: Auf einer hauptpolarisierten Abelschen Varietät kann nichtrelativistische Quantenmechanik betrieben werden. Die Gitterdarstellung wird detailliert betrachtet. Der harmonische Oszillator wird in der Gitterdarstellung berechnet und illustriert. Es zeigt sich, daß die Eigenfunktionen des harmonischen Oszillators in der Gitterdarstellung systematisch aus der mit einem Exponentialfaktor multiplizierten Riemannschen Thetafunktion für die zugrundeliegende hauptpolarisierte Abelsche Varietät hervorgehen. Die vollständigen Eigenfunktionensysteme der Impuls- sowie der Ortsoperatoren werden in der Gitterdarstellung aufgestellt und führen zu einer deutlichen Verbesserung des Galerkinverfahrens für die Berechnung der Bandstruktur des Kristallelektrons

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms

Stand und Perspektiven der Erfassung sozialwissenschaftlicher Publikationen: Erfahrungen aus der Pilotstudie Forschungsrating Soziologie

Der Beitrag bezieht die Erfahrungen von GESIS aus der Publikationserhebung im Rahmen der Pilotstudie Forschungsrating Soziologie auf die Qualität und Validität der Informationen in SOLIS und sowiport sowie auf die allgemeinere Fragestellung, wie die Fachinformation für die Sozialwissenschaften den Anforderungen der Fachdisziplin und deren Evaluation entsprechen kann. Die Erhebung von Veröffentlichungen gehört zu den konsensuellen Bereichen wissenschaftlicher Leistungsmessung. Die Soziologie zeichnet sich durch ein Publikationsverhalten mit hoher Interdisziplinarität und breiter Streuung hinsichtlich der Publikationstypen und -organe sowie der geographischen Verteilung aus. Daraus ergeben sich besondere Herausforderungen für die möglichst umfassende Dokumentation und Erschließung. Das Forschungsrating des Wissenschaftsrats war – quasi im Nebeneffekt – damit auch ein besonderer Selbsttest für das integrierte Datenbank- und Portalangebot von GESIS, da hieraus eine Vorlagemenge erstellt wurde. Die teilnehmenden Wissenschaftlerinnen und Wissenschaftler konnten ihre Literaturlisten in einem Online-System einsehen, korrigieren und ergänzen. Die Analyse der Vorlagemenge und Nachmeldungen gibt Aufschlüsse über Streuungseffekte, woran sich Fragen und Vorschläge zur effizienten Integration von Informationen unter Nutzung der technischen Möglichkeiten anschließen.   The article refers to GESIS’ experiences from the publication survey within the context of the Pilot Study for the Research Rating in Sociology (Pilotstudie Forschungsrating Soziologie) on the quality and validity of information contained in SOLIS and sowiport, as well as the more general question as to how the Specialized Information for the Social Sciences (Fachinformation für die Sozialwissen- schaften) should meet the demands of the discipline as well as those of recurrent evaluations. The survey of publications belongs to the mutual areas of academic performance measurement. Sociology is characterized by a publication behaviour which displays high interdisciplinarity and a broad spread with respect to the types and organs of publications, as well as their geographic distribution. This results in special challenges for the most comprehensive documentation and indexing. Thus, the research rating of the German Council of Science and Humanities (Wissen- schaftsrat) was – as a sort of side-effect – also a self-test for GESIS’ databases and integrated portal from which preliminary publication lists were generated. The participating scientists could read, correct and complete their publication lists online. The analysis of these lists and their amendments yields insights on diffusion effects, followed by questions on and suggestions for the efficient integration of information through utilization of technical possibilities

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes

Breaking the Waves: Modelling the Potential Impact of Public Health Measures to Defer the Epidemic Peak of Novel Influenza A/H1N1

BACKGROUND: On June 11, 2009, the World Health Organization declared phase 6 of the novel influenza A/H1N1 pandemic. Although by the end of September 2009, the novel virus had been reported from all continents, the impact in most countries of the northern hemisphere has been limited. The return of the virus in a second wave would encounter populations that are still nonimmune and not vaccinated yet. We modelled the effect of control strategies to reduce the spread with the goal to defer the epidemic wave in a country where it is detected in a very early stage. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a deterministic SEIR model using the age distribution and size of the population of Germany based on the observed number of imported cases and the early findings for the epidemiologic characteristics described by Fraser (Science, 2009). We propose a two-step control strategy with an initial effort to trace, quarantine, and selectively give prophylactic treatment to contacts of the first 100 to 500 cases. In the second step, the same measures are focused on the households of the next 5,000 to 10,000 cases. As a result, the peak of the epidemic could be delayed up to 7.6 weeks if up to 30% of cases are detected. However, the cumulative attack rates would not change. Necessary doses of antivirals would be less than the number of treatment courses for 0.1% of the population. In a sensitivity analysis, both case detection rate and the variation of R0 have major effects on the resulting delay. CONCLUSIONS/SIGNIFICANCE: Control strategies that reduce the spread of the disease during the early phase of a pandemic wave may lead to a substantial delay of the epidemic. Since prophylactic treatment is only offered to the contacts of the first 10,000 cases, the amount of antivirals needed is still very limited

Development of a risk assessment tool for contact tracing people after contact with infectious patients while travelling by bus or other public ground transport: a Delphi consensus approach

Background: Tracing persons who have been in contact with an infectious patient may be very effective in preventing the spread of communicable diseases. However, criteria to decide when to conduct contact tracing are not well established. We have investigated the available evidence for contact tracing with a focus on public ground transport aiming to give guidance in what situations contact tracing should be considered. Methods: Relevant infectious diseases suitable for contact tracing in ground transport and a set of disease-specific epidemiological criteria were defined through literature search and structured multistep expert consultations. We developed continuous scales for each criterion to be rated for its relevance to contact tracing in ground transport. We used the Delphi method with an international expert panel to position the values of criteria on the respective scales. Results: The study led to the development of the ‘Contact Tracing-Risk Assessment Profile’ (CT-RAP), a decision-making instrument, taking into account pathogen-specific as well as situation-specific criteria. This report describes the methodology of this instrument and presents two examples of ready-to-use CT-RAP for tuberculosis and for meningococcal disease in public ground transport. Discussion: The systematic and transparent use of the CT-RAP for tuberculosis and meningococcal disease is likely to facilitate reasonable, efficient and user-friendly decisions with respect to contact tracing. New CT-RAPs for additional pathogens and different settings such as schools and kindergartens are being planned

Interaction between intravenous thrombolysis and clinical outcome between slow and fast progressors undergoing mechanical thrombectomy: a post-hoc analysis of the SWIFT-DIRECT trial.

BACKGROUND In proximal occlusions, the effect of reperfusion therapies may differ between slow or fast progressors. We investigated the effect of intravenous thrombolysis (IVT) (with alteplase) plus mechanical thrombectomy (MT) versus thrombectomy alone among slow versus fast stroke progressors. METHODS The SWIFT-DIRECT trial data were analyzed: 408 patients randomized to IVT+MT or MT alone. Infarct growth speed was defined by the number of points of decay in the initial Alberta Stroke Program Early CT Score (ASPECTS) divided by the onset-to-imaging time. The primary endpoint was 3-month functional independence (modified Rankin scale 0-2). In the primary analysis, the study population was dichotomized into slow and fast progressors using median infarct growth velocity. Secondary analysis was also conducted using quartiles of ASPECTS decay. RESULTS We included 376 patients: 191 IVT+MT, 185 MT alone; median age 73 years (IQR 65-81); median initial National Institutes of Health Stroke Scale (NIHSS) 17 (IQR 13-20). The median infarct growth velocity was 1.2 points/hour. Overall, we did not observe a significant interaction between the infarct growth speed and the allocation to either randomization group on the odds of favourable outcome (P=0.68). In the IVT+MT group, odds of any intracranial hemorrhage (ICH) were significantly lower in slow progressors (22.8% vs 36.4%; OR 0.52, 95% CI 0.27 to 0.98) and higher among fast progressors (49.4% vs 26.8%; OR 2.62, 95% CI 1.42 to 4.82) (P value for interaction <0.001). Similar results were observed in secondary analyses. CONCLUSION In this SWIFT-DIRECT subanalysis, we did not find evidence for a significant interaction of the velocity of infarct growth on the odds of favourable outcome according to treatment by MT alone or combined IVT+MT. However, prior IVT was associated with significantly reduced occurrence of any ICH among slow progressors whereas this was increased in fast progressors

OXPHOS Supercomplexes as a Hallmark of the Mitochondrial Phenotype of Adipogenic Differentiated Human MSCs

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. Recently, an increasing number of data has highlighted the role of mitochondria for cellular differentiation processes. Metabolic differences between stem cells and mature derivatives require an adaptation of mitochondrial function during differentiation. In this study we investigated alterations of the mitochondrial phenotype of human mesenchymal stem cells undergoing adipogenic differentiation. Maturation of adipocytes is accompanied by mitochondrial biogenesis and an increase of oxidative metabolism. Adaptation of the mt phenotype during differentiation is reflected by changes in the distribution of the mitochondrial network as well as marked alterations of gene expression and organization of the oxidative phosphorylation system (OXPHOS). Distinct differences in the supramolecular organization forms of cytochrome c oxidase (COX) were detected using 2D blue native (BN)-PAGE analysis. Most remarkably we observed a significant increase in the abundance of OXPHOS supercomplexes in mitochondria, emphasizing the change of the mitochondrial phenotype during adipogenic differentiation