Research Projects
This section introduces all ongoing projects of the Enzymology & Metabolism group listed under 4 main topics:
- Metabolite/Protein Damage and Repair
- Rare Disease Research
- Metabolomics Approaches applied to Enzyme Discovery and Disease Research
- Technological Platforms
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Figure: Cover picture proposition for Paczia et al. 2019 Biochemistry paper |
Metabolite/Protein Damage and Repair
| NAD(P)HX formation and repair |
| In 2011, we discovered a widely conserved enzymatic repair mechanism for hydrated forms of NADH and NADPH, two central cofactors of cell metabolism. The hydration of NAD(P)H can be catalysed as a side reaction of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or can proceed spontaneously at elevated temperatures, leading to the formation of two epimers of NAD(P)H hydrates designated S-NAD(P)HX and R-NAD(P)HX. Those hydrated derivatives cannot act as enzyme cofactors and have been shown to inhibit dehydrogenases. To cope with these damaged forms of NAD(P)HX, organisms have evolved a repair system that comprises an ATP-dependent S-NAD(P)HX dehydratase (NAXD) and an NAD(P)HX epimerase (NAXE). Mutations in NAXD or NAXE lead to a severe infantile neurometabolic disorder. To elucidate the molecular mechanism of this disorder, we aim in this project to better understand how and under which circumstances NAD(P)HX is formed in the cell and what cellular functions are most affected by the presence of NAD(P)HX. We are therefore investigating the consequences of NAD(P)HX repair deficiency in various models, starting from the simple yeast and human HAP1 cell models to more complex iPSC-derived neuronal cell lines and brain organoids, as well as zebrafish models. In-depth phenotyping of these models is performed at molecular (e.g. metabolomics, transcriptomics) and cellular (e.g. viability, mitochondrial function) levels. The ultimate aim is to pinpoint critical perturbations induced by NAXD and/or NAXE deficiency and to conceive and test strategies to prevent or correct these perturbations. The focus is on small molecule based approaches with therapeutic potential.
Video abstract for a paper about NAXD deficiency: Contacts:
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| GDP-glucose formation and repair |
| Another recently discovered enzyme potentially involved in metabolite repair is GDP-glucose phosphorylase (GDPGP1). The latter degrades GDP-glucose, a nucleotide sugar that has no known function in mammalian cells and that seems to be erroneously formed by GDP-mannose pyrophosphorylase, the enzyme that normally forms GDP-mannose (GDP-Man). GDP-Man is an important nucleotide sugar used for protein N-glycosylation in the cell. Surprisingly, mammalian GDP-Man pyrophosphorylase has been relatively poorly studied. Purification of this enzyme from mammalian tissues revealed that it is composed of two different subunits that are encoded by two distinct genes, GMPPA and GMPPB. While the isolated GMPPB protein has been shown to catalyse GDP-Man formation, the role and function of the GMPPA subunit remains obscure. In collaboration with Marcelo Guerin’s group at CIC bioGUNE, we currently work on producing, isolating and characterizing the GMPPA-GMPPB protein complex. The relevance of this project is underlined by the fact that defects in both the GMPPA and GMPPB proteins have been found to lead to rare glycosylation disorders. Similarly, GDP-glucose phosphorylase deficiency could be causally involved in glycosylation disorders of currently unknown genetic origin. Contacts: Collaborator:
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| 2-Hydroxyglutarate metabolism and function in yeast |
| D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG) are metabolites that accumulate in several types of inherited neurometabolic diseases and certain forms of cancer such as glioblastomas and acute myeloid leukaemias. In each of these diseases, mutations in specific dehydrogenases for L-2HG, D-2HG, or isocitrate have been shown to cause 2HG accumulation. However, the intracellular pathways leading to 2HG formation and the roles of 2HG in disease are still unclear. We recently uncovered the metabolic reactions leading to 2HG formation and degradation in Saccharomyces cerevisiae, an organism that produces exclusively the D-enantiomer of this dicarboxylic acid. Our results show that in yeast, D-2HG metabolism links the main serine synthesis pathway to the mitochondrial respiratory chain. Using an mQTL approach, we search for additional genes involved in controlling D-2HG levels in this model organism. In addition, in collaboration with Lasse Sinkkonen’s group, we investigate the epigenetic effects of 2HG accumulation (and potential consequences thereof) in yeast. This work could lead to the identification of molecular drug target candidates to be tested for the treatment of certain forms of cancer and severe neurometabolic disease.
Figure: Cover picture for the 2016 JBC paper by Becker-Kettern et al.Contact:
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| Metabolite repair and fluorometabolite annotation in SinFonia |
| SinFonia, an EU-funded H2020 project, aims to integrate the nonnative element fluorine into the metabolism of Pseudomonas putida to produce novel fluorinated polyhydroxyalkanoates (PHAs), ideally in such a way that bacterial growth will become dependent on this incorporation. Fluorine is common in industrial chemicals with versatile applications from electrical insulation to waterproofing, yet it is seldom present in biological systems. The current production processes for fluorochemicals often negatively affect the environment, and there is a great need for more sustainable and less harmful alternatives. If successful, SinFonia will provide a less hazardous and more sustainable solution to synthesizing fluorochemicals. Our role, in collaboration with the Environmental Cheminformatics Group, is to identify fluorinated and undesirable metabolites in engineered bacteria using non-targeted mass spectrometry. Engineered systems may lack sufficient metabolite repair capacity, which we aim to counteract by screening for metabolite damages in our cell factories and envisaging strategies to repair them. Metabolite repair may be particularly important in SinFonia due to the load of adding heterologous pathways and a nonnative element to create new-to-nature products
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| Role of a protein repair methyltransferase in calcium signalling |
| PCMT1 is a highly conserved repair enzyme that recognizes spontaneously formed isoaspartyl residues in proteins and, through methylation, facilitates their reconversion back into the normal aspartyl precursors. PCMT1 deficiency leads to accumulation of high levels of damaged proteins, especially in the brain, and massive seizures in a knockout mouse model. In addition, PCMT1 overexpression led to a longer life span in worms and flies. Other studies suggest roles for PCMT1 also in neurodegenerative disease and cancer. In this project, we have established and phenotyped zebrafish and mouse hippocampal cell models with PCMT1 deficiency. Our studies indicate that PCMT1 plays an important role in supporting normal calcium signalling in the brain and our models can now be used to investigate the underlying molecular mechanisms. More generally, they can contribute to gain a deeper understanding of the physiological function of this intriguing protein repair enzyme that seems to play a negative role in cancer (anti-apoptotic) and a positive role in aging (increases life span).
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Rare Disease Research
| Modeling Batten disease in zebrafish |
| Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), known as Batten disease, is an autosomal recessive, rare neurodegenerative disorder that affects mainly children from the age of 5 years on. The most common cause for this disease is a mutation in the CLN3 gene, which results in accumulation of autofluorescent material (ceroid lipofuscins) in the lysosomes. The CLN3 protein is conserved among species and several model organisms for JNCL have been created, including yeast and mouse models. Despite these efforts, the protein function and consequently the pathophysiological disease mechanism remain unclear. In the present work, we created two cln3-deficient zebrafish lines using a CRISPR/Cas9 approach. Those models are currently analysed using behavioral assays and metabolomics, in order to identify disease-relevant phenotypes that can be exploited for disease mechanism elucidation and for the development of in vivo drug screening approaches. Contact: Funding:
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| Towards small-molecule therapies for ATP13A2 deficiencies |
| Mutations in ATP13A2 (also designated PARK9) were described in 2006 to cause the Kufor-Rakeb Syndrome, a rare early-onset form of Parkinsonism with dementia. Interestingly, in 2012, a different mutation in the same gene was found to lead to neuronal ceroid lipofuscinosis (also known as Batten disease) and in 2017, yet other ATP13A2 mutations were associated with Spastic Paraplegia-78. The link between these rare neurodegenerative diseases is further supported by studies in animal models, but the molecular mechanism(s) underlying the connection between ATP13A2 deficiency and disease pathogenesis is not yet understood. Nevertheless, these findings indicate that ATP13A2 plays a crucial role in supporting healthy neuronal cells. In this project, we developed a drug screening pipeline to accelerate the discovery of potential therapeutic compounds for ATP13A2 associated disorders. We took advantage of the fact that ATP13A2 is highly conserved from yeast to humans and we developed a high-throughput drug screening strategy for ATP13A2 deficiencies in yeast based on a decreased zinc resistance phenotype. We have applied it to implement a primary screen of 2560 drugs. For validation of the positive hits, we generated two stable ATP13A2 knockout zebrafish lines that showed increased sensitivity to manganese toxicity, especially in the central nervous system. Based on phenotypic rescue assays, we validated 2 hits from the primary yeast screen, N-acetylcysteine and furaltadone, in zebrafish. Currently we are testing these two drugs in ATP13A2 patient-derived cells. We aim in the future to keep exploiting our unique combination of ATP13A2 models for further investigation of related disease mechanisms as well as small molecule-based therapeutic approaches.
Figure: Graphical Abstract Scheme from 2019 ATP13A2 paperContacts: Collaborators:
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| Towards small-molecule therapies for Zellweger Syndrome |
| Zellweger syndrome (ZS) is a rare peroxisomal biogenesis disorder. It affects several organs (including brain, liver and kidneys) and symptoms typically appear in the newborn period. Mutations in the PEX1 gene are the most common cause for ZS. There is no cure for ZS and good disease models are rare. Our project is focused on development of a robust high-throughput drug screening approach in PEX1-deficient ZS patient-derived cell lines as well as the development of a pex1-deficient vertebrate model. The overall aim is to discover compounds that can alleviate the underlying cause of ZS, namely peroxisomal dysfunction, by first screening thousands of compounds in patient-derived cells and potentially advancing to a whole organism model for validation, in our case zebrafish (Danio rerio). In the human cell line screens, our readout is subcellular localization of catalase via immunostaining and fluorescence imaging. We collaborate with the LCSB Automation and Imaging platforms to perform the imaging-based drug screens in 384-well plate format followed by automated data analysis using MATLAB scripts. The pex1 mutant zebrafish model is being generated using CRISPR/Cas9 technology. This is a very exploratory part of the project and phenotypes in this model, if viable, will have to be analysed carefully before positive hits from the cell-based screens can be validated in zebrafish through potential phenotypic rescue assays. Contact: Funding:
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Metabolomics Approaches applied to Enzyme Discovery and Disease Research
| Comparative genomics- and metabolomics-based strategies for enzyme function discovery |
| In collaboration with the Bioinformatics Core, we identified more than 600 and 2000 putative enzyme genes among the about 2000 and 6500 genes of unknown function in yeast and human, respectively. Prioritising putative enzyme genes with predicted roles in metabolism and/or disease, we develop, in collaboration with the Metabolomics platform hosted by our group and with the Environmental Cheminformatics Group, targeted and non-targeted LC-MS-based approaches to compare metabolite profiles of control cells and cells with specific gene deletions. Metabolites whose levels differ significantly between these cells help to predict endogenous reactions catalysed by the enzymes of interest. Those predictions can then be validated on recombinant protein level through appropriate enzymatic assays. This approach has already allowed to identify a new eukaryotic D-ribulokinase. With this project, we aim to address a major post-genomic challenge that can only be tackled by a community-wide effort: progress from knowing the code to understanding the message by decreasing the number of genes of unknown function. Collaborator:
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| Untargeted and targeted approaches to identify metabolites involved in colon cancer development and progression |
| Hydrogen sulfide (H2S) and persulfides have been shown to affect signalling pathways and metabolic functions involved in carcinogenesis. H2S is produced in mammalian cells through the transsulfuration pathway, but intestinal cells are also exposed to H2S produced by commensal gut bacteria. The sulfur oxidation pathway, involving the SQRDL and ETHE1 enzymes, plays a key role in catabolism of H2S in mammalian cells. The aims of the project comprise (1) elucidation of the mammalian sulfur oxidation pathway and the potential role of alterations in this pathway in colorectal cancer, (2) investigation of metabolomic profile changes, and more specifically in sulfur metabolism, in colorectal cancer development and progression, and (3) analysis of the influence of the gut microbiome on sulfur metabolism in the host intestinal epithelium. Gaining a deeper insight into the molecular mechanisms of the microbiome-host crosstalk is essential for the development of dietary recommendations, notably in the context of colorectal cancer. Contact: Collaborators:
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| Search for novel PD biomarkers originating from increased molecular damage and/or deficient molecular quality control systems |
| The Parkinson associated protein DJ-1 has recently been proposed to have a robust deglycase activity towards nucleotides and amino acids modified by the reactive dicarbonyl metabolites, glyoxal and methylglyoxal. Protein and DNA glycation adducts produced by exposure to glyoxal and methylglyoxal were also shown to be repaired in vitro by DJ-1. Glyoxal and methylglyoxal can be formed physiologically by lipid peroxidation and as by-product of glycolysis, respectively. Glycation adducts of deoxyguanosine (dG) have previously been shown to reach similar cellular levels than 8-oxodG, a commonly used marker of DNA oxidation. Both glycated and oxidized dG derivatives can be measured in biological samples, including plasma and urine. Stable isotope dilution LC-MS/MS is the analytical method of choice, but immunoassays also exist to measure both types of DNA damage markers (8-oxodG CEdG ELISA kits). Given the recent findings concerning DJ-1 function, we propose here to evaluate glycated dG derivatives as biomarkers for PD. This is further motivated by the observation that diabetes mellitus is a risk factor for PD and other previous findings indicating a link between protein glycation and PD. In this project, analyses will be performed on DJ-1 mutant cell models as well as PD patient derived material. In collaboration with IBBL, different types of biospecimens (plasma, urine, CSF, fibroblast extracts) as well as pre-analytical sample processing procedures will be optimized for detection of the markers of interest. Cell culture experiments and LC-MS method development and analyses will be performed at LCSB. Ideally, the project will conclude with a biomarker validation phase at IBBL. Contact: Collaborators:
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Technological Platforms
| Yeast Platform |
| Our group has built up a platform for using yeast as a model organism, notably for functional genomics purposes. Yeast is the simplest eukaryotic organism, but, due to its ease of manipulation and amenability to genetic modifications, has proven invaluable in the elucidation of conserved molecular mechanisms underlying important cell functions such as mitochondrial biogenesis, cell division, and cell death. Many human disease genes have counterparts in yeast, which can therefore also be used for the dissection of molecular mechanisms of disease and for drug screens. Gene deletion collections in Saccharomyces cerevisiae for all non-essential genes are hosted by the platform as well as a collection of natural yeast strains and humanized yeast models (alpha-synuclein overexpression strains). Production and subsequent purification of recombinant proteins of interest by overexpression in yeast can be performed. Yeast-specific metabolite and RNA extraction protocols for subsequent metabolomics and transcriptomics analyses, and high-throughput phenotypic screens and lifespan assays have been developed to allow detailed functional analyses of genes of interest and to assist metabolic modelling efforts. Currently, a new design for microfluidics-based determination of yeast replicative lifespan is being tested.
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| Metabolomics platform |
| The Metabolomics Platform is specialized in developing and applying mass spectrometry-based comprehensive analysis methods of small molecules in complex biological samples. The Metabolomics Platform supports a wide range of cutting-edge metabolomic approaches for several sample types, including mammalian cells, bacteria, yeast, body fluids (e.g.cerebrospinal fluid, plasma, serum, urine), soft tissues (e.g.brain, liver, lung, heart, kidney) and others (e.g.Dried Blood Spots). >> More information about the metabolomics platform |
