Poster Abstracts – Session 1

Agnes B. Petersen1, Anita Solem1, Gerd Inger Sætrom1, Håvard Sletta2, Finn L. Aachmann1, Anne Tøndervik2,1, Jochen Schmid2, & Gaston Courtade1

(1) Department of Biotechnology and Food Science, Norwegian University of Science and Technology. Trondheim, Norway, (2) Department of Biotechnology and Nanomedicine, SINTEF Industry. Trondheim, Norway

Alginate epimerases convert β-D-mannuronate (M) to its C-5 epimer α-L-guluronate (G) in alginates. Alginates are linear anionic polysaccharides produced by brown seaweed and some bacteria. In alginates M and G are organized in blocks of M (polyM), blocks of G (polyG), and in alternating blocks of MG (polyMG). [1]

Alginate epimerases are modular, Ca²⁺-dependent enzymes that work in a processive manner [1] The alginate epimerase AlgE1 from Azotobacter vinelandii consists of two catalytically active A-modules (A₁ and A₂) and four carbohydrate binding R-modules (R₁-R₄) (Figure 1) [2]. Previous studies show that A₁ introduces G to both polyMG and polyM, while A₂ creates MG-blocks from polyM, when the A-modules and their subsequent R-modules are expressed separately [2]. The present study seeks to better understand how the modules function together, and how they influence substrate binding and enzyme processivity. The main methods used were mutational studies combined with NMR spectroscopy.

This study consists of three parts. In the first part of the study, the two A-modules were inactivated, which confirmed the specificity of the A-modules. To enable mutation, a small three amino acid residue motif was inserted in the beginning of A₂, which disrupted their processiveness and substrate binding.

The second part of the study investigated what happens when the relative position of A₁ and A₂ are interchanged, and when A₂R₄ are switched to the N-terminal of the enzyme. Both modifications changed the mode of action of AlgE1.

Lastly, the A-modules of AlgE1 were replaced with the A-module of AlgE7, which is similar in structure, but performs both epimerisation and lyase reactions. This showed that AlgE1 processes along the alginate chain with the C-terminal end first (see Figure 1).

Overall, the study widens the understanding of the role of the two catalytic modules of AlgE1 and how they function together.

References:

1. Petersen AB, Tøndervik A, Gaardløs M, Ertesvåg H, Sletta H, and Aachmann FL (2023) Mannuronate C-5 Epimerases and Their Use in Alginate Modification. Essays in Biochemistry, 67(3): 615-627
2. Ertesvåg H, Høidal HK, Skjåk-Bræk G, and Valla S (1998) The Azotobacter vinelandii Mannuronan C-5-Epimerase AlgE1 Consists of Two Separate Catalytic Domains. Journal of Biological Chemistry, 273(47) 30927-30932

Ayla Steffensen Coder, Mateu Monserrat Canals & Ute Krengel

Department of Chemistry, University of Oslo, Sem Sælands vei 26, 0371 Oslo

The pathogenic bacteria Vibrio cholerae cause millions of cholera infections and hundreds of thousands of recorded deaths globally each year [1]. An adhesin called N-acetylglucosamine binding protein (GbpA) is used by V. cholerae to both colonize the small intestines of mammals, and to adhere to chitin in plankton and shellfish  [2-4]. This adhesin has lytic polysaccharide monooxygenase (LPMO) activity [3, 5], which breaks down complex carbohydrates, including chitin. GbpA’s adhesive and chitinase activity enables the survival of V.cholerae in an aqueous environment by promoting biofilm formation and supplying the bacteria with a readily available carbon source [6]. How this protein interacts with the bacterial surface at the molecular level is poorly understood. The aim of this project is to determine the mechanism by which this adhesin binds to the bacterial surface. A combination of different methods has been and will be used, including binding assays, negative stain EM, and NMR.

A deeper understanding of the molecular mechanisms is of interest in more than one way. They may inform new treatments for cholera or other infections, and may even lead to improved ways for biofuel production from underutilized carbon sources such as chitin. 

References:

1. Organization, W.H. Cholera. [Webpage] 2022 11 December 2023 [cited 2022 23 July 2024]; Available from: https://www.who.int/news-room/fact-sheets/detail/cholera.
2. Bhowmick, R., et al., Intestinal Adherence of Vibrio cholerae Involves a Coordinated Interaction between Colonization Factor GbpA and Mucin. Infection and Immunity, 2008. 76(11): p. 4968-4977.
3. Wong, E., et al., The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLOS Pathogens, 2012. 8(1): p. e1002373.
4. Tamplin, M.L., et al., Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters. Appl Environ Microbiol, 1990. 56(6): p. 1977-80.
5. Loose, J.S.M., et al., A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Letters, 2014. 588(18): p. 3435-3440.
6. Islam, M.S., et al., Biofilm Acts as a Microenvironment for Plankton-Associated Vibrio cholerae in the Aquatic Environment of Bangladesh. Microbiology and Immunology, 2007. 51(4): p. 369-379.

Davide Luciano1, Jochen Schmid2, & Gaston Courtade1

 (1) Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Trondheim, Norway, (2) Institute of Molecular Microbiology and Biotechnology, University of Münster, Münster, Germany

Keywords: Glycosyltransferase, GTB, Xanthan Gum, biased MD

Exopolysaccharides are a diverse class of molecules with a wide range of industrial applications. Xanthan gum, a heteropolysaccharide synthesized by the Gram-negative bacterium Xanthomonas campestris,[1] is a significant contributor to this class of polymers. Its versatile mechanical properties make it useful in various fields such as the food, material, and pharmaceutical industries.[2] Modifications of the polysaccharide are being studied to modulate its properties for more specific applications.[3] In this study, we present a computational approach to identify key residues in the active site of GumK, a glycosyltransferase involved in xanthan biosynthesis, that influence the dynamics and activity of the enzyme. We describe the dynamics of the two domains and the residues involved in the binding of the natural substrates. Our computational protocol is applicable to other glycosyltransferases within the pathway and can elucidate enzyme selectivity. This insight can inform bioengineering strategies to produce new variants of xanthan gum.

References:

1. Anke Becker and Federico Katzen and Alfred Pühler and Luis Ielpi (1998) Xanthan gum biosynthesis and application: a biochemical /genetic perspective. Applied Microbiology and Biotechnology, 50: 145-152
2. Chaturvedi Surabhi, Kulshrestha Sanchita, Bhardwaj Khushboo and Jangir Rekha (2021) A Review on Properties and Applications of Xanthan Gum. Microbial Polymers: Applications and Ecological Perspectives: 87-107
3. Patel, Jwala and Maji, Biswajit and Moorthy, N. S. Hari Narayana and Maiti, Sabyasachi (2020) Xanthan gum derivatives: review of synthesis, properties and diverse applications. RSC Adv, 10(45): 27103-27136

Gabriela Ruiz-Velarde, Valeriia Kalienkova, Andrea J. Lopez & Petri Kursula

Department of Biomedicine, University of Bergen, Jonas Lies Vei 91, 5009, Bergen, Norway

The myelin sheath is a specialized multilayer wrapped dozens of times around the axons, which is facilitated by myelin-specific proteins that participate in the stacking of the lipid membranes. One of such proteins is MAL (Myelin and Lymphocyte Protein), which is predominantly localized in the compact myelin. MAL has been associated with glycosphingolipid-rich microdomains in the myelin and other tissues, suggesting that it might play a role in the formation and maintenance of the myelin membrane.

Currently, no structural information is available for MAL, in part due to its small size. The protein measures ~17 kDa and comprises only 4 transmembrane helices which are almost entirely embedded in the membrane, making crystallization and single particle cryo-EM challenging. We aim to obtain high-resolution cryo-EM structures of MAL using membrane mimicking tools, such as nanodiscs. In addition, binding partners need to be implemented to increase the size and stability of the protein and thereby enable structure determination. Solving the structure of MAL in a lipid environment will help in better comprehending myelin formation.

We have been able to express and purify MAL from insect cells. Similar to other heterologously produced myelin proteins (PLP and DM20), MAL forms oligomeric assemblies in detergent. Furthermore, we have successfully reconstituted MAL into salipro lipid particles, and have implemented the ALFA-tag as a C-terminal extension of the last transmembrane helix. The ALFA-tag is recognized by the specific nanobody (nbALFA), which can potentially serve as a fiducial for single-particle cryo-EM. These results are a promising start for sample preparation for cryo-EM.

Greta Nardini1, Kristian Hovde Liland2, Sileshi Gizachew Wubshet3, Nils Kristian Afseth3 & Kenneth Aase Kristoffersen1

(1) NMBU – Norwegian University of Life Science, KBM Faculty, P.O. Box 5003, NO-1432, Ås, Norway, (2) NMBU – Norwegian University of Life Science, REALTEK Faculty, P.O. Box 5003, NO-1433, Ås, Norway, (3) Nofima – Norwegian Institute of Food, Fisheries and Aquaculture Research, P.O. Box 210, NO-1431, Ås, Norway

Enzymatic Protein Hydrolysis (EPH) is a well-established and versatile technology for the valorization of protein from food industry side-streams. Low-value cuts from poultry, a protein-rich biomass, are transformed into high-value protein hydrolysates with different physicochemical properties. One example is collagen-enriched hydrolysates used in food products, pharmaceuticals, and cosmetics.^[1]

Process monitoring and feed-forward control mechanisms are crucial to optimize yield and product quality from EPH processes.^[2] The amino acid composition is an important process parameter, and along with the molecular weight distribution (MWD), it is a key parameter in determining the hydrolysates’ physicochemical properties. In poultry side-stream valorization, collagen solubilization is another key parameter and can be followed using Hydroxyproline, an amino acid almost exclusive to collagen.^[3]

The gold standard for amino acid composition analysis is the chromatographic method.^[4] Recently, however, there has been a growing interest in spectroscopic techniques which are green, rapid, and non-destructive, making them ideal for real-time measurements.^[2,5,6] Fourier Transform Infrared spectroscopy (FTIR), a vibrational spectroscopy technique, has successfully been applied to monitor industrial processes to assess relevant analytical parameters like MWD and collagen content.^[7,8] While vibrational spectroscopy is not suitable for predicting the amino acid composition, Nuclear Magnetic Resonance (NMR) spectroscopy is. NMR offers deeper insights because it is superior to FTIR in probing metabolites and low molecular components, it also provides more reliable quantitative (qNMR) data.^[9,10]

In this study, the performance of FTIR and NMR spectroscopy has been compared with classical methods for investigating collagen content in EPH samples from poultry side-streams. 

References:

1. K. A. Kristoffersen et al., Food Chem 2022, 382, DOI 10.1016/j.foodchem.2022.132201.
2. S. G. Wubshet et al., Food Bioproc Tech 2018, 11, 2032–2043.
3. R. E. Neuman et al., Journal of Biological Chemistry 1950, 184, 299–306.
4. European Commission Regulation, No 152/2009 of 27 January 2009.
5. S. Shin et al., Food Chem 2021, 352, DOI 10.1016/j.foodchem.2021.129329.
6. M. S. Eissa et al., TrAC 2024, 170, DOI 10.1016/j.trac.2023.117435.
7. I. Måge et al., LWT 2021, 152, DOI 10.1016/j.lwt.2021.112339.
8. K. A. Kristoffersen et al., Spectrochim Acta A 2023, 301, DOI 10.1016/j.saa.2023.122919.
9. T. Tchipilov et al., Methods Protoc 2023, 6, DOI 10.3390/mps6010011.
10. T. Riemer et al., Biomacromolecules 2012, 13, 2110–2117.

Hanne Øye1, Monica Hellesvik1,2, Thomas Arnesen1,2,3, & Henriette Aksnes1

(1) Department of biomedicine, University of Bergen, Norway, (2) Department of biological sciences, University of Bergen, Norway, (3) Department of surgery, Haukeland University Hospital, Norway

3D cell culture assays are becoming increasingly popular due to their higher resemblance to tissue environment. These provide an increased complexity compared to the growth on 2D surface and therefore allow studies of advanced cellular properties such as invasion. We report here on the use of 3D Matrigel cell preparations combined with a particular gentle and informative type of live-cell microscopy: quantitative digital holographic microscopy (DHM), here performed by a commercial software-integrated system, currently mostly used for 2D cell culture preparations. By demonstrating this compatibility, we highlight the possible time-efficient quantitative analysis obtained by using a commercial software-integrated DHM system, also for cells in a more advanced 3D culture environment. Further, we demonstrate an example making use of this advantage by performing quantitative DHM analysis of Matrigel-trapped single and clumps of suspension cells. For this, we benefited from the autofocus functionality of digital phase holographic imaging to obtain 3D information for cells migrating in a 3D environment. We demonstrate that it is possible to quantitatively measure tumorigenic properties like growth of cell clump (or spheroid) over time, as well as single-cell invasion out of cell clump and into the surrounding extracellular matrix. Overall, our findings highlight several possibilities for 3D digital holographic microscopy applications combined with 3D cell preparations, therein studies of drug response or genetic alterations on invasion capacity as well as on tumor growth and metastasis.

Hemanga Gogoi, Dario Segura-Peña, & Nikolina Sekulic

Centre for Molecular Medicine Norway, University of Oslo

The chromosomal passenger complex (CPC) is a macromolecular assembly comprising four subunits: Aurora B kinase, Inner Centromere Protein (INCENP), Survivin, and Borealin. This complex plays a role in the regulation of chromosomal segregation during mitosis. Additionally, Shugoshin 1 (Sgo1) ensures chromosome stability as it protects the cohesin, through recruitment of two protein complexes with opposite enzymatic activities, the Protein phosphatase 2A (PP2A) and the CPC. The enzymatic component of the CPC is the serine/threonine protein kinase Aurora B. Localization of Aurora B to the inner centromere is critical for proper chromosome segregation and therefore chromosome stability. Sgo1, plays an important role in the recruitment of the CPC to the inner centromere but the biochemical and structural details of the Sgo-CPC interaction are not understood.  Here we undertake a biochemical and structural approach to understand this critical interaction, base in Hydrogen deuterium exchange, mutagenesis, and Fida-Bio experiments, we found that borealin is the subunit of the CPC that is more engaged in interaction with Sgo1.  In addition, we show in-vitro that both the CPC and PP2A can interact simultaneously with Sgo1 and propose a model for the formation of this ternary complex.

Irene Fausk Crnic, Natalia Mojica, & Ute Krengel

Department of Chemistry, University of Oslo

Cholera is an acute diarrhoeal disease caused by the bacterium Vibrio cholerae¹, which releases the bacterial toxin, cholera toxin (CT), inside the host intestine. The cholera toxin is similar in structure and function to the heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC), which causes the less severe diarrhoea known as traveller’s diarrhoea. Both toxins belong to the AB₅ family of toxins, characterized by a catalytically active A subunit and a pentamer of B subunits (CTB/LTB) important for binding to receptors on the host cell². The B-pentamers also bind to other molecules, including lipopolysaccharides (LPS), which are an integral part of the outer membrane of Gram-negative bacteria. They consist of a hydrophobic part (lipid A) attached to the outer membrane, an inner and outer saccharide core, and an O-antigen³. LPS is also an important component of outer membrane vesicles (OMVs), which are the main delivery systems of LT toxins by ETEC².LT is attached to OMVs through the B-pentamer, which binds directly to LPS, but little is known about the detailed molecular interaction of LTB-LPS.

This project aim is to investigate the molecular interaction between the lipopolysaccharides and the B subunit of the heat-labile enterotoxin by NMR and ELISA. 

References:

1. WHO. Diarrhoeal disease. World Health Organization, 2017. https://www.who.int/newsroom/fact-sheets/detail/diarrhoeal-disease (accessed 25.04.2024)
2. Horstman, A. L.; Kuehn, M. J. Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway. J. Biol. Chem. 2002, 277 (36), 32538–32545. https://doi.org/10.1074/jbc.M203740200.
3. Horstman, A. L.; Bauman, S. J.; Kuehn, M. J. Lipopolysaccharide 3-deoxy-d-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins. J. Biol. Chem. 2004, 279 (9), 8070–8075. https://doi.org/10.1074/jbc.M308633200.

José M. Godoy Muñoz1, Lasse Neset1, Sigurbjörn Markússon1, Sarah Weber1, Oda C. Krokengen1, Aleksi Sutinen2, Eleni Christakou1, Andrea J. Lopez1, Clive R. Bramham1, & Petri Kursula1,2

(1) Department of Biomedicine, University of Bergen, Bergen, Norwa^y, (2) Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu, Finland

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity. To promote the characterisation of its function and structure, nanobodies targeting Arc have been developed. Two anti-Arc nanobodies, named E5 and H11, have been selected for further study. These nanobodies selectively bind the human Arc N-lobe (Arc-NL), which contains a multi-peptide binding pocket that mediates multiple molecular functions of Arc.

In this study, we aimed to characterise the Arc-NL-nanobody complexes of E5 and H11 from structural and functional perspectives. The structures of the complexes were solved at atomic resolution using X-ray crystallography. Interestingly, both nanobodies bind the multi-peptide binding pocket of Arc-NL. As shown by isothermal titration calorimetry, the nanobodies bind Arc-NL with nanomolar affinity and displace a peptide derived from TARPγ2, the endogenous substrate of Arc with the highest known affinity. Altogether, these results suggest that E5 and H11 can be competitive inhibitors of Arc-NL.

We have provided a thorough biochemical characterisation of E5 and H11. Both nanobodies could be used to target Arc-dependent synaptic plasticity and support the development of diagnostic and treatment tools for Arc-related disorders.

Juliana Miranda Tatara1, Victoria Queiroz2,Klara Stensvåg1, Jônatas Abrahão2, & Gabriel Magno De Almeida1

(1) The Arctic University of Norway, Tromsø, Norway, (2) Universidade Federal de Minas Gerais, Brazil

Giant viruses are part of the nucleocytoplasmic large DNA viruses (NCLDVs) group, and they were first described in 2003 [1], leading to a revolution in microbiology. Recent metagenomic research revealed that giant viruses are ubiquitous [2], and their genome size can reach up to 2.5Mb [3]. Such characteristics lead to a remarkable biotechnology potential coded by their still mysterious genomes. The arctic region, such as the Nordic Sea, have been described as hotspots for finding giant viruses [4]. Herein, we aimed to collect different samples above the arctic circle and use them to isolate giant viruses using Acanthamoeba sp. as hosts, and further explore different compounds produced during infection using one of our isolates as model. So far, we collected over 250 samples around the north of Norway and Nordic Sea, including: marine and freshwater environments, urban, sewage and deep-sea vent samples. All of them were prepared and used for virus isolation by mixing samples and cells, following culturing steps until cytopathic effect (CPE) appeared. Six different samples presented CPE and the presence of a giant virus was confirmed by Transmission Electron Microscopy images. Virus species are still to be confirmed through sequencing analysis, yet morphological features suggest viruses to be part of the marseilleviridae and mimiviridae families. Preliminary results from metabolomics analysis pointed to different compounds being produced during a Norwegian marseillevirus infection in amoeba. Subsequent steps are virus species identification and characterization, and the description of these diverse compounds produced during the infection.

 References:

1. La Scola, Bernard et al. “A giant virus in amoebae.” Science (New York, N.Y.) vol. 299,5615 (2003): 2033. doi:10.1126/science.1081867
2. Schulz, Frederik et al. “Giant virus diversity and host interactions through global metagenomics.” Nature vol. 578,7795 (2020): 432-436. doi:10.1038/s41586-020-1957-x
3. Philippe, Nadège et al. “Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes.” Science (New York, N.Y.) vol. 341,6143 (2013): 281-6. doi:10.1126/science.1239181
4. Gao, Chen et al. “Viral Characteristics of the Warm Atlantic and Cold Arctic Water Masses in the Nordic Seas.” Applied and environmental microbiology vol. 87,22 (2021): e0116021. doi:10.1128/AEM.01160-2 Abstract text (Calibri 12 point).

Liza Nguyen, Emil Lindbäck, & Magne O. Sydnes

UiS

Bacteria have always been a threat to human health, but the hazard and mortality rates have been increased with the emergence of resistance strains.¹ Their mechanism of defence and natural evolution have compromised the effectiveness of antibiotics, to the extent that there may be no viable medicine on the market to counter bacterial infection in the future.²

Dual action-based molecules are one of the approaches that can be explored to design new drugs for various diseases, including cancer,³ diabete,⁴ and alzheimer.⁵ In the context of bacterial resistance, several hybrid medicines have been synthesized during the past decades, as part of an effort to overcome and find a solution to the global bacterial resistance.⁶

The Marie Curie project “Stop Spread Bad Bugs” (SSBB) aims to develop and test new dual action-based molecules with antibacterial properties. The linkage of a known active scaffold (Ciprofloxacin) with new oligosaccharides offers a combination of their respective pharmaceutical activities and the novelty of the structure is making it more difficult for bacteria to develop simultaneous resistance mechanisms against both moieties

References:

1. Baindara, P.; Mandal, S. M. Protein Pept. Lett. 2019, 26, 324-331.
2. World Health Organization,Antibacterial agents in clinical development : an analysis of the antibacterial clinical development pipeline, including tuberculosis, 2017.
3. Rou, R.; Ria, T.; RoyMahaPatra, D.; Hossain Sk, U. ACS Omega, 2023, 19, 16532-16544.
4. Woolston, C., Nature, 2013, 14062.
5. Cheong, S.L.; Tiew, J.K.; Fong, Y.H.; Leong, H.W.; Chan, Y.M.; Chan, Z.L.; Kong, E.W.J. Pharmaceuticals, 2022, 15, 1560.
6. Bremner, J. B.; Ambrus, J. I.; Samosorn, S. Curr. Med. Chem., 2007, 14, 1459-1477.
7. Roemhild, R.; Bollenbach, T.; Andersson, D. I. Nat. Rev. Microbiol. 2022, 20, 478-490.

Maria Wilhelmsen Hoff1, Peik Haugen1, Terje Vasskog1, Njål Rauø2, Lindsey Martinsen3, Johann Eksteen4 & Gøril Laugsand5

(1) UiT Norges arktiske universitet, (2) PHARMAQ, (3) Amicoat, (4) Ard Innovation and (5) Alliance Healthcare

Sustainable technological solutions that maximize the utilization of renewable biological resources are essential to the development of the bioeconomy^1,2. The key principle of the bioeconomy is the use of natural resources and recycling of these resources. “Green microbiology” is presented as a solution to mitigate the detrimental environmental effects seen across several large industries. These industries include food and energy production, as well as waste management. Microorganisms not only requires less amounts of natural resources for production but are easy to dispose of. A main challenge in the development of sustainable microbial processes is the cost of upscaling³.

By combining the use of a sequential batch reactor (SBR), mainly used in wastewater treatment, with a mixed microbial community (MMC), operational costs are substantially reduced. This is largely due to removing the need for sterile conditions⁴. In addition, MMCs has the potential to utilize complex feedstocks due to the metabolic diversity. This allows for further reduction of operational costs and has the potential to increase the value of underutilized materials^5,6.

Large quantities of organic side streams with low value are produced in the aquaculture industry⁴. Simultaneously the demand for seafood increases, and with it the need for sustainable feedstocks which ensure both fish welfare and sufficient nutritional quality⁷.

A challenge with MMCs is the often lengthy selection time. In this project, a feast and famine regime with uncoupled carbon and nitrogen feeding was implemented for internal and external selective pressure^4,5. The aim is to establish a stable SBR operation enriched in lipid storing bacteria for production of a bacterial meal with a lipid composition suitable as a fish feed additive.

References:

1. European Commission (2020). Bioeconomy. Bioeconomy | Research and Innovation (europa.eu)
2. Forskningsrådet, Innovasjon Norge og Siva (2019). Bioøkonomi – felles handlingsplan for forskning og innovasjon | Norges Forskingsråd (www.forskingsradet.no/publikasjoner)
3. A. A. Akinsemolu 2023, Environmental Advances 14:100440.
4. Marreios et al. 2023, Journal of Environmental Chemical Engineering, 11:110100.
5. Oliveira et al. 2017, New Biotechnology, 37(A), 69-79.
6. De Groof et al. 2019, Molecules, 24(3):398.
7. Sprague et al. 2016, 2006–2015. Scientific Reports 6:21892.

Mateu Montserrat-Canals1,2#, Kaare Bjerregaard-Andersen2§#, Henrik Vinter Sørensen2§, Eirik Kommedal3M, Gabriele Cordara2, Gustav Vaaje-Kolstad3, & Ute Krengel2

(#) These authors contributed equally, (1) Centre for Molecular Medicine Norway, University of Oslo, NO-0318 Oslo, Norway, (2) Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway, (3) Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), NO-1433 Ås, Norway

Despite major efforts towards its eradication, cholera remains a major health threat and economic burden in many low- and middle-income countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, e.g., on zooplankton. N-acetyl glucosamine binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides V. cholerae with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca²⁺, Mg²⁺ or K⁺ binding close to its carbohydrate-binding surface. In addition to X-ray crystal structures of cation-LPMO complexes (to 1.5 Å resolution), we explored how the presence of ions affects the stability and activity of the protein. Calcium and magnesium ions were found to bind to GbpA specifically, with calcium ions—abundant in natural sources of chitin—having the strongest effect on protein stability. When the cation-binding site was rendered non-functional, a decrease in activity was observed, highlighting the importance of the structural elements stabilized by calcium. Our findings suggest a cation-binding site specific to GbpA and related LPMOs that may fine-tune binding and activity for the different substrates during environmental survival and host infection.

Mina Gravdahl1, Olav A. Aarstad1, Agnes B. Petersen1, Stina G. Karlsen1, Ivan Donati2, Mirjam Czjzek3, Ove Alexander Høgmoen Åstrand4, Philip D. Rye4, Anne Tøndervik5, Håvard Sletta5, Finn L. Aachmann1 & Gudmund Skjåk-Bræk1

(1) Norwegian Biopolymer Laboratory, Department of Biotechnology and Food Science, Norwegian University of Science and Technology, Trondheim, Norway, (2) Department of Life Sciences, University of Trieste, Trieste, Italy, (3) Statiotation Biologique de Roscoff (SBR), Sorbonne Université, Roscoff, France, (4) AlgiPharma AS, Sandvika, Norway, (5) Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway.

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-L-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 ˚C. Finally, this method has been demonstrated to be superior in the production of α-L-guluronate oligosaccharides with a lower content of β-D-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Nadia Aftab, Jonathan Hira1, Bhupender Singh1, Johanne U. Ericson1, Mona Johannessen1, & Christian S. Lentz1​

Research Group for Host-Microbe Interaction, Department of Medical Biology, UiT – The Arctic University of Norway, Tromsø, Norway​

Bacterial pathogens have evolved a variety of adaptation strategies to thrive in ever changing environment  (1). Phenotypic heterogeneity, often acts as a short-term adaptive mechanism, enabling an isogenic population to cope better with environmental challenges (2). An example of this is the presence of distinct sub-populations with varying ability to adapt and grow in new milieu where some bacteria can flourish in novel nutritional conditions, while others cannot (3). This variability in growth is a critical aspect of bacterial physiology, playing a vital role in their ability to colonize diverse niches within a host environment. Given this background, we hypothesize that by cultivating isogenic bacteria under different growth conditions, we can identify and quantify the sub-populations that vary in their adaptability. Therefore, we established a simplistic invitro model that led to systematically study and characterize these growth phenotypes in Staphylococcus aureus, an important human pathogen. Our results showed a great deal of variability across different sub-population within a clonal population.  Further investigations are underway to identify the molecular factors contributing to these distinct phenotypes and to explore their mechanisms of action, aiming to enhance our understanding of this adaptive phenomenon.

References:

1. Martino ME, Joncour P, Leenay R, Gervais H, Shah M, Hughes S, et al. Bacterial Adaptation to the Host’s Diet Is a Key Evolutionary Force Shaping Drosophila-Lactobacillus Symbiosis. Cell Host Microbe. 2018;24(1):109-19.e6.
2. Kundu K, Weber N, Griebler C, Elsner M. Phenotypic heterogeneity as key factor for growth and survival under oligotrophic conditions. Environmental Microbiology. 2020;22(8):3339-56.
3. Reyes Ruiz LM, Williams CL, Tamayo R. Enhancing bacterial survival through phenotypic heterogeneity. PLoS Pathog. 2020;16(5):e1008439.

Robin Jeske & Richard A. Engh

Department of Chemistry (NORSTRUCT), UiT – The Arctic University of Norway, Tromsø

The thermodynamics and kinetics of ligand (L) binding to enzymes (E) in biophysical studies and drug design applications is typically characterized assuming a simple association reaction scheme:                                                       

which is associated with a Gibbs energy of binding (ΔG_bind), an equilibrium constant, and on and off rates (k_on, k_off) of binding. This may be misleading in a biological context, as it obscures important physical properties of the binding process, including especially  structural multiplicity or disorder and the diversity of chemical environments in molecularly crowded spaces. A more complete scheme may be abbreviated as:

Here, the brackets indicate sets of differing structures whose statistical distributions depend on the changing biological environment, including the proximity of the binding partner. The “#” sign marks a set of higher energy transition state type complexes, and the lower energy final bound state may also be a set of structural conformations. Binding involves a trajectory across a total energy landscape that encodes the process into a reciprocal dance of the binding partners (with their individual average monomeric energy landscapes modulated by proximities of each other, and of other species on the molecularly crowded “dance floor” of the biological environment).

The disorder corresponds to the entropy of the specific averaged states along the reaction coordinate. The figure at right illustrates the entropies of rapidly interconverting structures of a single state (A) and of more distinct structural states, likely with slower interconversion rates (B), and likely with lowered enthalpy. Detailed structural and biophysical studies are needed to characterize entropy across the reaction coordinate.

This has practical implications for peptidic drug design, important for our efforts to design peptide ligands of DYRK1A and other protein kinases. Because peptides are highly flexible compared to typical small molecules, entropy considerations are especially important. Prioritizing compounds that modelling programs predict to have the best binding enthalpies may not be the best approach. We hypothesize that compounds predicted to have multiple good binding poses should be given special weight. Such compounds have lower entropic penalties of binding, but would also have potentially faster on-rates and also would be less susceptible to errors introduced by the simulation method.

Siri T. Sola1, Lise Madsen2, Marie Austdal3, Rolf K. Berge4 & Magne O. Sydnes5

(1) University of Stavanger (UiS)/Tomega AS, (2) University of Bergen(UiB), (3) Stavanger University Hospital, (4) Tomega AS,(5)UiB

Therapeutic options for mitochondrial disorders are currently limited. Consequently, there is a growing interest in discovering safe and effective approaches to address mitochondrial dysfunction. Among these, small-molecule therapies show significant promise for enhancing mitochondrial performance. (1) To prevent pathogenic processes, one approach is to use synthetic versions of mitochondrial fatty acids (MTFAs) which can be orally ingested to increase the mitochondria functioning. A structurally modified fatty acid might be resistant to both β-oxidation and ω-oxidation. One type of structurally modified fatty acid comes from Berge and his group, where the modified fatty acid, tetradecylthioacetic acid (TTA), has been synthesized and investigated (Figure 1). (2)  

TTA was found to have a significantly positive effect on the fatty acid composition in cells. In the current project the aim is to further develop TTA to have an even greater effect on liver and brain health. By synthetic methods we have coupled TTA with another health improving molecule and tested the effect the compound in cells and in rats. (Figure 2)

Primary analysis, show that the TTA analogue effect on fatty acid composition is not significantly different from the results obtained with TTA itself (Figure 2).

References:

1. Meng, L., & Wu, G. (2023). Recent advances in small molecules for improving mitochondrial disorders. RSC Advances, 13, 20476-20485.
2. Jorgensen, M. R., Bhurruth-Alcor, Y., Rost, T., Bohov, P., Muller, M., Guisado, C., . . . Skorve, J. (2009). Synthesis and analysis of novel glycerolipids for the treatment of metabolic syndrome. J Med Chem, 52(4), 1172-1179.

Szymon Szostak & Reidar Lund

Department of Chemistry, University of Oslo, Norway

Peptoids (N-substituted polyglycines) are a relatively new group of synthetic polymers, synthesized for the first time in 1992,^[1,2] designed to mimic peptides, while bearing some advantages like enzymatic stability or higher side chain diversity.^[3] Differently from peptides, in peptoids the side chain is attached to the nitrogen atom instead of the α-carbon position in the polyglycine backbone. That small change results in the absence of backbone chirality and internal hydrogen bonding, whereas tertiary structure depends entirely on side groups’ interactions.^[4] 

In this work, we present a new class of monodisperse poly(ethylene glycol)₄₅-peptoid (mPEG45-peptoid) conjugates that self-assemble into stable micelles with amorphous inner core, crystalline outer core, and diffuse PEG shell. We employed Small Angle X-ray Scattering (SAXS) with model analysis, Matrix-Assisted Laser Desorption/Ionization – Mass Spectrometry (MALDI-MS), Pendant Drop Tensiometry (PDT), and Differential Scanning Calorimetry (DSC) for detailed characterization of the compounds and self-assembled structure in biologically relevant pH range. The results give important insight into the process of sequence-dependent peptoid self-assembly showing the possibility of multi-layered nanoparticles which can be potentially used for an adjustable encapsulation system, in which the composition of the core can be altered to interact most efficiently with a desired molecule. The outer crystalline shell provides additional thermodynamic stability and an additional protective layer for the cargo and environment.

References:

1. R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, Proc. Natl. Acad. Sci. 1992, 89, 9367–9371.
2. R. N. Zuckermann, Pept. Sci. 2011, 96, 545–555.
3. A. Battigelli, Biopolymers 2019, 110, e23265.
4. N. Gangloff, J. Ulbricht, T. Lorson, H. Schlaad, R. Luxenhofer, Chem. Rev. 2015, 116, 1753–1802.

Tobias Rindfleisch1, Jarl Underhaug2, Hanne Antila3 & Markus Miettinen4

(1) Computational Biology Unit and Department of Chemistry, University of Bergen, (2) Department of Chemistry, University of Bergen, (3) Department of Biomedicine, University of Bergen, (4) Computational Biology Unit and Department of Chemistry, University of Bergen

Keywords: Molecular Microscope, MD simulations, IDPs, NMR relaxation, Force field development

Intrinsically disordered proteins (IDPs) are defined by a lack of a specific 3D structure in aqueous solution. They behave like flexible and strongly dynamic random coils, but demonstrate a characteristic distribution over the conformational space, the so-called structural ensemble. However, IDPs perform important and specific functions at the cellular level and at the super cellular level of the organism.

Molecular dynamics (MD) simulations of IDPs represent a complex problem, because none of the common IDP-specific force fields – a model in MD which defines and parameterizes the interactions of atoms – are able to describe the dynamics and thus the flexible motions of disordered proteins accurately. In consequence, the development of a force field, which is capable of reproducing the dynamics for IDPs correctly, as confirmed by validation against experimental NMR data, is of special importance.

The key-idea behind this strategy is to combine the accuracy and precision of NMR experiments with the highly intuitive visualization of MD simulations, such that NMR data can be finally interpreted via the full-atomistic representation of MD – which ultimately describes the creation of a molecular microscope.

This project is motivated by the findings of Oh et al. (2012) that dipeptides behave similarly to disordered proteins, suggesting that these elementary building blocks can be used to test and, if needed, calibrate an IDP-specific MD model.

A gradient-free evolutionary approach will be used to automatically optimize a previously selected and well performing force field; here the force field parameters are iteratively adjusted to obtain an improved match between the resulting protein dynamics in MD simulations and in NMR experiments (relaxation times, homo and hetero nuclear Nuclear Overhauser Effect). Importantly, the corresponding NMR observables can be computed from the MD simulations directly, without assuming an intervening model. The NMR experiments are performed at the Norwegian NMR Platform.

Trond-André Kråkenes¹, Kristine Kippersund Brokstad¹, Aurora Martunez^1,2,3 & Svein Isungset Støve^1,2,3

(1) Department of Biomedicine, University of Bergen, (2) Neuro-SysMed, Department of Neurology, Haukeland university Hospital, (3) K.G. Jebsen Center for Translational Research in Parkinson’s Disease, University of Bergen

INTRODUCTION: Dopamine (DA) and other monoamine neurotransmitters are sequestered into synaptic vesicles by vesicular monoamine transporter 2 (VMAT2) at the presynaptic terminals. VMAT2 is a crucial regulator of DA homeostasis and is a potential drug target for neuronal disorders, such as Parkinson’s disease and involuntary movement disorders. VMAT2 variants have been shown to cause brain monoamine vesicular transport disease, which is a severe, rare infantile-onset disorder that mimics cerebral palsy. We aim to find VMAT2 activators or stabilizers that can rescue misfolded, dysfunctional VMAT2 variants, such as P237H and P387L, and be used in the treatment of monoamine vesicular transport disease.

METHODS: Screening for VMAT2 modulators was done in HEK293 cells permanently transfected to express human VMAT2 with an mCherry fusion tag. Cells in 96 well plates were treated with compounds from the Prestwick Chemical library® (PCL; 1520 compounds, most FDA- and EMA approved) for 48 h. The inhibition or activation of VMAT2 was determined by quantifying the decreased or increased uptake of a fluorescent VMAT2 substrate in treated cells, respectively. The chaperone effect was determined by measuring the increase in protein amount by quantification of fluorescence from the mCherry fusion tag.

RESULTS: We have established a functional high-throughput cellular screening approach combining the effect of the compounds on uptake activity of a fluorescent VMAT2 substrate in cells and their effect on the stability and thus half-life of the VMAT2 in cells. Using this monitoring approach, the screening of the PCL library resulted in the identification of several novel VMAT2 modulators that will be assessed for their effect on substrate transport and protein expression level on disease causing VMAT2 variants.

CONCLUSION: We have identified small pharmacological chaperones that modulate the activity of VMAT2 in cells with a potential therapeutic effect on VMAT2 variants that cause brain monoamine vesicular transport disease.

Zuzanna Samol1, Magne O. Sydnes2, & Erik Agner3

(1) Polypure AS, Norway, Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Norway, (2) Department of Chemistry, University of Bergen, Norway, (3) Polypure AS, Norway

Capsaicin, a well-known spicy compound found in chili peppers, binds selectively to one of the vanilloid receptors, TRPV1 (transient receptor potential vanilloid 1). When capsaicin is delivered in a high enough dose to the receptor, it can provide pain relief. Since these receptors are prevalent in sensory neurons, they are an interesting target for the treatment of chronic neuropathic pain.¹

Capsaicin is often applied topically through creams and patches. However, these medications have several drawbacks. Creams deliver low quantities of the compound, requiring multiple reapplications throughout the day. High-concentration patches, due to their pungency and skin-burning effects, must be applied with a local anesthetic. This results in limited efficacy and patient compliance.^2,3 Therefore, there is a need to develop topical formulations with enhanced drug loading capacity and minimized side effects.

Our work focused on improving the properties of the polymeric excipients used in hydrogels for drug delivery. The employed strategies included the synthesis of monodisperse PEG and PPG derivatives, which provided defined thermosensitive copolymers. We have also investigated the effect of incorporation high-purity PPG-8 oligomer into the hydrogel. This enhanced the gelation characteristics, as well as in vitro release of capsaicin. In combination, these approaches offer higher purity and reproducibility of the hydrogel matrix, as well as higher hydrophobic payloads with improved delivery.

References:

1. Yang F, Zheng J. Understand spiciness: mechanism of TRPV1 channel activation by capsaicin. Protein Cell. 2017;8(3):169-177. doi:10.1007/s13238-016-0353-7
2. Avila F, Torres-Guzman R, Maita K, et al. A Review on the Management of Peripheral Neuropathic Pain Following Breast Cancer. Breast Cancer (Dove Med Press). 2023;15:761-772. doi:10.2147/BCTT.S386803
3. Thouaye M, Yalcin I. Neuropathic pain: From actual pharmacological treatments to new therapeutic horizons. Pharmacol Ther. 2023;251:108546. doi:10.1016/j.pharmthera.2023.108546

Ema Albrechtova, Gabriele Cordara, & Ute Krengel

Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway

Cytolethal distending toxins (CDTs) are a class of genotoxins produced by various Gram-negative bacteria, known for their ability to cause DNA damage and genomic instability, potentially leading to oncogenesis. CDT is a heterotrimeric AB₂ toxin consisting of a catalytic “A” subunit (CdtB) with DNase activity and two cell-binding “B” subunits (CdtA and CdtC).

As a first step to analyze the ATP-induced disassembly mechanism of CDT, we plan to study the interaction between ATP and CdtB. For this, we will use various structural biology techniques. This poster gives an introduction to our project.

The research is expected to significantly advance our understanding of bacterial toxin biology, offering potential pathways for therapeutic intervention against CDT-induced cellular damage. The insights gained from this study could lead to novel strategies for targeting CDT-related pathologies, ultimately contributing to improved clinical outcomes.