University of Groningen

Fructose-1,6-bisphosphate and its role on the flux-dependent regulation of metabolismBley Folly, Brenda

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Citation for published version (APA):Bley Folly, B. (2018). Fructose-1,6-bisphosphate and its role on the flux-dependent regulation ofmetabolism. [Groningen]: University of Groningen.

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Chapter 6

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SummaryMetabolism describes the biochemical reactions that occur in a living organism in order to maintain life. The survival of living organisms depends on the regulation of their metabolism in response to environmental changes by sensing external and internal signals. In order to understand how the regulation of metabolism is orchestrated, we explored a novel concept of metabolic regulation. In this novel concept, the availability of extracellular nutrients causes alterations in the intracellular metabolic fluxes that are recognized by flux signaling metabolites, such as fructose-1,6-bisphosphate (FBP), whose levels correlate with the flux through glycolysis. Flux signaling metabolites than induce cellular regulation mechanisms, for instance, by modulating the activity of tran-scription factors, which in turn regulate gene expression in a flux-dependent manner.

In this thesis, we investigated the role of FBP as a flux-signaling metabolite and its par-ticipation in the regulation of metabolism. In Chapter 1, we review the current knowl-edge about the regulatory network of FBP with proteins from various cellular processes across different organisms, and the phenotypic effects caused by altered FBP levels. We also review different methods available to identify protein-metabolite interactions, and investigated a novel mass spectrometry technique1 developed to study alterations in protein conformation caused by the interaction with metabolites. In this method, it was observed that the conformation of several proteins changed in presence of FBP, which suggests that FBP interacts with these proteins.

In order to confirm whether the reported alterations in protein conformation were due to a direct interaction with FBP, in Chapter 2 and 3 we biochemically tested some of these putative interactions. In Chapter 2, we analyzed the interaction between FBP and the glycolytic enzyme hexokinase 2 (Hxk2). We observed no alteration in the stability of Hxk2 in presence of FBP, and also no indication of a direct binding. The activity of the enzyme was also not influenced by FBP at any concentration tested. In order to explain the conformational changes previously observed in Hxk2, we explored the pos-sibility of secondary effectors, such as metal ions. In fact, we found that in presence of zinc, the stability and the activity of Hxk2 were impaired. We also observed that the effects of zinc on Hxk2 were restored by FBP acting as a chelator. FBP is a key metabolite of the glycolytic pathway, and also a flux-signaling metabolite. Therefore, it could be conceived, that FBP could play an indirect regulatory role by chelating metal ions, which would globally modulate the activity of enzymes in a glycolytic flux-dependent manner.

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Towards a biochemical validation of the putative interactions identified by the novel mass spectrometry method, in Chapter 3 we analyzed seven other enzymes (Asc2, Pgi1, Pck1, Mdh2, Erg10, Icl1, and Pdc1) that also showed structural changes in presence of FBP. Also with these proteins, we did not observe any alteration in the stability, nor found indications of direct binding with FBP. For Pgi1 and Mdh2 we further investigated the role of FBP on the enzyme activity, but also here no differences were identified. Therefore, we concluded that the conformational changes previously observed in the structure of the studied proteins were not due to a direct interaction with FBP. The hypothesis generated for Hxk2 in Chapter 2 could also explain the conformational changes observed in these proteins. In this hypothesis, FBP would act as a chelator, and interact with metal ions responsible for activation or inhibitions of the studied proteins, causing indirect alterations in protein structure.

With the increasing recognition of FBP as a metabolite that signals the glycolytic flux, in Chapter 4 we investigated the role of FBP in the flux-depending regulation of the bacterial transcription factors CggR and Cra. The interaction between FBP and Cra is still a point of debate in the literature, since no solid biochemical evidence has been presented so far. We observed that the stability of Cra was not affected by FBP, and no other indication of a direct interaction was observed. Functional experiments also showed no influence of FBP in the interaction of Cra and its DNA operator. Therefore, we have provided experimental prove that FBP does not interact with Cra, nor regulate the activity of this transcription factor.

Although the interaction between CggR and FBP is well established, there are still controversies about the concentration range in which FBP regulates the interaction between CggR and its DNA operator. In Chapter 4, by assessing the interaction of CggR and FBP, we found that millimolar concentrations of FBP bind to CggR, and considerably increase the stability of the protein. Functional analysis of CggR also confirmed that millimolar concentrations of FBP were required for the regulation of CggR’s activity as transcription factor. The concentrations range of FBP required for the regulation of CggR’s activity corroborates with the physiological concentration of FBP. Therefore, FBP, by acting as a flux signaling metabolite, provides an essential link between flux signals and gene expression regulation by modulating the activity of CggR.

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References 1. Feng, Y, Franceschi, G, Kahraman, A, Soste, M, Melnik, A, Boersema, P J, Laureto, P P, Nikolaev, Y, Oliveira,

A P, & Picotti, P. Global analysis of protein structural changes in complex proteomes. Nat. Biotechnol. (2014). doi:10.1038/nbt.2999

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SamenvattingMetabolisme omschrijft de biochemische reacties die plaatsvinden in levende organis-men om in leven te blijven. De overleving van levende organismen hangt af van de regu-latie van hun metabolisme als reactie op veranderingen door middel van het waarnemen van externe en interne signalen. Om te begrijpen hoe de regulatie van metabolisme is georganiseerd, hebben we een nieuw metabolisch regulatieconcept onderzocht. In dit nieuwe concept, veroorzaakt de beschikbaarheid van extracellulaire nutriënten veranderingen in de intracellulaire metabolische fluxen. Deze worden herkend door flux signalerende metabolieten, zoals fructose-1,6-bisfosfaat (FBP), waarvan de hoe-veelheden correleren met de flux door de glycolyse. Flux signalerende metabolieten induceren cellulaire regulatiemechanismen, bijvoorbeeld, middels het moduleren van de activiteit van transcriptiefactoren, welke dan beurtelings de gen expressie reguleren op een flux-afhankelijke manier.

In dit proefschrift onderzochten we de rol van FBP als een flux-signalerend metaboliet en zijn participatie in de regulatie van metabolisme. In hoofdstuk 1 geven we een overzicht van de huidige kennis over het regulatienetwerk van FBP in combinatie met eiwitten van verschillende cellulaire processen in verschillende organismen, en de fenotypische effecten veroorzaakt door veranderende FBP niveaus. We geven ook een overzicht van verschillende methoden die beschikbaar zijn voor het identificeren van eiwit-metaboliet interacties, en onderzoeken een nieuwe massaspectrometrie techniek1 die is ontwik-keld voor het bestuderen van veranderingen in eiwitconformaties veroorzaakt door de interactie met metabolieten. In deze methode is geobserveerd dat de conformatie van verschillende eiwitten verandert in de aanwezigheid van FBP, wat suggereert dat FBP interacties heeft met deze eiwitten.

Om te bevestigen of de gerapporteerde verandering in eiwitconformaties een gevolg is van een directe interactie met FBP hebben we, in hoofdstuk 2 en 3, sommige van deze vermeende interacties biochemisch getest. In hoofdstuk 2 hebben we de interacties tussen FBP en het glycolyse enzym hexokinase 2 (Hxk2) geanalyseerd. We observeerden geen verandering in de stabiliteit van Hxk2 in de aanwezigheid van FBP, en eveneens geen indicatie van een directe binding. Bij alle geteste concentraties werd de activiteit van het enzym niet beïnvloed door FBP. Om de eerder geobserveerde confirmatie veranderingen in Hxk2 te verklaren, onderzochten we de mogelijkheid van secundaire effectoren, zoals metaalionen. We vonden dat de stabiliteit en activiteit van Hxk2 ver-zwakt in de aanwezigheid van zink. We observeerden ook dat de effecten van zink op

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Hxk2 werden hersteld doordat FBP werkte als een chelator. FBP is een sleutelmetaboliet van de glycolytische route, en ook een flux-signalerend metaboliet. Daarom kan worden opgevat dat FBP een indirecte regulatorische rol kan spelen middels het binden van metaalionen, welke globaal de activiteit van enzymen moduleren in een glycolytische flux-afhankelijke manier.

Voor een biochemische validatie van de vermeende interacties, geïdentificeerd middels een nieuwe massaspectrometrie methode, hebben we in hoofdstuk 3, zeven andere enzymen (Asc2, Pgi1, Pck1, Mdh2, Erg10, Icl1 en Pdc1) geanalyseerd die ook structurele veranderingen lieten zien in de aanwezigheid van FPB. Ook bij deze eiwitten obser-veerden we geen enkele verandering in de stabiliteit, en eveneens vonden we geen indicaties van directe binding met FBP. Voor Pgi1 en Mdh2 deden we verder onderzoek naar de rol van FBP op de enzymactiviteit, maar ook hier vonden we geen verschillen. Daarom concluderen we dat de eerdere geobserveerde verandering in conformatie gevonden in de structuur van de bestudeerde eiwitten niet kwam door een directe interactie met FBP. De hypothese die ontwikkeld was voor Hxk2 in hoofdstuk 2, kan ook de conformatie veranderingen verklaren, geobserveerd in deze eiwitten. In deze hypothese gedraagt FBP zich als een chelator, welke interacteert met metaalionen die verantwoordelijk zijn voor de activatie of inhibitie van de bestudeerde eiwitten, welke indirecte veranderingen in de eiwitstructuur veroorzaakt.

Met de toenemende erkenning van FBP als een metaboliet dat de glytolytische flux sig-naleert, onderzoeken we in hoofdstuk 4 de rol van FBP in de flux-afhankelijke regulatie van de bacteriële transcriptiefactoren CggR en Cra. De interactie tussen FBP en Cra is nog steeds een discussiepunt in de literatuur, aangezien er geen solide biochemische bewijzen zijn gepresenteerd tot nu toe. We observeerden dat de stabiliteit van Cra niet wordt beïnvloed door FBP, en er was geen andere indicatie dat er een directe inter-actie is. Functionele experimenten laten ook zien dat er geen invloed is van FBP op de interactie van Cra en zijn DNA operator. Derhalve hebben we experimenteel bewijs verschaft dat FBP geen interactie heeft met Cra, noch de activiteit reguleert van deze transcriptiefactor.

Hoewel de interactie tussen CggR en FBP goed is onderzocht, zijn er nog steeds con-troversies over de concentratierange waarin FBP de interactie tussen CggR en zijn DNA operator reguleert. In hoofdstuk 4 hebben we de interactie tussen CggR en FBP beoordeelt. We vonden dat millimolaire concentraties van FBP binden aan CggR en de

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stabiliteit van het eiwit aanzienlijk verbeteren. Functionele analyse van CggR bevestigt dat millimolaire concentraties van FBP nodig zijn voor de regulatie van CggR’s activiteit als transcriptiefactor. De concentratierange van FBP die nodig is voor de regulatie van CggR’s activiteit, komt overeen met de fysiologische concentraties van FBP. Daarom voorziet FBP, werkend als een flux signalerend metaboliet, een essentiële link tussen flux signalen en genexpressie regulatie door middel van het moduleren van de activiteit van CggR.

Referenties 1. Feng, Y, Franceschi, G, Kahraman, A, Soste, M, Melnik, A, Boersema, P J, Laureto, P P, Nikolaev, Y, Oliveira,

A P, & Picotti, P. Global analysis of protein structural changes in complex proteomes. Nat. Biotechnol. (2014). doi:10.1038/nbt.2999

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ResumoO metabolismo descreve as reações que ocorrem em um organismo a fim de manter a vida. A sobrevivência dos organismos depende da regulação do metabolismo de acordo com alterações no ambiente, através da detecção de sinais externos e internos. Com o intuito de entender de que maneira a regulação metabólica é orquestrada, nós investigamos um novo conceito de regulação do metabolismo. Neste novo conceito, alterações na disponibilidade dos nutrientes extracelulares resultam em alterações nos fluxos metabólicos internos, os quais são reconhecidos por metabólitos de sinalização de fluxo. Um exemplo é o metabólito fructose-1,6-bisfosfato (FBP), cuja concentração está correlacionada com o fluxo através da glicólise. Os metabólitos de sinalização de fluxo são capazes de induzir mecanismos de regulação celular, como por exemplo, atra-vés da regulação da atividade de fatores de transcrição, os quais, por sua vez, regulam a expressão gênica de acordo com o fluxo metabólico.

Na presente tese, investigamos a função de FBP como metabólito de sinalização de fluxo, e a sua participação na regulação do metabolismo. No capítulo 1, revisamos o conhecimento atual sobre a rede regulatória de FBP, que se dá pela interação com proteínas de diversos processos celulares, bem como os efeitos fenotípicos causados pela alteração da concentração intracelular de FBP. Nós também analisamos os dife-rentes métodos disponíveis para identificar interacões entre proteínas e metabólitos, e averiguamos uma nova técnica de espectrometria de massa1 desenvolvida para estudar alterações na estrutura de proteínas que ocorrem devido à interação com metabólitos. Neste método, foi observado que várias proteínas sofreram alterações conformacionais quando em presença de FBP, o que sugere a interação destas proteínas com FBP.

Com o objetivo de confirmar se tais alterações conformacionais foram de fato causadas pela interação direta com FBP, no capítulo 2 e 3 nós analisamos bioquimicamente algumas dessas interações. No capítulo 2, a interação entre FBP e a enzima glicolítica hexoquinase 2 (Hxk2) foi estudada. Nenhuma alteração na estabilidade de Hxk2 foi observada quando em presença de FBP. Nenhuma indicação de interação direta com FBP foi tampouco observada. A atividade da enzima também não foi influenciada por FBP em nenhuma das concentrações testadas. A fim de explicar as alterações confor-macionais previamente observadas em Hxk2, a possibilidade de efetores secundários, como íons metálicos, foi explorada. De fato, observamos que na presença de zinco, a estabilidade e a atividade da enzima Hxk2 foram prejudicadas. Porém, FBP, agindo como quelante, foi capaz de restaurar os efeitos causados por zinco em Hxk2. FBP é um

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metabólito chave na via glicolítica, bem como um metabólito de sinalização de fluxo. Portanto, é possível conceber que FBP atue na regulação do metabolismo de maneira indireta, chelando íons metálicos e modulando a atividade de diferentes enzimas de acordo com o fluxo metabólico.

Com o intuito de validar bioquimicamente as interações putativas identificadas pelo método de espectrometria de massa mencionado acima, no capítulo 3, consideramos sete outras enzimas que sofreram alterações estruturais em presença de FBP (Asc2, Pgi1, Pck1, Mdh2, Erg10, Icl1, and Pdc1). A estabilidade destas proteínas não foi alte-rada em presença de FBP, e nenhuma indicação de interação direta com este metabólito foi encontrada. A atividade enzimática de Pgi1 e Mdh2 foi analisada na presença de FBP, e nenhuma alteração foi observada. Desta forma, concluímos que as alterações conformacionais previamente observadas na estrutura das proteínas estudadas não foram causadas por interação direta com FBP. A hipótese gerada para Hxk2 no capítulo 2 também poderia explicar as alterações conformacionais observadas nestas proteí-nas. Nesta hipótese, FBP atuaria como um chelante e interagiria com íons metálicos responsáveis pela ativação e inibição das proteínas estudadas e causaria alterações na estrutura destas proteínas de maneira indireta.

Com o crescente reconhecimento de FBP como um metabólito de sinalização do fluxo glicolítico, no capítulo 4 pesquisamos acerca da função de FBP na regulação fluxo-dependente dos fatores de transcrição Cra e CggR. A interação entre FBP e Cra ainda é um ponto de debate na literatura, uma vez que nenhuma evidência bioquímica foi apresentada até agora. Nossas análises mostraram que a estabilidade de Cra não foi afetada por FBP, e também nenhuma outra indicação de interação direta foi observada. Experimentos funcionais mostraram que a presença de FBP não influencia a interação de Cra e seu DNA operador. Desta forma, nosso trabalho fornece evidências experimen-tais sólidas de que FBP não interage com Cra, e não regula a atividade deste fator de transcrição.

Embora a interação entre CggR e FBP seja bem estabelecida, ainda existem controvér-sias a respeito da concentração na qual FBP regula a interação entre CggR e o DNA operador. Ainda no capítulo 4, através da avaliação da interação entre CggR e FBP, pudemos observar que concentrações milimolares de FBP se ligam a CggR, e aumentam consideravelmente a estabilidade da proteína. Análises funcionais de CggR também confirmaram que concentrações milimolares de FBP são necessárias para a regulação

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da atividade de CggR como fator de transcrição. É interessante notar que a concentra-ção de FBP necessária para a regulação da atividade de CggR correlaciona-se com a concentração fisiológica de FBP. Portanto, FBP, ao atuar como metabólito de sinalização de fluxo, estabelece uma ligação essencial entre os sinais do fluxo e a expressão gênica através da regulação da atividade de CggR.

Referências 1. Feng, Y, Franceschi, G, Kahraman, A, Soste, M, Melnik, A, Boersema, P J, Laureto, P P, Nikolaev, Y, Oliveira,

A P, & Picotti, P. Global analysis of protein structural changes in complex proteomes. Nat. Biotechnol. (2014). doi:10.1038/nbt.2999