Poster Abstracts – Session 2

Amalie Skogvold, Heidi T. Hillier & Ingar Leiros

UiT – The Arctic University of Norway, Tromsø

ω-Transaminases are biocatalysts highly sought after in the pharmaceutical industry as a green alternative for the production of valuable chiral amines. The DABA ω-transaminases EctB and DoeD catalyze the forward and reverse transaminase reactions in the bacterial ectoine biosynthesis and degradation pathways, respectively. Ectoine is a highly valuable compound used in both the cosmetics and pharmaceutical industries due to its many novel properties, such as acting as a protein stabilizer, DNA protector, and membrane, cell, and skin protectant. It is primarily produced by a method called bacterial milking, but more recent research has focused on heterologous production using non-halophilic bacteria such as Escherichia coli. Despite the high market demand for ectoine, research on characterizing the enzymes in the ectoine biosynthesis and degradation pathways has been limited. Recently, we solved the first crystal structure of DoeD and completed a biochemical and biophysical characterization of the transaminase, including exploring the substrate scope for other potential uses of the enzyme as a pharmaceutical biocatalyst. Previous work by the Ectoine Research Group includes solving the crystal structure of EctB from the model organism for ectoine production, Chromohalobacter salexigens DSM 3043 (Hillier et al., 2020). The group aims to characterize all the core enzymes involved in ectoine synthesis, including EctB, EctA, and EctC, as well as DoeD from the ectoine degradation pathway. Future work will also prioritize the rational design to improve the operational stability and efficiency of the transaminases, especially EctB and DoeD.

References:

1. Hillier HT et al. (2020) FEBS J 287, 4641-4658.

Daniel Haga Hasselstrøm , Osman Gani1 & Trond Vidar Hansen 

Department of Pharmacy, Section for Pharmaceutical Chemistry, University of Oslo, PO Box 1068 Blindern, N – 0316 Oslo, Norway

The G protein-coupled receptor (GPCR) GPR32 plays a crucial role in mediating the anti-inflammatory and tissue-repairing actions of several specialized pro-resolving mediators (SPMs) such as resolvin D1 and lipoxin A4, which act as potent agonists at this receptor.^1,2

No experimental structures of GPR32 have been solved, necessitating the use of computational means to investigate the stereoselective binding of these potent fatty acid metabolites at GPR32 and synthetic agonists such as C2A.3

This poster presents our current work on elucidating the binding modes of resolvins and synthetic agonists at GPR32 by employing state-of-the-art computational methods such as molecular dynamics simulations, free energy calculations and residue interaction analysis.

References:

1. Krishnamoorthy, Sriram, et al. “Resolvin D1 binds human phagocytes with evidence for proresolving receptors.” Proceedings of the National Academy of Sciences 107.4 (2010): 1660-1665.
2. Krishnamoorthy, Sriram, et al. “Resolvin D1 receptor stereoselectivity and regulation of inflammation and proresolving microRNAs.” The American journal of pathology 180.5 (2012): 2018-2027.
3. Chiang, Nan, et al. “Identification of chemotype agonists for human resolvin D1 receptor DRV1 with pro-resolving functions.” Cell chemical biology 26.2 (2019): 244-254.

Eva Smorodina1, Oliver Crook2, Rahmad Akbar1, Puneet Rawat1, Dario Segura Pena3, Nikolina Sekulic3, Ole Magnus Fløgstad3, Khang Lê Quý1, Brij Bhushan Mehta1, Johannes Loeffler4, Monica Fernandez-Quintero4, Hannah Turner4, Andrew B. Ward4, & Victor Greiff1

(1) Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway, (2) Department of Statistics, University of Oxford, Oxford, (3) Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Faculty of Medicine, University of Oslo, Oslo, Norway, (4) Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, USA

Antibodies are key therapeutics but the principles behind diverse paratopes binding to the same epitope remain unexplained. An insufficient understanding of the structural rules behind antibody-antigen binding, due to a lack of experimentally resolved structures, leads to the current inability to characterize antibody variants binding in silico. Here we propose a rule-based antibody design that relies on a thorough understanding of epitope-paratope interactions, in contrast to generative design based on millions of trials and errors. We identified the epitope of five affinity-verified Trastuzumab variants using cryo-EM and position-resolved HDX-MS. Rigid models alone are insufficient for accurate antibody-antigen modeling while molecular dynamics simulations with computational analysis of the complex conformations succeed in replicating and complimenting experimental findings. Structural parameters calculated based on geometry, surface, and biochemical properties were able to distinguish between high and low binders. We highlight the possibilities of AI in antibody and antibody-antigen structure modeling, demonstrating the limitations of various language-based models to predict and understand antibody variants. Overall, our study explains the binding mechanisms of the variant sequences, showing how antibodies with diverse sequences share similar antigen-binding rules.

Gloria Gamiz-Arco, Mary Dayne Sia Tai, Kunwar Jung-KC & Aurora Martinez

Department of Biomedicine, University of Bergen, 5020 Bergen, Norway

Tyrosine Hydroxylase Deficiency (THD) is a rare genetic disorder characterized by severely low dopamine levels due to variants in tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine. Patients with THD present a phenotype ranging from dystonia to severe encephalopathy. Presently, the standard medication to address dopamine deficiency in these patients is the dopamine precursor levodopa (synthetic L-Dopa). While it is relatively effective in managing motor dysfunction, especially in less severe forms of THD, it often comes with side effects and tends to lose effectiveness over time. Effective disease-modifying therapies for THD are currently unavailable, making the development of alternative treatments a priority.

Recently, the interaction of TH with the cochaperone DNAJC12 has been reported. TH and DNAJC12 form a high-affinity complex which increases the stability of TH and delays its aggregation. We hypothesize that the loss of integrity and catalytic efficiency in THD patients might be compensated by its interaction with DNAJC12, and we propose the stabilization of the TH:DNAJC12 complex as an innovative target to develop potential treatments for THD. To achieve that, our research is focused on searching for pharmacological chaperones using high-throughput screening (HTS). The HTS performed with the Prestwick Chemical library® (1520 compounds, most FDA- and EMA approved) has revealed a promising compound (Hit 1) that binds both to TH and the TH:DNAJC12 complex, stabilizing it, and delaying TH aggregation, with minor effect on its catalytic activity.

The discovery of Hit 1 largely proofs the pharmacological chaperone concept and the value of stabilizing the TH:DNAJC12 complex as a new therapeutic strategy to develop potential treatments for THD and other dopamine deficiencies.

Greta Daae SandsdalenI1, Maryam Imam2, Ole Morten Seternes3, Adele Williamson4 & Hanna-Kirsti Schrøder Leiros1

(1) Biomolecular and Structural Chemistry, Department of Chemistry, UiT The Arctic University of Norway, (2) The Norwegian College of Fishery Science, UiT The Arctic University of Norway, (3) Department of Phamacy, UiT The Arctic University of Norway, (4) The University of Waikato, New Zealand

The CRISPR-Cas genome editing system has revolutionized molecular biology, providing an array of biotechnological tools for carrying out precision genome modification and regulation. One limitation of the system at present is that most available tools are developed from and optimized for mesophilic organisms, which limits their utility in cold living organisms. This paucity of available knowledge on psychrophilic CRISPR-Cas systems and affiliated genome editing tools is particularly problematic for researchers of cold-blooded eukaryotes, where mismatched thermal preferences of the CRISPR components hinder application efficiency.

This project is one of three interdisciplinary components of the UiT Strategic-Funded ‘FISH&CRISPR Innovative strategies to improve salmon health’ which aims to establish a platform for the development of a low-temperature CRISPR-Cas genome editing system optimized for salmonids.

In this project, the main goal is to discover and develop one or more CRISPR-associated endonucleases for efficient and precise genome editing at low temperatures. A second aim is to gain insights on CRISPR-systems across cold-adapted bacteria through bioinformatics analysis.

Our findings reveal a low prevalence of CRISPR-Cas systems in cold-adapted bacteria, compared to mesophilic and thermophilic species, where only 17.7% of the analyzed genomes contained CRISPR operons. Further, five CRISPR endonucleases were selected for experimental characterization. Currently, one of them shows promise for genome editing applications in low-temperature conditions.

The identification and initial characterization of Cas endonucleases from cold-adapted bacteria mark a pivotal step towards establishing a CRISPR-Cas platform optimized for salmonids. This advancement could significantly impact genomic studies and biotechnological applications for cold-adapted organisms, aligning with the goals of enhancing salmon health and aquaculture sustainability.

Helena Hradiská & Bjørn Olav Brandsdal

Hylleraas Centre for Quantum Molecular Sciences, Department of Chemistry, UiT The Arctic University of Norway, Tromsø, Norway

Laccase is a multicopper oxidase, which has found its use in many fields of industry and is also a promising biocatalyst in organic synthesis and bioremediation. To explore its possible application even further, one can search for homologs or engineer laccase to be efficient under extreme conditions such as low temperature. Even though the majority of produced laccase comes from fungi, this work focuses on bacterial laccases because they have been less studied in the past and cold-adapted bacterial homologs were recently found.

To model chemical reactions, one needs to run quantum mechanical (QM) calculations, however those are computationally demanding, especially for enzymes. Empirical valence bond (EVB) method allows us to run QM only for the reaction in solution and then use the calibration for the reaction in the enzyme and its homologs to determine reaction and activation Gibbs free energies. With EVB we can also obtain Arrhenius plots that inform about entropic and enthalpic contributions, thus about the temperature dependence of enzyme activity.

References:

1. Åqvist, J., Isaksen, G. V., & Brandsdal, B. O. (2017). Computation of enzyme cold adaptation. Nature Reviews Chemistry, 1(7), 14, Article 0051. https://doi.org/10.1038/s41570-017-0051
2. den Boer, D., de Heer, H. C., Buda, F., & Hetterscheid, D. G. H. (2023). Challenges in Elucidating the Free Energy Scheme of the Laccase Catalyzed Reduction of Oxygen. Chemcatchem, 15(1), 27. https://doi.org/10.1002/cctc.202200878
3. Silva, C. S., Durao, P., Fillat, A., Lindley, P. F., Martins, L. O., & Bento, I. (2012). Crystal structure of the multicopper oxidase from the pathogenic bacterium Campylobacter jejuni CGUG11284: characterization of a metallo-oxidase. Metallomics, 4(1), 37-47. https://doi.org/10.1039/c1mt00156f
4. Warshel, A., & Weiss, R. M. (1980). An empirical valence bond approach for comparing reactions in solutions and in enzymes. Journal of the American Chemical Society, 102(20), 6218-6226. https://doi.org/10.1021/ja00540a008

Ida Marie Stephansen1 & Fernando Pérez-García1*

(1) Department of Biotechnology and Food Science, Norwegian University of Science and Technology, 7491 Trondheim, Norway

This project aims to meet the increasing demand for sustainable resources driven by a growing population. New renewable and alternative feedstocks are continuously sought in microbial biotechnology, and seaweed presents a promising solution. In particular, we aim to engineer the bacterium Corynebacterium glutamicum, which is commonly used as an industrial workhorse for the large-scale production of amino acids[1]. By applying molecular and synthetic biology tools, we will enable the utilization of seaweed-derived sugars like mannitol, xylose, or galactose by C. glutamicum strains[2]. Our approach will focus on co-cultivation, where each C. glutamicum strain will be engineered to utilize a specific sugar. We hypothesize that this method may improve the carbon conversion yield compared to classic monoculture approaches[3]. To support the concept of circular bioeconomy, utilization of seaweed sugars will be coupled with the production of added-value compounds like amino acids.  Finally, to prove the potential of this idea, the newly established co-cultures will be scaled up in bioreactors using red and brown seaweed hydrolysates as the carbon source. Hence, this research seeks to contribute to sustainable production by tapping into the vast potential of ocean resources.

References:

1. Wolf, Sabrina, et al. “Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being.” Essays in biochemistry 65.2 (2021): 197-212.
2. Zhong, Haowei, et al. “The structural characteristics of seaweed polysaccharides and their application in gel drug delivery systems.” Marine drugs 18.12 (2020): 658.
3. Pérez-García, Fernando, et al. “Dynamic co-cultivation process of Corynebacterium glutamicum strains for the fermentative production of riboflavin.” Fermentation 7.1 (2021): 11.

Jan Benedict Spannenkrebs, Agnes Beenfeldt Petersen, & Prof. Johannes Kabisch

NTNU, Institute for Biotechnology and Food Science

Bacillus subtilis can form metabolically dormant endospores in response to deteriorating environmental conditions such as poor nutrient availability. In a process called sporulation, a Bacillus subtilis cell divides asymmetrically. In one half of the cell a copy of the DNA is enclosed in multiple membrane and proteinaceous layers. Once sporulation concludes, the mother cell lyses, releasing the metabolically dormant spore into the environment. The formed spore provides protection for the DNA against extreme / harmful conditions, including high temperatures, desiccation, radiation and irritating chemicals. The spore core, which contains the DNA, is encased in an inner and outer coat. The outermost layer, designated the “crust,” is primarily composed of six proteins (CotV, W, X, Y, Z, and CgeA)^[1].

These crust proteins as well as others from different spore layers have already been used to create fusion proteins with proteins of interest, for example enzymes or antigens. For this, a copy of the fusion proteins DNA is inserted into B. subtilis under the control of a sporulation specific promoter. These fusion proteins self-assemble into the respective spore layer during sporulation. This system has for example been used for the display of a photodecarboxylase for the transformation of lipids to hydrocarbons^[2], as well as for the display of the receptor binding domain of SARS-CoV-2^[3]. The immobilization of proteins in this manner greatly facilitates downstream purification. Due to the spores’ size, purification is possible by comparatively low-tech means like repeated centrifugation and washing.

A novel candidate class of enzymes for spore display are Alginate epimerases such as the processive AlgE-type. Alginates are linear polysaccharides comprised of linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G). The G-blocks can chelate divalent cations like Ca²⁺, resulting in a hydrogel. The properties of the hydrogel can be modified by altering the ratio and sequence of the M and G blocks, making them an interesting product for example the food and pharmaceutical industry^[4]. Alginate epimerases are enzymes capable of epimerizing M into G blocks, thus modifying the alginates properties.

Join this talk to get an insight into recent developments in spore displayed enzymes in our lab, including the display of epimerases and how we can measure their activity in real-time.

References:

1. Bartels, J., Blüher, A., López Castellanos, S., Richter, M., Günther, M. and Mascher, T. (2019), The Bacillus subtilis endospore crust: protein interaction network, architecture and glycosylation state of a potential glycoprotein layer. Mol Microbiol, 112: 1576-1592
2. Karava, M.; Gockel, P.; Kabisch, J. Bacillus Subtilis Spore Surface Display of Photodecarboxylase for the Transformation of Lipids to Hydrocarbons. Sustainable Energy Fuels 2021, 5 (6), 1727–1733.
3. A. Vetráková, R. Kalianková Chovanová, R. Rechtoríková, D. Krajčíková, I. Barák, Bacillus subtilis spores displaying RBD domain of SARS-CoV-2 spike protein, Computational and Structural Biotechnology Journal, Volume 21, 2023, Pages 1550-making 1556
4. Petersen AB, Tøndervik A, Gaardløs M, Ertesvåg H, Sletta H, Aachmann FL. Mannuronate C-5 Epimerases and Their Use in Alginate Modification. Essays in Biochemistry. 2023;67(3):615-27.

Joseph Lumba, Maria Psarrou, Kristine Marie Halvorsen, & Solon Economopoulos

Department of Chemistry, NTNU, Gløshaugen, 7030 Trondheim, Norway

Squaraine dyes have shown particular promise as a class of dyes exhibiting ultrastrong absorption properties that routinely cover a large part of the visible spectrum and even extend to the near-IR region.^1,2,3 On the other hand, the behavior of squaraine-based dyes in solution or thin films is subject to severe aggregation phenomena.⁴ Therefore, detailed knowledge of the squaraine dye interactions in homogenic aggregates and with other species can be of importance for the design of new materials of desired properties.

In this study, a triad comprised of quinoline, squaraine and pyrene chromophores is synthesized and characterized. The resulting chromophore follows a Donor-Acceptor-Donor D-A-D architecture and exhibits very desirable optical characteristics for optoelectronic applications such as solar cells. The chromophores have been studied using steady state absorption and emission spectroscopy. To study the interaction of the light harvester with a well-known electron accepting material, exfoliated graphene nanoparticles are introduced in dilute solutions of the triad and the subsequent formation of H-type and J-aggregates is examined. Picosecond time-resolved fluorescence was used to try and probe the behavior of the observed interactions, while electrochemistry was used to rationalize the thermodynamically favorable pathways for energy or charge transfer in such complex nanoensembles.

Kunwar Jung-KC1,2, Svein I Støve1,3, & Aurora Martinez1,2

(1) Department of Biomedicine, University of Bergen, Norway, (2) KG Jebsen Centre for Parkinson’s Disease (DECODE-PD), University of Bergen, Norway, (3) Neuro-SysMed, Department of Neurology, Haukeland University Hospital, Norway

Parkinson’s disease (PD) is a prevalent neurodegenerative movement disorder such as bradykinesia with resting tremor or rigidity, affecting approximately 2% of individuals aged 60 years and older. Mitochondrial dysfunction is strongly implicated in the aetiology of idiopathic and genetic PD. Since mitochondria are highly multifunctional organelles, their integrity is essential for neuronal function and survival. This study employs a cell-based screening approach to identify small molecule compounds capable of alleviating mitochondrial dysfunction in PD, specifically targeting complex I of the electron transport chain. Utilizing SHSY5Y cells as a model system, a systematic cell-based screening of Prestwick chemical library including 1520 FDA-approved drugs was conducted to identify compounds modulating complex I levels. The investigation identified a promising hit compound (Hit1), whose impact on mitochondrial level and function was assessed. Further assessment by SDS-PAGE indicated significantly increased levels of complex I in SHSY5Y cells upon treatment with Hit1. In addition, a significant enhancement in total oxidative phosphorylation (Oxphos) complexes in SHSY5Y cells was observed following treatment with Hit1. Also, complex I activity substantially increased, suggesting a potential therapeutic avenue for mitigating mitochondrial dysfunction in PD. The results indicate the potential of Hit1 as a drug-repurposing candidate for PD treatment, specifically addressing the mitochondrial dysfunction due to reduced complex I activity. This study also underscores the importance of leveraging cell-based screening techniques to identify novel compounds targeting specific disease pathophysiology, offering insights into potential avenues for therapeutic interventions. Overall, these findings contribute insights into the development of targeted interventions for PD, opening avenues for further preclinical and clinical investigations.

Magnus Philipp, Carina Dietz, & Johannes Kabisch

Department of Biotechnology and Food Science, NTNU

Bacterial spores are among the most resilient biological systems in nature. In addition, they are biologically inactive, easy to handle and can be purified by simple centrifugation. These characteristics make spores an excellent target for biotechnology research and application. On an industrial scale, spores of the Bacillus genus are produced at several tons per year as seed additives to promote plant growth and act as pathogen antagonists. In research, the spore-surface-display technology has been developed that allows for the simultaneous production and immobilization of proteins which to date has mostly been applied for catalysis applications and the display of antigens and antibody formats. The display of the protein on the outside of the spore is achieved by genetic modification that introduces a synthetic gene encoding for a fusion of a spore surface protein and a protein of interest, controlled by a sporulation dependent promoter, resulting in protein display after sporulation.

In our current project we aim to expand the applications of Bacillus subtilis spores by developing them into a novel type of programmable biomaterial. To this end we display load-bearing proteins like e.g. spider silk, squid ring teeth protein and the Bacillus subtilis amyloid TasA on the surface of these spores. Load-bearing proteins are notoriously difficult to produce in heterologous systems due to their repetitive amino acid sequences and hydrophobic nature. However, displaying them on top of the spore negates problems such as inclusion body formation and allows the spores to serve as a seeding point for forming cheap biomaterials with novel properties. Once a baseline formula for a biomaterial has been developed, we aim to use existing knowledge such as the ability to use spores in 3D printing, hydrogels or concrete to further explore the properties of spore-based materials. In addition, we aim to further functionalize these materials by addition of upconverting nanoparticles and inorganic dyes to enable oxygen sensing and the use of light dependent enzymes.

Mary Dayne Sia Tai1, Marte Innselset Flydal2, Gloria Gamiz-Arco1, Trond-André Kråkenes1, Christer Flagtvedt Didriksen1, Juha Pekka Kallio1 & Aurora Martinez1

(1) Department of Biomedicine, University of Bergen, Bergen; (2) Department of Medical Genetics, Haukeland University Hospital

Hyperphenylalaninemia (HPA) primarily results from pathogenic variants in the PAH gene, which encodes phenylalanine hydroxylase (PAH), the enzyme responsible for converting L-Phe to L-Tyr. Variants of DNAJC12 also cause hyperphenylalaninemia along with dystonia, intellectual disability and neurotransmitter deficiencies in patients without any variants in PAH, in other tetrahydrobiopterin (BH4) dependent hydroxylases or in enzymes involved in BH4 synthesis or regeneration. As an Hsp40 protein, DNAJC12 binds to its client proteins, such as PAH, and presents them to Hsp70 for proper protein folding and homeostasis. However, the mechanism by which DNAJC12 binds to PAH is currently unknown. Human DNAJC12 and PAH, wild-type (WT) and HPA-associated variants, were recombinantly expressed in E. coli and purified before in vitro complex reconstitution. Biophysical and biochemical methods such as analytical size exclusion chromatography (SEC), native PAGE,  immunoblotting and dynamic light scattering (DLS) were used to confirm complex formation and investigate the effect of complex formation on the stability of PAH. DNAJC12 and PAH form a complex that can be purified for further characterization. Results from SDS-PAGE, native PAGE and immunoblotting confirm the co-migration of DNAJC12 and PAH in non-denaturing conditions. By monitoring the time-dependent self-aggregation of PAH and HPA associated variants over time using DLS, DNAJC12 was also found to significantly delay PAH aggregation in vitro. Removal of an evolutionarily conserved octapeptide sequence in DNAJC12 was found to abolish its ability to bind to PAH, indicating the significance of this motif for DNAJC12 client binding. DNAJC12 recognizes and binds PAH through an evolutionarily-conserved octapeptide sequence. The binding of DNAJC12 stabilizes PAH, preventing its self-aggregation over time.

Md Abu Hanif 1, Ingrid Quist-Løkken2, Clara Andersson-Rusch2,3, & Toril Holien1,2,3,4

(1) Department of Biomedical Laboratory Science, NTNU – Norwegian University of Science and Technology, Trondheim, Norway, (2) Department of Clinical and Molecular Medicine, NTNU – Norwegian University of Science and Technology, Trondheim, Norway, (3) Department of Hematology, St. Olav’s University Hospital, Trondheim, Norway, (4) Department of Immunology and Transfusion Medicine, St. Olav’s University Hospital, Trondheim, Norway

Background: Multiple myeloma is a heterogenous cancer which requires new drugs to overcome relapse and resistance. Selinexor, an inhibitor of the XPO1 cargo protein, has been approved myeloma and cancers. Inhibiting XPO1 with Selinexor could block major tumor suppressor proteins and trap mRNAs inferring oncoproteins, leading to apoptosis/cell death. BMPs activate canonical SMADs, causing apoptosis and cell growth arrest in myeloma cells. SMADs are believed to depend on XPO1 for nuclear-cytoplasmic transport.

Aim: We hypothesize that combining Selinexor and BMPs could potentiate apoptosis in multiple myeloma by increasing the retention of SMAD proteins in the nucleus. Moreover, Selinexor has severe side effects which urges for identification of new biomarkers. This can be easily done by investigating association of Selinexor with BMP-SMAD activity.

Methods: Dose response curves exhibiting cell viability were generated where myeloma cell lines were treated with Selinexor and Eltanexor followed by combination with BMP6, and Activin B. Flow cytometry was used with annexin V-FITC to identify apoptosis. Primary CD138+ myeloma cells were treated with Selinexor and BMP6 alone or combination of them. Additionally, BRE-LUC reporter assay and immunoblot was used to identify SMAD activity and protein expression respectively. We also plan to explore localization and abundance of SMADs using various microscopic techniques

Results: Selinexor mediated inhibition of XPO1 reduced cell viability in myeloma cell lines where Eltanexor has a more potent effect. Combining Selinexor and BMP6 leads to synergistic cell death in INA6 cell lines in different assays. Selinexor also potentiates BMP6 in primary CD138+ myeloma cells. We plan to use different microcopy techniques to determine localization and abundance of SMAD proteins.

Conclusion: Our results indicate that Selinexor treatment cooperates with BMP-SMAD activity to induce apoptosis in multiple myeloma. It would decipher the mechanistic relevance of the synergistic cell death approach in multiple myeloma. This detailed mechanistic knowledge could help to identify biomarkers for Selinexor efficacy to predict patient response to it.

Morten Rese1, Gijs van Erven2,3, Romy Veersma3, Gry Alfredsen4, Vincent G. H. Eijsink1, Mirjam A. Kabel3, & Tina R. Tuveng1

 (1) Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1433 Ås, Norway, (2) Wageningen Food and Biobased Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands, (3) Wageningen University & Research, Laboratory of Food Chemistry, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands, (4) Norwegian Institute of Bioeconomy Research, Department of Wood Technology, P.O. Box 115, NO-1431, Ås, Norway

Wood-degrading brown-rot fungi primarily target carbohydrates, while lignin becomes partially modified and of potential interest for targeted lignin valorization[1]. Here, we report a comprehensive comparison of  lignin conversion by a brown-rot basidiomycete, Gloeophyllum trabeum, growing on a hardwood and a softwood substrate. By harnessing the latest advancements in analytical methodologies, we show that G. trabeum not only degrades polysaccharides efficiently and selectively but may also remove more lignin from wood than previously reported. Structure-wise, brown-rotted lignin appeared substantially C_α-oxidized, O-demethylated, depleted in interunit linkages, and enriched in diagnostic substructures indicative of C_α-C_β, β-O and O-4 bond cleavages in the β-O-4 aryl ether linkage. These findings enhance our understanding of lignin conversion by brown-rot fungi, revealing previously unknown aspects of this process. Specifically, despite the well-documented differences in lignin structure between hardwood and softwood[2], G. trabeum attacks the same bonds in the lignin structures, resulting in similar chemical modifications regardless of the wood substrate. Furthermore, we show that G. trabeum enhances the antioxidant capacity of the lignin, and that the residual lignin can be separated into low- and high-molecular weight fractions with distinct properties. This highlights the biotechnological potential of brown-rot fungi for developing lignin-based antioxidant or resin products.

References:

1. D. J. Yelle, J. Ralph, F. Lu, and K. E. Hammel, “Evidence for cleavage of lignin by a brown rot basidiomycete,” Environmental Microbiology, vol. 10, no. 7, pp. 1844-1849, Jul. 2008.
2. W. Boerjan, J. Ralph, and M. Baucher, “Lignin biosynthesis,” Annual Review of Plant Biology, vol. 54, pp. 519-546, Jun. 2003.

Natalia Mojica1, Flore Kersten1,2, Albert Serrano3, Joel B. Heim1, Gabriele Cordara1, Ken Teter3, & Ute Krengel1

(1) Department of Chemistry, University of Oslo, (2) Centre for Molecular Medicine Norway (NCMM), University of Oslo, (3) Burnett School of Biomedical Sciences, University of Central Florida

Cholera toxin (CT) and heat-labile enterotoxin (LT) are two similar AB5 toxins responsible for the diarrhea characteristic of Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) infections. They consist of a catalytically active A1 subunit, an A2 linker, and a pentamer of cell-binding B-subunits ¹. Both toxins bind to the same GM1 surface receptor on the host cells and have similar levels of enzymatic activity, yet CT is more potent than LT, making cholera the more severe disease. The difference in toxicity has been attributed to structural differences near the C-terminus of the A2 linker (amino acid residues 226-236) ², but the underlying molecular mechanism remains unknown. Recently, we showed that toxin disassembly by protein disulfide isomerase (PDI), which is a key event in the intoxication process, is more efficient for CT than for LT³. We hypothesized that the difference in toxin disassembly is related to the positioning of the A1 subunit relative to the B-pentamer³ (Figure 1).

Here, we determined the crystal structures of two cholera toxin variants where either one (D229E) or four (D229E, I230V, T232I, H233Y) amino acid residues in the critical A2 linker sequence were substituted for the residues present in LTA2 (Figure 1; colored residues within the pores of the grey pentamers). The results of this structural analysis will be presented here.

References:

1. Heggelund, J. E., Bjørnestad, V. A., and Krengel, U. (2015) Vibrio cholerae and Escherichia coli heat-labile enterotoxins and beyond, in The Comprehensive Sourcebook of Bacterial Protein Toxins, pp 195–229. Elsevier Ltd.
2. Rodighiero, C., Aman, A. T., Kenny, M. J., Moss, J., Lencer, W. I., and Hirst, T. R. (1999) Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin. Journal of Biological Chemistry 274, 3962–3969.
3. Serrano, A., Guyette, J. L., Heim, J. B., Taylor, M., Cherubin, P., Krengel, U., Teter, K., and Tatulian, S. A. (2022) Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin. Sci Rep 12, 34.

Simen Jervell Lund1, Fernando Pérez-García1, & Trygve Brautaset1*

(1) Department of Biotechnology and Food Science, Norwegian University of Science and Technology, 7491 Trondheim, Norway

This project addresses the rising demand for sustainable resources due to population growth. In microbial biotechnology, there’s a continuous search for renewable and alternative feedstocks in order to avoid food-competitive materials and fossil-based substrates (Perez-Garcia et al. 2022^[1]). Our goal is to engineer the bacterium Corynebacterium glutamicum, a widely used industrial workhorse for large-scale amino acid production (Wolf et al. 2021^[2]). Through the application of molecular and synthetic biology tools, we will enable the use of carbohydrate containing substrates such as spent grain from breweries, side streams from potato industry, and agricultural harvest residues.  We will develop C. glutamicum strains for the degradation of the polymer starch into glucose, and for the utilization of the sugar pentoses xylose and arabinose as well as the sugar hexoses mannose and galactose (Wendisch et al. 2016^[3]).  To advance the concept of a circular bioeconomy, our approach will couple the utilization of carbohydrates with the production of high-value compounds such the amino acids and amino acids derivatives. To demonstrate the viability of this strategy, the newly developed microbial strains will be tested in lab-scale bioreactors using real hydrolysates from the aforementioned substrates as the carbon source. This research aims to contribute to sustainable biotechnology by integrating new and renewable biomass resources into biotechnological processes.

References:

1. Pérez-García, F., Brito, L. F., Irla, M., Jorge, J. M., & Sgobba, E. (2022). Promising and sustainable microbial feedstocks for biotechnological processes. Frontiers in Microbiology, 13, 973723.
2. Wolf, S., Becker, J., Tsuge, Y., Kawaguchi, H., Kondo, A., Marienhagen, J., … & Wittmann, C. (2021). Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being. Essays in biochemistry, 65(2), 197-212.
3. Wendisch, V. F., Brito, L. F., Lopez, M. G., Hennig, G., Pfeifenschneider, J., Sgobba, E., & Veldmann, K. H. (2016). The flexible feedstock concept in industrial biotechnology: metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources. Journal of Biotechnology, 234, 139-157.

Susanne Hansen Troøyen, Davide Luciano, & Gaston Courtade

Department of Biotechnology and Food Science, Norwegian University of Science and Technology (NTNU). Trondheim, Norway

Several carbohydrate-active enzymes contain carbohydrate-binding modules (CBMs) that regulate enzymatic activity by localizing the catalytic domain towards the surface of insoluble substrates such as cellulose [1]. Recently, some CBMs have also been shown to have affinity for non-natural substrates such as polyethylene terephthalate (PET) [2, 3]. CBMs are thought to act as anchors on the substrate surface, allowing the enzyme to perform its activity inside a radius limited by the length of the flexible linker connecting the two domains. However, as illustrated by the Sabatier principle, the CBM should not stay attached at the same position too long – otherwise it would limit the catalytic efficiency of the enzyme. The dynamics of exchange between the free and bound state of the binding module is thus an important, but largely unexplored property of these proteins. NMR spectroscopy offers an opportunity to study protein exchange processes through carefully chosen experiments such as dark state exchange saturation transfer (DEST), solvent paramagnetic relaxation enhancement (sPRE) and relaxation rate measurements. We present here our ongoing investigation into CBM binding dynamics and identification of their substrate-binding site. In combination with affinity assays and kinetic experiments, we anticipate that these insights will contribute to developing our understanding of CBM binding mechanisms.

References:

1. Courtade, G., Forsberg, Z., Heggset, E. B., et al. (2018) The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation. J Biol Chem 293(34), 13006 –13015.
2. Weber, J., Petrović, D., Strodel, B. et al. (2019) Interaction of carbohydrate-binding modules with poly(ethylene terephthalate). Appl Microbiol Biotechnol 103, 4801–4812.
3. Rennison, A. P., Westh, P., and Møller, M. S. (2023) Protein-plastic interactions: The driving forces behind the high affinity of a carbohydrate-binding module for polyethylene terephthalate. Sci Total Environ 870, 161948.

Xuan Thang Nguyen1,2, Emily Martiensen1,2, Marie Rogne1,2, Bernd Thiede3 & Ragnhild Eskeland1,2

(1) Institute of Basic Medical Sciences, Medical Faculty, University of Oslo, Norway, (2) Centre for Cancer Cell Reprogramming (CanCell), Centre of Excellence, (3) Department of Biosciences, University of Oslo, Norway.

Transcription factors, as vital regulatory proteins, bind to DNA, orchestrating the activation or deactivation of genes and governing the transcription of messenger RNA (mRNA) crucial for protein synthesis in all cells and organisms. Mutated or dysregulated transcription factors can significantly impact cell growth, dominated by the protein network, and can lead to cancer progression. Our study, which investigates the network of transcription factor regulation in osteosarcoma, has the potential to emphasize the need for further investigation significantly. We used a combination of proteomics and transcriptomics analysis to map transcription factor networks in three different aggressive of human osteosarcoma cell lines. Our findings on master regulators and pioneer transcription factors associated with higher aggressiveness in osteosarcoma, including the RUNX, HGM, and FOX family’s transcription factors, could inspire new directions for drug screening and potential treatment strategies.

Tonje Reinholdt Haugen1, Laura Erntsen1, Bjørn Olav Brandsdal1,2

(1) Hylleraas Centre for Quantum Molecular Sciences, Department of Chemistry, UiT The Arctic University of Norway, Tromsø, Norway

Antimicrobial peptides (AMP) are a ubiquitous part of the host defense systems of many organisms, ranging from fungi, and bacteria to mammals. The have proven to have a wide range of use, showing not only antimicrobial properties, but also showing antiviral and antifungal properties. AMPs can be classified as oligopeptides with less than 100 amino acids  and a cationic charge and they were first reported in 1939, the same year Florey and Chain started their work purifying penicillin. In this study, 120 different cyclic hexapeptides were screened and the top 4 were further analyzed using both computational and experimental methods. This poster will focus on the computational part of the analysis.

The computational analysis has done by using molecular dynamics at atomistic level with the 4 different peptides in water and in combination with two models of the inner membrane of E. coli. To prevent the simulations to be too computationally demanding, a small patch of about 100-150 lipids per lipid layer was used in the simulations. The two bilayer models used in the computational analysis were a POPE and POPG lipid bilayer with 3:1 ration and a DMPC and DMPG lipid bilayer with a 1:20 ratio. The second model was constructed as a more direct comparison with experimental results. Comparing the models with and without the lipid bilayer, also lets us investigate what conformational differences there may be in the peptides depending on environment.

References:

1. Bahar, A. A.; Ren, D. Antimicrobial Peptides. Pharmaceuticals 2013, 6 (12), 1543–1575. https://doi.org/10.3390/ph6121543.
2. Hong, C.; Tieleman, D. P.; Wang, Y. Microsecond Molecular Dynamics Simulations of Lipid Mixing. Langmuir 2014, 30 (40), 11993–12001. https://doi.org/10.1021/la502363b.
3. Sarre, B. R. MD Simulations Reveal How Synthetic Antimicrobial Peptides Interact with Membrane Models. 2014, No. December.

Yomkippur Perez1, Erik Agner2, & Magne Olav Sydnes3

(1) University of Stavanger, (2) Polypure, (3) University of Bergen

Polyethylene glycol (PEG) stands as a premier biocompatible polymer extensively employed in medical applications. Its usage spans across a wide spectrum of pharmaceutical products, including oral drugs, topical medications, and most prominently, in the COVID-19 vaccines. Industrially produced PEG typically exhibits polydispersity, consisting of single PEG chains with atleast 10 different lengths. Conversely, monodisperse (uniform) PEGs comprise a single PEG chain length, albeit they are less common and available in smaller quantities at a higher cost.

In this study, we pioneered methods for producing monodisperse PEG derivatives essential to the pharmaceutical industry. Specifically, we established pathways for synthesizing high molecular weight monodisperse PEGs over 1000 Da. We also developed synthetic routes and purification methods for in-demand monodisperse PEG-lipids, such as DMG-PEG 2000, utilized in Moderna’s COVID-19 vaccine, Spikevax®. Additionally, we created a diverse repository of PEG-peptides, evaluating them as potential candidates for pancreatic cancer vaccines.

Luca Streitlein, Mateu Montserrat Canals & Ute Krengel

Universitetet i Oslo, Department of Chemistry

Chorismate mutase is a key enzyme in the shikimate pathway, where it catalyses the pericyclic Claisen rearrangement of chorismate to prephenate. It plays a central regulatory role in the pathway by directing the synthesis towards phenylalanine and tyrosine instead of tryptophane. As the shikimate pathway is absent in mammals, better understanding of the structure and function of chorismate mutase could lead to its exploitation as a target for new antibiotics, fungicides and herbicides.

The focus of this study lies on the characterization of the chorismate mutase from Corynebacterium glutamicum, which has limited activity on its own and requires
activation by a partner enzyme, D-arabino-heptulosonate-7-phosphate
synthase. Our collaborators at the ETH, led Dr. Peter Kast (ETH), have
recently created enzyme variants of chorismate mutase with directed
evolution that are highly active on their own. Structural characterization will be performed by single crystal X-ray diffraction and comparison of the conformations adopted by the enzymes, to deepen our understanding of the activity and regulation of chorismate mutase.