Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis.

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress

No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis

The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation. However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon, and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form. Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner-Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the 'Warburg effect' of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism.We acknowledge funding from the European Commission (Brussels) Role ofMitochondria in Conserved Mechanisms of Aging (MIMAGE) Project (Contract 512020, to M.B.), the Cancer Research Programme Grant (C197/A3514 to K.M.B.), Cancer Research UK and ERC Grants 322842-METABOp53 (supporting E.C.), the Wellcome Trust (RG 093735/Z/10/Z to M.R.), the ERC (Starting grant 260809 to M.R.), the German Research Foundation DFG (PR 1527/1-1 to A.P.), and the Austrian Science Fund (FWF) S9302-B05 (to M.B.). V.O.-S. is supported by Consejo Nacional de Ciencia y Tecnologia (CONACyT) Mexico postdoctoral fellowship 203450, M.A.K. by the FWF (Austria) by an Erwin Schroedinger postdoctoral fellowship (J 3341). M.R. is a Wellcome-Trust Research career development and Wellcome-Beit prize fellow.This is the final published version. It is also available from Wiley at http://onlinelibrary.wiley.com/doi/10.1111/brv.12140/abstract

The impact of acute nutritional interventions on the plasma proteome

Context: Humans respond profoundly to changes in diet, while nutrition and environment have a great impact on population health. It is therefore important to deeply characterize the human nutritional responses. Objective: Endocrine parameters and the metabolome of human plasma are rapidly responding to acute nutritional interventions such as caloric restriction or a glucose challenge. It is less well understood whether the plasma proteome would be equally dynamic, and whether it could be a source of corresponding biomarkers. Methods: We used high-throughput mass spectrometry to determine changes in the plasma proteome of i) 10 healthy, young, male individuals in response to 2 days of acute caloric restriction followed by refeeding; ii) 200 individuals of the Ely epidemiological study before and after a glucose tolerance test at 4 time points (0, 30, 60, 120 minutes); and iii) 200 random individuals from the Generation Scotland study. We compared the proteomic changes detected with metabolome data and endocrine parameters. Results: Both caloric restriction and the glucose challenge substantially impacted the plasma proteome. Proteins responded across individuals or in an individual-specific manner. We identified nutrient-responsive plasma proteins that correlate with changes in the metabolome, as well as with endocrine parameters. In particular, our study highlights the role of apolipoprotein C1 (APOC1), a small, understudied apolipoprotein that was affected by caloric restriction and dominated the response to glucose consumption and differed in abundance between individuals with and without type 2 diabetes. Conclusion: Our study identifies APOC1 as a dominant nutritional responder in humans and highlights the interdependency of acute nutritional response proteins and the endocrine system

The difference between rare and exceptionally rare: molecular characterization of ribose 5-phosphate isomerase deficiency

Ribose 5-phosphate isomerase (RPI) deficiency is an enzymopathy of the pentose phosphate pathway. It manifests with progressive leukoencephalopathy and peripheral neuropathy and belongs, with one sole diagnosed case, to the rarest human disorders. The single patient was found compound heterozygous for a RPI frameshift and a missense (RPI(Ala61Val)) allele. Here, we report that two patient-derived cell lines differ in RPI enzyme activity, enzyme concentration, and mRNA expression. Furthermore, we present a transgenic yeast model, which exhibits metabolite- and enzyme-activity changes that correspond to the human syndrome and show that the decrease in RPI activity in patient cells is not fully attributable to the residue exchange. Taken together, our results demonstrate that RPI deficiency is caused by the combination of a RPI null allele with an allele that encodes for a partially active enzyme which has, in addition, cell-type-dependent expression deficits. We speculate that a low probability for comparable traits accounts for the rareness of RPI deficiency

A time-resolved proteomic and prognostic map of COVID-19.

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

Die Rolle der Pyruvatkinase in der Synchronisierung von Energie- und Redoxstoffwechsel

The metabolic network responds rapidly to changing conditions in order to self-adapt and ensure cellular survival. Metabolic modules such as pathways of carbohydrate metabolism can gain activity when required by varying demands. In Grüning et al. 2011 we demonstrated a metabolic feedback loop that synchronizes energy- and redox metabolism in respiring yeast cells. The activity of the glycolytic enzyme PYK plays a crucial role in the regulation of mitochondrial respiration and simultaneous PPP activation. Coordinated activation of both processes protects yeast cells from oxidative damage by ROS produced through the respiratory chain. However, the question for the connecting mechanism between low PYK activity and elevated mitochondrial respiration is still unanswered. Also, elucidating the sources that fuel mitochondria when PYK activity is low will be subject of continuative projects. In cancer cells, an isoform switch of pyruvate kinase (PKM1 and PKM2) was stated to be causative for increased fermentation and lowered respiration - the tumor-typical Warburg effect. In Bluemlein et al. 2011 we could refute this postulation. The comprehensive LC-MRM dataset did not confirm an exchange in PKM isoforms, but an upregulation of PKM1 and PKM2 in human malignant cells. These data point to importance of the total PKM expression level for cancer cell metabolism. Future studies have to elucidate the mechanisms that require PKM upregulation in proliferating cells, and therefore provide an advantage for growing cells. Metabolic rearrangements such as caused by varying PYK activities or oxidative stress lead to wide- ranging effects on the whole cellular reaction network. In Krüger et al. 2011, regulatory importance of PPP activation in timing of the transcriptional oxidative-stress program could be proven. Therefore, the PPP does not only provide the cell with NADPH as immediate reaction to counter-act the oxidative stress situation, fast changes in PPP metabolite levels also function as mediator for the stress signal to regulatory components of the transcriptional machinery which follow in time. The sensors that are stimulated by changes in line with PPP activation are still to be found. Disruptions in central pathways of the metabolic network can lead to severe physiological dysfunctions in humans. Deficiency of the PPP enzyme RPI was reported as the rarest inheritable human disease to date. In Wamelink et al. 2010 we revealed the molecular RPI-deficiency pathogenesis, to our knowledge, with a single diagnosed patient, the rarest disease on earth. A combination of two mutations that cause cell-type dependent reduced RPI expression levels and decreased specific RPI activity lead to the manifestation of RPI-deficiency symptoms. Finally, investigating metabolic transitions gives insight into fundamental biological organizational principles that mostly affect not only the metabolome, but also other hierarchical layers such as the transcriptome and proteome. Regarding the cell as complex reaction network, that dynamically reacts to changing conditions, has always to be considered in tackling biological questions.Das metabolische Netzwerk reagiert schnell auf sich ändernde Bedingungen, um sich an die neuen Umstände anzupassen und so das Überleben der Zelle zu ermöglichen. Metabolische Module, wie Stoffwechselwege des zentralen Kohlenhydratstoffwechsels, können an Aktivität gewinnen, wenn dies durch die sich ändernden Bedingungen erforderlich ist. In dem Manuskript Grüning et al. 2011 zeigen wir einen metabolischen feedback-loop in atmenden Hefezellen, der den Energie- mit dem Redox-Stoffwechsel koordiniert. Das glykolytische Enzym Pyruvatkinase (PYK) spielt eine entscheidende Rolle in der Regulierung der mitochondrialen Atmung und der zeitgleichen Aktivierung des Pentosephosphat Weges (PPP). Durch die mitochondriale Atmungskette kommt es zu einer erhöhten Produktion freier Sauerstoffradikale (ROS). Die koordinierte Aktivierung von Zellatmung und PPP schützt Hefezellen jedoch vor Schäden, die durch ROS verursacht werden können. Allerdings konnte bislang nicht geklärt werden, wie ein Wechsel in der PYK Aktivität die mitochondriale Atmung reguliert. Außerdem wird die Frage nach den Nährstoffen, die eine erhöhte mitochondriale Atmung bei geringer PYK Aktivität ermöglichen, Gegenstand zukünftiger Arbeit sein. In Krebszellen wurde ein Wechsel von Pyruvatkinase Isoformen (PKM1 und PKM2) als ursächlich für den Tumor-typischen Warburg Effekt, eine Reduzierung der Zellatmung und Erhöhung der Fermentation, erklärt. In dem Manuskript Bluemlein et al. 2011 konnten wir diese Behauptung widerlegen. Ein umfassender Datensatz, der mittels LC-MRM erstellt wurde, konnte keinen Austausch beider Isoformen während der Tumorigenese bestätigen. Vielmehr kommt es, im Vergleich zu gesunden Kontrollen, zu einer Hochregulierung beider Isoformen in Tumorzellen. Diese Daten verweisen auf eine entscheidende Rolle des PKM Expressionslevels im Krebsstoffwechsel. In zukünftigen Studien soll geklärt werden, welche zellulären Mechanismen eine PKM Hochregulierung erfordern und dadurch Krebszellen einen Proliferationsvorteil verschaffen. Metabolische Verschiebungen, wie sie z.B. durch eine Veränderung der PYK Aktivität oder oxidativen Stress verursacht werden, haben weitreichende Konsequenzen für das gesamte zelluläre Reaktionsnetzwerk. In Krüger et al. 2011 konnten wir die regulatorische Bedeutung der PPP Aktivität für das anti-oxidative transkriptionelle Stressprogramm der Zelle beweisen. Es konnte gezeigt werden, dass der PPP nicht nur NADPH bereitstellt, um sofort auf die oxidative Stresssituation zu reagieren - die schnellen Änderungen der PPP- Metabolitkonzentrationen fungieren auch als Signale für regulatorische Komponenten der Transkriptionsmaschinerie, welche für die längerfristige Anpassung an oxidativen Stress benötigt wird. Die Sensoren, die durch eine erhöhte PPP Aktivität aktiviert werden, bleiben Fokus zukünftiger Forschung. Störungen des zentralen Kohlenhydratstoffwechsels können zu schweren physiologischen Beeinträchtigungen im Menschen führen. Die Defizienz des PPP Enzyms RPI gilt bislang, mit einem diagnostizierten Patienten, als die seltenste Erbkrankheit. In Wamelink et al. 2010 konnten wir die Ätiologie dieser Krankheit entschlüsseln. Die Manifestierung der RPI-Defizienz Symptome basiert auf der Kombination zweier Mutationen, die zu einer Zelltyp- spezifischen Reduzierung des Expressionslevels und zu einer Reduzierung der spezifischen Enzymaktivität führen. Abschließend lässt sich sagen, dass die Untersuchung metabolischer Verschiebungen Einblicke in fundamentale biologische organisatorische Prinzipien bietet. Diese Verschiebungen betreffen dabei nicht nur das Metabolom, sondern ebenfalls andere hierarchische Ebenen der Zelle wie das Transkriptom oder Proteom. Bei der Untersuchung biologischer Fragestellungen sollte daher die Zelle stets als komplexes Reaktionsnetzwerk, welches sich dynamisch an ändernde Bedingungen anpasst, betrachtet werden

Monogenic Disorders of ROS Production and the Primary Anti-Oxidative Defense

Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the cellular anti-oxidant defense mechanisms, plays a critical role in the pathogenesis of various human diseases. Redox metabolism, comprising a network of enzymes and genes, serves as a crucial regulator of ROS levels and maintains cellular homeostasis. This review provides an overview of the most important human genes encoding for proteins involved in ROS generation, ROS detoxification, and production of reduced nicotinamide adenine dinucleotide phosphate (NADPH), and the genetic disorders that lead to dysregulation of these vital processes. Insights gained from studies on inherited monogenic metabolic diseases provide valuable basic understanding of redox metabolism and signaling, and they also help to unravel the underlying pathomechanisms that contribute to prevalent chronic disorders like cardiovascular disease, neurodegeneration, and cancer

Global analysis of cytosine and adenine DNA modifications across the tree of life.

Funder: Medical Research CouncilInterpreting the function and metabolism of enzymatic DNA modifications requires both position-specific and global quantities. Sequencing-based techniques that deliver the former have become broadly accessible, but analytical methods for the global quantification of DNA modifications have thus far been applied mostly to individual problems. We established a mass spectrometric method for the sensitive and accurate quantification of multiple enzymatic DNA modifications. Then, we isolated DNA from 124 archean, bacterial, fungal, plant, and mammalian species, and several tissues and created a resource of global DNA modification quantities. Our dataset provides insights into the general nature of enzymatic DNA modifications, reveals unique biological cases, and provides complementary quantitative information to normalize and assess the accuracy of sequencing-based detection of DNA modifications. We report that only three of the studied DNA modifications, methylcytosine (5mdC), methyladenine (N6mdA) and hydroxymethylcytosine (5hmdC), were detected above a picomolar detection limit across species, and dominated in higher eukaryotes (5mdC), in bacteria (N6mdA), or the vertebrate central nervous systems (5hmdC). All three modifications were detected simultaneously in only one of the tested species, Raphanus sativus. In contrast, these modifications were either absent or detected only at trace quantities, across all yeasts and insect genomes studied. Further, we reveal interesting biological cases. For instance, in Allium cepa, Helianthus annuus, or Andropogon gerardi, more than 35% of cytosines were methylated. Additionally, next to the mammlian CNS, 5hmdC was also detected in plants like Lepidium sativum and was found on 8% of cytosines in the Garra barreimiae brain samples. Thus, identifying unexpected levels of DNA modifications in several wild species, our resource underscores the need to address biological diversity for studying DNA modifications