2024 Program (September - December)

The latest developments in biosourced polymers for regenerable filtration show great potential in the brewing sector. The innovation focuses on a polymer designed through micronization, atomization, and screening, with recent results meeting industry standards for cycle time, pressure, turbidity, and stability. PA11 and PA12 polymers, structurally similar to PVPP, are the most promising candidates, targeting polyphenols and potentially proteins in the filtration process. Success will depend on ensuring filtrate quality, cost efficiency, and minimizing the environmental footprint

 

Flavouring substances are found in almost all food products, either naturally occurring or added on purpose. However, some of them raise toxicological concerns due their reactive chemical group. This is the case of certain alpha,beta-unsaturated carbonyls, some of them being genotoxic while also being naturally formed during thermal food preparation. This research investigated the occurrence of these compounds in a series of commercial sweet snacks and throughout chocolate production process, from raw cocoa beans to finished chocolate bars.

In a world facing increasing food demand and extreme climate change-related environmental cues, it is of global interest to understand the cellular mechanisms that allow plants to cope with biotic and abiotic stresses and, when necessary, to reallocate their resources under such conditions.

Macroautophagy (hereafter autophagy) is an essential major catabolic pathway that allows eukaryotic cells to recycle superfluous or toxic cellular components. Under non-stressful conditions, a basal level of autophagy acts as a quality control, ensuring cellular homeostasis.

Moreover, under a/biotic stress, autophagy becomes a paramount survival response. This highly conserved and regulated pathway involves the de novo formation of a double membrane organelle called autophagosome (AP). AP engulfs defined cargo, ranging from protein aggregates to large defective organelles, and deliver them to the plant cell vacuole for degradation. However, the protein composition of AP is still largely unknown, more so in plant where key regulators responsible of cargo selection, membrane recognition, tethering and fusion of AP with the vacuole are yet to be identified and characterized. Identifying functional proteins as well as autophagosome cargoes requires the purification of these organelles.

However, plant AP purification is challenging due to their relatively short cellular lifetime between their formation and fusion with the lytic compartment. Our implemented purification strategy is first of all to aggregate them with mitochondria to concentrate them in the cytosol and then to affinity purify these autophagosome-mitochondria complexes.

This co-purification strategy will allow us to answer several essential questions such as: i) Are the content and regulatory factors associated with AP specific to a given cell-type and/or stress-dependent? ii) What are the proteins responsible for the fusion of autophagosomes with the lytic compartment of the plant cell?

 

Type 2A protein phosphatases (PP2A) play crucial roles in the regulation of many cellular, biochemical processes in eukaryotes. One of the principal goals of our research group is to understand, how these enzymes interfere with the organization and dynamics of cytoskeleton and endomembranes in plants. The interactions between microtubules, microfilaments and the ER is important for the integrated functioning of a cell. We have found several evidence for the involvement of PP2A (and the related phosphatase, PP1) in these processes. For most of the experiments, Arabidopsis was used as a model. Its mutants in catalytic and regulatory subunits of PP2A and treatments of wild-type plants with the protein phosphatase inhibitor microcystin-LR were used as tools. For these plants we have found defective organization, dynamics and functioning of microtubules, ER, vacuoles, mitochondria, plastids.

We currently investigate the biochemical background of all these alterations. Also, we have found several evidence that regulatory and catalytic subunits of PP2A interfere with oxidative stress phenomena including the activities of superoxide dismutases. Our future plan is to conduct research for a better understanding of the interference of PP2A with ROS generating systems like RBOHD. Our studies may have practical applications, since more knowledge on the regulation of subcellular dynamics and PP2A- oxidative stress interactions may be applied in crop improvement.

 

During meiosis, DNA double strand breaks (DSBs) initiate recombination, crucial for proper chromosome segregation. These breaks are regulated by overlapping pathways and are linked to chromosome organization, though the exact relationship is unclear. Recent findings suggest that RMM proteins (Rec114, Mei4, Mer2) may drive DSB formation through phase separation, creating specialized nuclear compartments. The project aims to study these structures by identifying proteins interacting with RMM complexes in yeast using proximity labeling and mass spectrometry. Additionally, the 3D organization of chromatin within these compartments will be analyzed to understand their role in DSB formation and chromosome structure.

Biomolecular condensation has emerged as a widespread mechanism to organize intracellular compartments and impacts a variety of biological processes by facilitating the kinetics and specificity of biochemical reactions. This project aims to investigate a newly-discovered condensation mechanism in vitro using quantitative methods based on atomic force microscopy (AFM). During meiosis, cells introduce high levels of DNA double-strand breaks (DSBs) throughout their genome. These breaks are essential for sexual reproduction because they trigger a genome-wide mechanism of DNA recombination that is required for the pairing and subsequent segregation of homologous chromosomes. DSB formation requires ten proteins, organized as three sub-complexes: the Spo11 core complex, MRX and RMM. Our laboratory recently found that the RMM subgroup (composed of proteins Rec114, Mei4 and Mer2) undergoes DNA-driven condensation, suggesting a new model of assembly of the meiotic DSB machinery, where RMM nucleoprotein condensates provide a cluster that recruits the other partners prior to DSB formation.
We set up AFM-based force spectroscopy approaches to quantify protein-protein and protein-DNA interactions involved during DNA-driven condensation using the DSB protein, Mer2. For example, we immobilized DNA substrates or Mer2 protein on a surface, and tethered Mer2 protein or an accessory partner of the DSB machinery, Spp1, on an AFM tip. By approaching the tip to the surface and retracting it, we can quantify the forces and binding kinetics of the interactions between each partner.
This work aims to provide quantitative insights into the assembly of the meiotic DSB machinery and to yield new tools to investigate molecular interactions that drive DNA-driven condensation

Biomolecular condensates are essentially discrete subcellular membrane-less compartments that concentrate proteins and nucleic acids, presumably to gain higher spatiotemporal control over complex biochemical reactions.
Thermodynamically, they resemble phase-transition systems, potentially gaining higher-order stability through multivalent intermolecular and intramolecular interactions, resulting in phaseseparated assemblies.

In the last ten years or so, phase separation has been implicated in several crucial physiological processes, such as embryonic development, stress response, signalling pathways, etc. Recently, phase separation exhibited by the Saccharomyces cerevisiae RMM complex was also implicated in meiotic DNA doublestrand break (DSB) formation.

The current study explores the phase separation phenomenon experienced by another S. cerevisiae meiotic DSB machinery subcomplex, the Mre11-Rad50-Xrs2 (MRX) complex.

Through a combination of biochemistry and genetics, it delves into the physical and biochemical nature of Mre11 condensates that are generated upon DNA damage in both vegetative and meiotic conditions. We further explored the interplay between Mre11 condensates and other components of the meiotic DSB machinery, such as the RMM complex and the Spo11 core complex. As an overarching goal, we attempt to solve the complex puzzle of how the process of meiotic DSB formation and repair is precisely orchestrated and how phase separation of the components of the DSB machinery contributes to it.

Ricinodendron heudelotii is a wild African tree widespread in sub-Saharan countries. It produces kernels, which are used as a sauce thickener in traditional dishes. Those kernels can also be pressed to extract an oil that is used in Africa as cosmetic or medical ointment. R. heudelotii oil is rich in an unconventional type of fatty acid: α-eleostearic acid, a member of the conjugated linolenic acid (CLnA) family.

CLnAs have demonstrated interesting properties regarding different diseases or conditions. In particular, they exert a strong antitumor activity, shown both in vitro and in vivo on mice and rats. However, CLnA-rich oils are highly prone to oxidation and therefore need to be extracted, processed, and stored with care.

The present work focusses on the precise characterization of R. heudelotii oil components, as well as on a better understanding of its oxidation dynamics via accelerated aging tests. Our results indicate that R. heudelotii oil contains up to 50% α- eleostearic acid, which explains its high sensitivity to oxidation. We also show that natural antioxidants such as green tea extracts help preserving the oil from adulteration.
Furthermore, we have demonstrated that the traditional dehulling processes of R. heudelotii seeds, which are widespread in Africa, have a significant impact on the rancidity of the oil and its safety as a food or cosmetic. Finally, experiments to characterize the effects of R. heudelotii oil on human skin are being carried out on an in vitro reconstructed human epidermis model.

Because of its peculiar composition, R. heudelotii oil has the potential to be better valued as a health-promoting food or topical agent, and could in turn become financially beneficial for the local producers in tropical Africa. Attention should be paid to its extremely high sensitivity to oxidation, which might be mitigated by careful processing of the seeds and through the addition of natural plant extracts to the oil.
 

The HOXA5 transcription factor plays critical roles in the central nervous system (CNS) formation during embryonic and fetal development. Our own data further support a functional requirement of HOXA5 in the mouse hindbrain after birth. Indeed, based on Hoxa5 expression in the postnatal brainstem and HOXA5 downstream targets we identified in this region, we hypothesized that this transcription factor plays a role in synaptogenesis processes during the first weeks after birth, a critical period for neural circuit plasticity.

To fully understand HOXA5 functions in the postnatal brain, we aim to study the HOXA5 transcriptional regulatory network at high resolution, including the identification of its DNA binding sites in neuronal cells. To achieve this goal, we performed genome-wide binding site mapping and motif analysis applying chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) using an in vitro neuronal model. The murine pluripotent P19 cells differentiated with retinoic acid were selected for their capacity to produce functional neurons in culture. As a prerequisite for this procedure, P19 cells were genetically modified using CRISPR-Cas9 to insert a FLAG tag into the coding sequence of Hoxa5, addressing the low affinity of existing anti-HOXA5 antibodies. This strategy was successfully applied and allowed to collect numerous data regarding HOXA5 molecular mechanisms, including its consensus binding site in immature and mature neurons and the presence of other binding sites in the vicinity, but also to identify functionally relevant direct target genes of HOXA5 in post-mitotic neurons. These data are currently being replicated and validated by alternative approaches and should help to establish models for the HOXA5’s mechanism of action.
 

An Adverse Outcome Pathway (AOP) is a structured framework that describes a consecutive sequence of perturbed biological events leading to an adverse health outcome, providing a mechanistic understanding of chemical toxicity. AOPs are pivotal in the next generation chemical risk assessment, supporting the transition towards human-based methodologies by replacing traditional animal testing. Owing to their mechanistic-based approach, AOPs have recently gained popularity in other scientific fields including biomedical research and nanotoxicology. We notably exploited the AOPs in the context of COVID-19 to organize the diverse and overwhelming data on the disease pathogenesis. This was the opportunity to describe intestinal outcomes in the framework, such as intestinal barrier disruption and alteration of gut microbiota. Both are unanimously recognized as central in many diseases, but currently overlooked in (nano)toxicology. This is true notably for nanoparticles that we are ingesting every day due to their widespread use by the food sector. Surprisingly, their effects towards our gut bacteria and barrier integrity remain unclear. Relying on AOPs, we are now investigating the impact of nanoparticles used as food additives, on the gut-liver axis and the potential role in fatty liver diseases in humans. By focusing on underlying mechanisms, AOPs facilitate the integration of evidence produced from in vitro, ex vivo, and in silico methodologies, enabling predictions of clinical outcomes. While many challenges remain, AOPs represent promising tools to assess potential gut-mediated hepatoxicity in humans.

 

In this talk we will discuss the role of roots in agrosystems and how they can be used to create more sustainable ones. In particular, we will see how their complexity and multiscale nature makes them hard to quantify, especially in the field. We will see how we can combine and leverage the latest computational tools to quantify relevant functions, despite this complexity. We will talk about roots, anatomies, architectures and also the role of soils.

 

Our research focuses on characterizing, comparing and understanding industrial yeasts, and using these insights to generate superior yeasts for fermentation processes, from beer and wine to precision fermentation for biofuels, lipids, proteins and chemical compounds.  

Over the past years, we collected and characterized thousands of yeast strains from various niches.  DNA analysis revealed the history and domestication of today’s yeasts, and also opened the doors to understanding and improving industrially-relevant phenotypes.   Using these resources, our team is producing several superior yeast variants with specific properties and aroma profiles for applications in the production of fermented beverages as well as various precision fermentation applications.  To do this, we use a combination of high-throughput breeding and screening, as well as genetic engineering.

In addition to generating superior industrial yeasts, we are also developing novel molecular tools to optimize genetic engineering.  A first technique, CoMuTER, uses a fusion between a base editor and Cas3 to induce random mutations in specific, large regions of genomes, for example to evolve complex biochemical pathways.  Secondly, we developed a new set of orthologous LoxP sits that allow independent shuffling of genomic elements or promoter modules.

Key references

• M. Schreurs, S. Piampongsant, M. Roncoroni , L. Cool, B. Herrera-Malaver, C. Vanderaa, F. Thesseling, L. Kreft, A. Botzki, T. Wenseleers and K.J. Verstrepen (2024). Predicting and improving complex beer flavor through machine learning.  Nature Communications. DOI: 10.1038/s41467-024-46346-0  (IF 16.6).

• A. Zimmermann, J.E. Prieto-Vivas, C. Bi, and K.J. Verstrepen (2024). Mutagenesis techniques for evolutionary engineering of microbes – exploiting CRISPR-Cas, oligonucleotides and recombinases.  Trends in Microbiology  DOI: 10.1016/j.tim.2024.02.006 (IF 15.9)
• C. Cautereels, J. Smets, J. De Saeger, L. Cool, Y. Zhu, A. Zimmermann, J. Steensels, A. Gorkovskiy, T. Jacobs and K.J. Verstrepen (2024). A set of 16 novel orthogonal LoxPsym sites allows multiplexed site-specific recombination in prokaryotic and eukaryotic hosts. Nature Communications. https://doi.org/10.1038/s41467-024-44996-8 (IF 16.6).
• C. Cautereels, J. Smets, J. De Saeger, L. Cool, Y. Zhu, A. Zimmermann, J. Steensels, A. Gorkovskiy, T. Jacobs and K.J. Verstrepen (2024). Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.  Nature Communications.  https://doi.org/10.1038/s41467-024-44997-7 (IF 16.6).
• A. Zimmermann, J.E. Prieto-Vivas, C. Cautereels, A. Gorkovskiy, J. Steensels, Y. Van de Peer and K. J. Verstrepen (2023).  CoMuTER, a Cas3-base editing tool for targetable in vivo mutagenesis.  Nature Communications. https://doi.org/10.1038/s41467-023-39087-z
• B. Gallone, J. Steensels, T. Prahl, L. Soriaga, V. Saels, B. Herrera, A. Merlevede, M. Roncoroni, K. Voordeckers, L. Miraglia, C. Teiling, B. Steffy, M. Taylor, A. Schwartz, T. Richardson, C. White, G. Baele, S. Maere, and K.J. Verstrepen (2016). Domestication and divergence of Saccharomyces cerevisiae beer yeasts. Cell. 166(6):1397-1410.e16. https://www.sciencedirect.com/science/article/pii/S0092867416310716 
• B. Gallone, J. Steensels, S. Mertens, M.C. Dzialo, J.L. Gordon, R. Wauters, F.A. Theßeling, F. Bellinazzo, V. Saels, B. Herrera-Malaver, T. Prahl, C. White, M. Hutzler, F. Meußdoerffer, P. Malcorps, B. Souffriau, L. Daenen, G. Baele, S. Maere and K.J. Verstrepen (2019). Interspecific hybridisation facilitates niche adaptation in beer yeast.  Nature Ecology and Evolution. https://doi.org/10.1038/s41559-019-0997-9

The last decade has seen the targeting of protein-protein interactions (PPIs) emerging as a new strategy of drug design. This strategy allowed to tackle challenging drug targets – sometimes called undruggable – whose active site cannot usually be drugged. From cancer therapy to anti-infectious agents, reports of successful therapeutics targeting PPIs are now numerous. Besides these therapeutic strategies, the last years witnessed the ever-growing importance of chemical biology, as well as a significant technical progress that has unlocked the study and targeting of more and more challenging protein interfaces. Among these new landscapes resides the targeting of self-assembling proteins. Indeed, so far, most molecules targeting PPIs have been designed to interact at heteromeric interfaces, ie surfaces between distinct protein chains.

The originality of our approach is to extend the methodology to homomeric interfaces, ie targeting PPIs between monomers of the same drug target in order to disrupt its quaternary structure. These numerous PPIs constitute an underexplored pool of potential targets for therapeutic interventions.¹

In this talk, the concept of homomeric disruption as a strategy to drug challenging/untractable targets will be illustrated with our recent works aiming at the targeting of lactate dehydrogenase (LDH) tetramerization site with (stapled)-peptides and small-molecules.^2-4

LDH plays a central role in cancer progression but is known to be very difficult and poorly druggable not only because of its highly hydrophilic and size-limited active site cavity but also because of its high cellular concentrations. So far, all the small-molecules developed against LDH eventually revealed to be poorly active and/or deprived of the required selectivity profile. Since LDH is active as a tetramer, we focused our research towards the development of molecules able to disrupt and/or prevent this tetramerization process. Our pivotal collaborative works led to (a) the delineation of hot spots at the LDH tetramerization site, (b) the design and synthesis of original (stapled)peptides capable of preventing LDH self-association and/or disrupting a preformed LDH tetramer, (c) the development of some chemical biology tools to interrogate LDH tetramerization using NMR spectroscopy (STD and WaterLogSy experiments), thermal shift, microscale thermophoresis, fluorescence spectroscopy and mass spectrometry experiments.

1.            Thabault, L.;  Liberelle, M.; Frederick, R., Targeting protein self-association in drug design. Drug Discov Today 2021, 26 (5), 1148-1163.

2.            Thabault, L.;  Brustenga, C.;  Savoyen, P.;  Van Gysel, M.;  Wouters, J.;  Sonveaux, P.;  Frederick, R.; Liberelle, M., Discovery of small molecules interacting at lactate dehydrogenases tetrameric interface using a biophysical screening cascade. Eur J Med Chem 2022, 230, 114102.

3.            Thabault, L.;  Liberelle, M.;  Koruza, K.;  Yildiz, E.;  Joudiou, N.;  Messens, J.;  Brisson, L.;  Wouters, J.;  Sonveaux, P.; Frederick, R., Discovery of a novel lactate dehydrogenase tetramerization domain using epitope mapping and peptides. J Biol Chem 2021, 100422.

4.            Thabault, L.;  Brisson, L.;  Brustenga, C.;  Martinez Gache, S. A.;  Prevost, J. R. C.;  Kozlova, A.;  Spillier, Q.;  Liberelle, M.;  Benyahia, Z.;  Messens, J.;  Copetti, T.;  Sonveaux, P.; Frederick, R., Interrogating the Lactate Dehydrogenase Tetramerization Site Using (Stapled) Peptides. J Med Chem 2020, 63 (9), 4628-4643.