PhD Poster Abstracts

Amalie Skogvold¹, Heidi Therese Hillier¹, Ingar Leiros¹

¹ Department of Chemistry, UiT – The Arctic University of Norway

ω-transaminases are biocatalysts highly sought after in the pharmaceutical industry, as a green alternative for production of valuable chiral amines. The DABA ω-transaminases EctB and DoeD catalyze the forward and reverse transaminase reaction in the bacterial Ectoine biosynthesis and degradation pathway, respectively. Ectoine is a highly valuable compound used both in the cosmetics and pharmaceutical industry due to its many novel properties such as cell protectant, protein stabilizer, skin and UV protectant. It is produced mainly by a method called bacterial milking, but more recent research is done on heterologous production using non-halophilic bacteria such as Escherichia coli. Despite the high marked demand of Ectoine, there is little research done on characterizing the enzymes in the Ectoine biosynthesis or degradation pathway. 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 (Hillier et al., 2020). The group aims to characterize all the core enzymes that synthesize Ectoine, including EctB, EctA and EctC, as well as DoeD which is a part of the Ectoine degradation pathway. Further work will also include rational design to improve operational stability and efficiency of especially the transaminases EctB and DoeD.

References:

1. Hillier, H. T., Altermark, B., & Leiros, I. (2020). The crystal structure of the tetrameric DABA‐aminotransferase EctB, a rate‐limiting enzyme in the ectoine biosynthesis pathway. The FEBS journal, 287(21), 4641-4658.

Andrea N. B. Englund¹, Eirin Landsem¹, Gustavo del Santos¹ and Åsmund Kjendseth Røhr¹

¹ Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway

Metalloenzymes catalyze a wide range of thermodynamically and kinetically difficult reactions in nature. Type-3 copper proteins as tyrosinases, phenolases and hemocyanins contain a binuclear copper center that can display several types of catalytic activity such as monooxygenation, oxidation of diphenols and proteolysis [1]. The diversity of the copper binding ligands observed in tyrosinase-like proteins indicate that there are still more enzyme activities to be discovered for these proteins. Of particular interest are enzyme variants that can activate, or be engineered to activate, small molecules like O₂, NO₂^– and CO₂.

Here we present a structural analysis of the oxidized and the reduced state of tyrosinase from Bacillus megaterium (BmTyr) with copper in the active site using X-ray crystallography. Our results indicate that the redox state have an effect on small molecule binding to the binuclear copper cluster. Furthermore, the structure of a copper-binding site mutant that naturally occurs in distant relatives of the BmTyr has been solved.

References:

1. [1] H. Claus, H. Decker, Bacterial tyrosinases, Syst. Appl. Microbiol. 29 (2006) 3–14. https://doi.org/10.1016/J.SYAPM.2005.07.012.

Bruna Schuck¹, Vipul Panchal¹, Ruth Brenk¹

¹ Department of Biomedicine, University of Bergen

Antibiotic resistance looms as a serious threat to our well-being, health care systems and, consequently, the global economy as we continue to overuse and misuse antibiotics. Therefore, the discovery and development of new antibiotics are urgently needed. The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway generates precursors of isoprenoids which are natural products essential for many human pathogens. As this pathway is absent in humans, its enzymes are attractive targets for new antibiotics.

In this project, we work with 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway, from Pseudomonas aeruginosa (PaDXR) and Mycobacterium tuberculosis (MtDXR). MtDXR is used mainly as a comparator since almost no data is available for PaDXR and much is already known about MtDXR. A plasmid for each was designed with a 6xHis-tag, Avi-tag, and TEV protease cleavage site. After a successful transformation in E. coli BL21(DE3), MtDXR, and E. coli Rosetta(DE3)pLysS, PaDXR, the proteins were expressed and purified. Subsequently, the process was optimized and the best buffer conditions for each protein were established. The enzymatic activity for PaDXR was confirmed by a DXS-DXR coupled assay.

Next, binding experiments using bio-layer interferometry (BLI) were performed. The conditions were optimized for PaDXR, using MgCl₂ as the salt, in order to reproducibly determine the binding of a series of known ligands (NADPH and NADP⁺, 1-deoxy-D-xylulose 5-phosphate (DXP), fosmidomycin and analogs). So far, only binding for the co-enzymes could be determined (NADPH, K_D = 11.9 ± 1.6 µM; NADP⁺, K_D = 42.0 ± 1.4 µM). As the molecular weight of fosmidomycin is close to the detection limit of BLI, the enzymatic assay is currently being used as an orthogonal binding assay. Further, enzymatic and BLI experiments with MtDXR are underway to characterize compound binding to this enzyme. Once the binding affinity of fosmidomycin could be asserted using the coupled assay, a fragment screening of in-house libraries available at the University of Bergen will be carried out.

Ezgi Basavci¹, Alvja Mali³, Erik Agner¹, Mangala Srinivas³, Magne Olav Sydnes²

¹ Polypure AS, Norway,
² University of Stavanger, Norway
³ Wageningen University and Research, The Netherlands

Magnetic resonance imaging (MRI) is a high-resolution and non-invasive preclinical and clinical imaging method using a strong magnetic field.¹ In this project we aim to develop a highly reproducible novel contrast agent (CA) for ¹⁹F MRI and study its physiochemical and biological properties. Since the ¹⁹F nucleus is extremely sensitive for MRI and the ¹⁹F signal is directly proportional to the amount of ¹⁹F present in the fluorine-based CAs, such as perfluoro-15-crown-5-ether (PFCE), making it possible to quantify precisely from the image, unlike conventional gadolinium (Gd³⁺) based CAs that alter the existing proton signal. PFCEs possess a substantial amount of ¹⁹F nuclei, but they are immiscible with lipophilic and hydrophilic solvents.

In this study, poly (lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polyester approved by and Drug Administration (FDA), is used as a versatile nanocarrier to encapsulate PFCE. However, hydrophobic PLGA polymer and PFCEs have some drawbacks as low clearance by the reticuloendothelial system (RES), and long half-life. Srinivas and co-workers reported that PFCE domains are stabilized by the polymeric matrix as a multicore structure which is extremely advantageous in terms of 15-fold faster removal from the body and resulting in a half-life of 16 days.² It has been proposed that attaching monodisperse and structurally well-defined polyethylene glycol (PEG) derivatives onto the surface of nanoparticles enhance biocompatibility and circumvent the opsonins.³ We are focusing on the reproducibility of nanoparticle production securing the same pharmaceutical quality between batches.

Currently, we are synthesizing and purifying heterobifunctional PEG amines followed by attaching them to the PFCE encapsulated PLGA nanoparticle. Physiochemical characterization of each product has been done by FTIR, DLS, Zeta, 1H NMR, and ¹⁹F MRI. It will be investigated the effect of PEG length and end group functionality on nanoparticles for modulating biodistribution and uptake by target cells. It is likely that further experiments will demonstrate the importance of oligomer purity and the impact of the heterobifunctional PEG design.

References

1. Ruiz-Cabello, J., Barnett, B. P., Bottomley, P. A. & Bulte, J. W. M. Fluorine (¹⁹F) MRS and MRI in biomedicine. NMR in Biomedicine vol. 24 114–129 (2011).
2. Koshkina, O. et al. Multicore Liquid Perfluorocarbon-Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking. Advanced Functional Materials 29, 110770, (2019).
3. Gajbhiye, K. R., Pawar, A., Mahadik, K. R. & Gajbhiye, V. PEGylated nanocarriers: A promising tool for targeted delivery to the brain. Colloids and Surfaces B: Biointerfaces vol. 187,114-129, (2020).

This work has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Actions grant agreement No 859908

Greta Daae Sandsdalen¹, Maryam Imam², Ole Morten Seternes³ and 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

The CRISPR-Cas genome editing system has revolutionized molecular biology, providing an array of biotechnological tools for carrying out precision genome modification and regulation in eukaryotic cells. One limitation of the system at present is that available tools are developed from and optimized for mesophilic organisms, which limits their utility in extremophilic organisms. Although thermophilic Cas homologs have been developed and verified for use at high temperatures, less attention has been given to the low-temperature end of the spectrum. This paucity of available psychrophilic genome editing tools is particularly problematic for researchers of cold-blooded eukaryotes such as fish biologists, as both the current enzymes and fish cells lose viability at elevated temperatures.

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 goal is to discover and develop one or more CRISPR-associated endonucleases for effective and precise genome editing at low temperatures. Candidates were selected by bioinformatics analysis of genomes of psychrophilic bacteria.

Currently we are focusing on optimizing CRISPR activity assays to confirm the cold-active nature of a Cas12a endonuclease in addition to optimization of purification protocols for the Cas9 target enzymes. The goal is that one or more of these Cas enzymes will be characterized, including biophysical properties, enzymatic properties, and structure. The ultimate goal is verification that the Cas/sgRNA complexes are able to perform efficient and accurate gene editing in salmon cell lines at low temperatures.

Jeanette Slettnes Grunnvåg, Christian Lentz, Kristin Hegstad*

*Research group for host-microbe interactions (HMI), Department of Medical Biology (IMB), UiT – The Arctic University of Norway, 9019, Tromsø, Norway.

A rise in antibiotic resistance among bacteria is making many concerned that we are
entering into a post-antibiotic era with untreatable bacterial infections and the
consequences this will have on human health. In the last two decades infection by
vancomycin resistant Enterococcus faecium (VRE) has been on the rise (1) , which is
worrisome as vancomycin is a last resort drug used to treat serious infection by
multiresistant bacteria. There is therefore a need to develop new treatment options that can
increase the susceptibility of VRE to antibiotics or anti-virulence agents. Many factors of E.
faecium pathogenicity have mostly been attributed through genomic investigations, but not
studied for their molecular function and their potential as drug targets.

In my PhD project, which will be outlined in the poster, we are investigating the proteome of
E. faecium by using a technique called activity-based protein profiling (ABPP), which can
decipher enzymatic activities and adaptations in biological samples (2-5) . Fundamental to
the technique are the chemical probes that bind active enzymes expressed by the bacteria,
and contains a reporter tag, such as a fluorophore for imaging, or a biotin molecule for
enrichment and identification. By using ABPP to investigate the proteome of E. faecium, my
PhD work will identify enzyme candidates that have the potential to be chemotherapeutic
targets against pathogenic E. faecium strains that are associated with the bacteria’s stress
response, antibiotic resistance, and infectiousness.

Keywords:

Activity-based probes, Enterococcus faecium, Serine hydrolases, Glycosidases, ATPases, Kinases, Penicillin binding proteins.

References:

1. Arias CA, Murray BE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol. 2012;10(4):266-78.
2. Lentz CS. What you see is what you get: activity-based probes in single-cell analysis of enzymatic activities. Biological Chemistry. 2020;401(2):233-48.
3. Speers AE, Cravatt BF. Activity-Based Protein Profiling (ABPP) and Click Chemistry (CC)-ABPP by MudPIT Mass Spectrometry. Curr Protoc Chem Biol. 2009;1:29-41.
4. Lentz CS, Sheldon JR, Crawford LA, Cooper R, Garland M, Amieva MR, et al. Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP). Nature Chemical Biology. 2018;14(6):609-17.
5. Keller LJ, Lentz CS, Chen YE, Metivier RJ, Weerapana E, Fischbach MA, et al. Characterization of Serine Hydrolases Across Clinical Isolates of Commensal Skin Bacteria Staphylococcus epidermidis Using Activity-Based Protein Profiling. ACS Infectious Diseases.
   2020;6(5):930-8.

Lisa Schroer¹, Erna Davydova¹ and Pål Falnes¹

¹ University of Oslo

Carnosine methyltransferase 1 (CARNMT1) is the responsible catalyst of the formation of anserine (π-methylation) from carnosine. Already in the original characterization of the MTase Drozak et al. have shown that CARNMT1, is a rather broad specific enzyme, methylating not only carnosine but also L-histidine itself and L-histidine-containing peptides¹.

Furthermore, it has been found that CARNMT1 is a highly conserved MTase, whereas the enzyme generating carnosine, is not. This, in addition to the promiscuous methylation of L-histidine containing peptides in vitro leads to the hypothesis that there are yet undiscovered substrates of CARNMT1, which can potentially include proteins ^1,2.

In this presentation, we followed up on this hypothesis and investigated the potential of CARNMT1 to methylate proteins and longer peptides as well. If you want to know if CARNMT1 indeed has a taste for proteins, come to my presentation and/or poster!

References:

1. Drozak, J. et al. UPF0586 protein C9orf41 homolog is anserine-producing methyltransferase. J. Biol.     Chem. (2015). doi:10.1074/jbc.M115.640037
2. Kwiatkowski, S., Kiersztan, A. & Drozak, J. Biosynthesis of Carnosine and Related Dipeptides inVertebrates. Curr. Protein Pept. Sci. (2018). doi:10.2174/1389203719666180226155657

Mateu Montserrat-Canals^1,2, Henrik Vinther Sørensen¹, Kaare Bjerregaard-Andersen¹, Esko Oksanen³, Ute Krengel¹

¹ Department of Chemistry, University of Oslo, Oslo, Norway;
² Norwegian Center for Molecular Medicine, University of Oslo, Oslo, Norway
³ European Spallation Source, Lund, Sweden

Many pathogenic bacteria require colonization factors for survival in the environment and attachment to their host. In this work, our focus is on N-acetylglucosamine binding protein A (GbpA) from Vibrio cholerae, which binds to multiple surfaces¹ and provides the bacteria with energy by degrading crystalline chitin with its lytic polysaccharide monooxygenase (LPMO) activity². Although there is abundant structural information on GbpA³, many questions regarding its structure-function relationship remain unanswered. We are using a combination of X-ray and neutron scattering techniques, together with quantitative binding studies, to fully resolve the molecular mechanisms of GbpA. Here, I will focus on the characterization of the LPMO domain of GbpA by X-ray and neutron crystallography.

References

1. Kirn, T. J., Jude, B. A. & Taylor, R. K. A colonization factor links Vibrio cholerae environmental survival and human infection. Nature 438, 863–866 (2005).
2. Loose, J. S. M., Forsberg, Z., Fraaije, M. W., Eijsink, V. G. H. & Vaaje-Kolstad, G. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett. 588, 3435–3440 (2014).
3. Wong, E. et al. The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. PLoS Pathog. 8, 1–12 (2012).

Menghour Huy¹, Gopalakrishnan Kumar¹

¹ Department of Chemistry, Bioscience, and Environmental Engineering, Faculty of Science and Technology, University

Natural astaxanthin synthesized from green microalgae (Haematococcus lacustris) withhold the numerous applications which potentially uses in nutraceutical and pharmaceutical industries ^[1]. There is two cultivations system to synthesized Natural astaxanthin production applied in the industries namely one-stage and two-stage cultivation. In one-stage cultivation, green microalgae are cultivated in one standard growth medium until the essential nutrient is fully consumed and astaxanthin starts accumulating but only less than 2% of astaxanthin content can be expected. For the two-stage cultivation system, biomass production is maintained at a nutrient-replete growth medium in the green vegetative stage whereas astaxanthin content is maximized in the red stage by switching to a nutrient-deplete growth medium. This happened due to the stress response of nutrient starvation of this strain upregulates its protein fraction to lipid fraction along with astaxanthin content^[2]. In this study, Haematococcus lacustis was culturing in standard BBM (Bold’s Basal Medium) in the green vegetative stage until it reached the stationary phase. The green cell culture was harvested by centrifugation and resuspended in three different types of nutrient-deplete growth medium for the red stage study. Three nutrient-deplete growth mediums were chosen for the study by removing nitrogen, phosphorus, and sulphate source from standard BBM for BBM (-N), BBM (-P), and BBM (-S), respectively. The red stage study was carried out for 14 days at 12h/12h of light/dark cycle illumination with 80 μmol.m^-2.s^-1. The result showed that by removing nutrient sources from the growth medium the astaxanthin content increased by around 2.2-fold, 1.7-fold, and 1.6-fold for BBM (-N), BBM (-P), and BBM (-S), respectively. This study proposed two-stage cultivation where biomass production should be maintained in standard BBM and where the red stage should be performed by switching to BBM (-N) to maximize the production.

References

1. Huy, M., Vatland A.K., Kumar, G., 2021. Nutraceutical productions from microalgal derived compounds via circular bioeconomy perspective. Bioresource Technology. 126575.
2. Han, D., Li, Y., Hu, Q., 2013. Astaxanthin in microalgae: Pathways, functions and biotechnological implications. Algae 28, 131–147.)

Nooshin Entezari Heravi¹, Ilke Pala-Ozkok¹

¹ Faculty of Science and Technology, Institute of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Stavanger, Norway

Chloroxylenol (PCMX) is a broad-spectrum antimicrobial widely used as an antiseptic for skin (e.g., hand soap) and disinfectant for non-living surfaces¹. The use of PCMX during the current global pandemic of Coronavirus (SARS-CoV-2) has increased due to its effectiveness in preventing and treating this disease^2,3. This may lead to an increase in PCMX concentrations entering the wastewater treatment plants (WWTPs) which serve as the final barrier before the discharge of PCMX into the receiving environments. The study aimed to deepen the understanding of PCMX’s impacts on nitrifying culture of activated sludge, i.e., the most widely used biological process in wastewater treatment systems⁴, to provide new insights into efficient control of this pollutant. The sludge from a 10 litter nitritation-denitritation SBR reactor was exposed for 30 days to 100 µg/L PCMX. Results showed that this level of PCMX significantly affected the biomass and touched the efficacy of nitrifiers. In the acute exposure to PCMX, the NH₄-N removal efficiency dropped by around 30.5 % and nitritation were completely inhibited. At the end of chronic experiment (30 days), ammonium oxidating bacteria (AOBs) and nitrite oxidating bacteria (NOBs) responded differently to this exposure; while NH₄-N removal efficiency was improved by only about 10% compared to the acute test, no inhibition effect was observed in NOBs indicating that this bacteria culture of the biomass was completely adapted itself to 100 µg/L PCMX after 30 days.

References

1. Choi, D., & Oh, S. (2019). Removal of chloroxylenol disinfectant by an activated sludge microbial community. Microbes and environments, 34(2), 129-135.
2. Tan, J., Kuang, H., Wang, C., Liu, J., Pang, Q., Xie, Q., & Fan, R. (2021). Human exposure and health risk assessment of an increasingly used antibacterial alternative in personal care products: Chloroxylenol. Science of the Total Environment, 786, 147524.
3. Nowak, M., Zawadzka, K., Szemraj, J., Góralczyk-Bińkowska, A., & Lisowska, K. (2021). Biodegradation of chloroxylenol by Cunninghamella elegans IM 1785/21GP and Trametes versicolor IM 373: Insight into Ecotoxicity and Metabolic Pathways. International journal of molecular sciences, 22(9), 4360.
4. Pala-Ozkok, I., Ubay-Cokgor, E., Jonas, D., & Orhon, D. (2019). Kinetic and microbial response of activated sludge community to acute and chronic exposure to tetracycline. Journal of hazardous materials, 367, 418-426.

Reza Talandashti¹, Nathalie Reuter¹

¹ Computational Biology Unit, Department of Chemistry, University of Berge, Bergen, Norway

STARD4 (steroidogenic acute regulatory protein-related lipid-transfer domain 4) is an evolutionarily conserved protein of START family domains that has non-vesicular cholesterol transfer activity from the plasma membrane (PM) to the endoplasmic reticulum (ER) membrane. Although the soluble form of STARD4 has been studied, the membrane-bound form of protein remains still unclear. The higher cholesterol transfer activity in the presence of anionic lipid suggested the possibility that anionic lipids confer membrane selectivity. To investigate the membrane selectivity of STARD4, we performed extensive computational studies. Results of molecular dynamics simulations show that STARD4 has two distinct binding modes: functional and non-functional binding models. Our results have shown that the presence of phosphoinositide lipids (PIP2) in the PM is necessary for protein to achieve the functional binding mode. Based on the results, a specific PIP2 binding site on β1 and β2 strands has been recognized which is the primary STARD4-PM interaction site and is composed of basic residues. There is a second interaction site on the Ω2 loop, which involves regulating protein orientation toward the lipid bilayers. In functional binding mode, the tilt angle of the C-terminal helix with membrane normal is slight, and Ω4 loop penetrates between lipid headgroups. Whereas, in non-functional binding mode, the C-terminal helix is parallel relative to the membrane XY plane and binding is superficial. Furthermore, results show that only the holo-structure of STARD4 obtains functional binding mode during interaction with ER membrane, which is in line with its biological function. These results suggest the self-regulating mechanism of STARD4 to get functional binding mode which improves our knowledge of membrane selectivity of peripheral membrane proteins in general.

Keywords

STARD4, membrane selectivity, phosphoinositide, molecular dynamics simulation.

Le Doujet Typhaine¹, Haugen Peik¹

¹ UiT – The Arctic University of Norway

The migrating Atlantic cod, a cold-water fish, comprises the world’s largest population of Atlantic cod. It is an interesting subject for microbiota/microbiome studies because of its distinct life cycle, migration pattern, feeding resources, and economic and cultural importance. The cod intestine is of particular interest for microbiome studies because it interacts directly with its environment, and helps in several processes including digestion. During evolution, its microbiota has evolved to exploit structural features and energy resources found in the gut, and over time, genes that benefit the microbes while minimizing negative impacts on the host are enriched. Given this assumption, the gut microbiota of marine fish is expected to contain bacteria with genes highly specialized for breaking down fibrous proteins (i.e., collagen, keratin, muscles), carbohydrates (e.g., chitin) and other macromolecules. Surprisingly, this obvious source of industry-relevant enzymes and small molecules is largely overlooked. Recently, we published the bacterial diversity as well as the functional profile and metaproteome of the gut of migrating Atlantic cod. Here, we describe how those results can be used as a platform for mining of commercially interesting cold-active enzymes (e.g. chitinase, collagenase, trypsin, aminopeptidase P). In addition, we will present Aliivibrio wodanis as a production host for expressing identified cold adapted enzymes in the gut of Atlantic cod.

Zuzanna Samol¹, Erik Agner²

¹ Polypure AS, Norway, Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Norway,
² Polypure AS, Norway

It is estimated that approximately 10% of the general population suffers from neuropathic chronic pain. However, currently available treatments are often based on centrally-acting drugs with systemic adverse effects with only short-term pain relief.

The main aim of the project is to develop and characterize a multifunctional hydrogel patch for the delivery of analgesics. This material will be based on PEG-PPG-PEG hydrogel matrix with incorporated PEGylated PLGA nanoparticles loaded with capsaicin. The focus will be on the chemical composition and purity of each biomaterial component. This approach could potentially lead to highly reproducible biomaterial with increased therapeutic efficacy and little to no side effects.

The current focus is on developing a suitable hydrogel matrix based on PEG-PPG-PEG triblock copolymers (poloxamers). These copolymers will be characterized with a specific length for each polymer block and strictly defined molecular weight. As a proof-of-concept, a triblock copolymer of PEG₁₅-PPG₆-PEG₁₅ has been synthesized and characterized. The future perspectives for this work include developing a library of monodisperse PEG-PPG-PEG triblock copolymers with varying lengths of the blocks and derivatization of the terminal groups with functional moieties.

This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions grant agreement No 956477.

Andrea J. López¹, Petri Kursula^1,2

¹ Department of Biomedicine, University of Bergen, Bergen, Norway
2 Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland

Myelin is an essential component of the nervous system. It is a specialized multilamellar membrane that is wrapped around axons. In the central nervous system (CNS), myelin is made by oligodendrocytes, while in the peripheral nervous system (PNS), it is formed by Schwann cells. In the CNS, myelin facilitates a rapid propagation of action potentials [1] and provides metabolic support to axons [2]. Recently, it has been shown that actin dynamics are essential for myelination in the CNS. Actin polymerization drives oligodendrocyte extension and ensheathment of the axons, while actin depolymerization causes myelin wrapping [3]. Moreover, the myelin basic protein (MBP), a main component of the CNS compact myelin, is indispensable for myelin wrapping and compaction [4]. Furthermore, MBP induces the bundling of actin filaments [5]. However, it is unknown if other structural elements of myelin are involved in actin dynamics. Here, using biochemical experiments and transmission electron microscopy, we show that some of the proteins that form the CNS and PNS compact myelin interact with filamentous α- actin. MBP forms bundles; it joins dozens of filaments together. Interestingly, MBP did not affect actin polymerization in the absence of NaCl, but, in the presence of NaCl, actin polymerization was reduced. On the other hand, structural proteins from the PNS show different behavior; myelin protein zero cytoplasmic extension (P0ct) and myelin protein 2 (P2) interacted exclusively with F-actin. Both proteins formed bundles with F-actin, which connect 2-3 filaments. These proteins have an essential effect during actin nucleation and elongation. In the presence of P0ct, the actin polymerization curve lacks the lag phase, and the elongation rate increases. Likewise, it showed a severing function that caused the disassembly of F-actin. In contrast, P2 enhanced actin polymerization. We are in the process of characterizing the polymerization kinetics of α-actin and the myelin proteins in detail. In the future, in vivo experiments are necessary to understand the role of these interactions during the myelination process.

References

1. Fields RD. A new mechanism of nervous system plasticity: Activity-dependent myelination. Nature Reviews Neuroscience. Nature Publishing Group; 2015. pp. 756–767.
2. Lee Y, Morrison BM, Li Y, Lengacher S, Farah MH, Hoffman PN, et al. Oligodendroglia metabolically support axons and contribute to neurodegeneration. Nature. 2012;487: 443–448.
3. Zuchero JB, Fu M meng, Sloan SA, Ibrahim A, Olson A, Zaremba A, et al. CNS Myelin Wrapping Is Driven by Actin Disassembly. Dev Cell. 2015;34: 152–167.
4. Readhead C, Popko B, Takahashi N, David Shine H, Saavedra RA, Sidman RL, et al. Expression of a myelin basic protein gene in transgenic shiverer mice: Correction of the dysmyelinating phenotype. Cell. 1987;48: 703–712. 5. Barylko B, Dobrowolski Z. Ca2+-calmodulin-dependent regulation of F-actin, myelin basic protein interaction. Eur J Cell Biol. 1984;35: 327–335.

Antonia Areali¹

¹ Norwegian University of Life Sciences

Secretion systems in bacteria are of high scientific importance, as various molecules can be secreted from bacteria to respond in different processes. Many factors can affect bacterial secretion systems and the export of proteins, therefore understanding of those factors is the key for a proper biotechnological exploitation of microbial secretion systems. Currently, several bacteria workhorses like Escherichia coli have been extensively studied and engineered to produce high value proteins. The purpose of this research is to study, engineer, optimize and exploit the flagellar secretion systems T3SS which is one of the most important secretion systems in bacteria and has shown a remarkably fast exportation of proteins to the medium. As proof-of-concept, within this project we will establish biotechnological production of high value proteins as, for instance, protein-based fish vaccines at different production scales.  

Eirin Landsem¹, Andrea Englund¹, Åsmund Kjendseth Røhr¹

¹ Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway

Utilizing the greenhouse gas carbon dioxide (CO₂) as a substrate to produce valuable chemicals is an alluring idea. There are still many avenues yet to be explored in the search for an optimal CO₂ reducingcatalyst. One such avenue is altering dinuclear metal sites like those found in type 3-copper proteins to enable them to activate and reduce CO₂. The inspiration for this idea is the similarities between the active site of these enzymes and an organometallic construct proven to be able to catalyse the reduction of CO₂ to oxalate¹.

Minor alterations in the active site or of the second sphere residues in metalloproteins can have drastic effects on the activity and specificity of these enzymes. An approach to investigate the effects of such residue alterations is to explore protein sequence space to identify native variants that display active site and second sphere motifs that are likely to be associated with altered functionality.

We have developed the program Traverse that combine structural information fom the PDB, multiple sequence alignments^2,3,4 and models predicted by Alphafold⁵ (AF2) to aid our search for novel metal binding motifs that may be able to activate CO₂. This program takes an input query sequence and the sequence position of residues indicated by the user (e.g., the metal ligating amino acids) and display the results as a sequence similarity network in Cytoscape⁶, with graphics that indicates number of, sequence placement and type of a-typical residues.

Here we present AF2 models of several candidates that are likely to display novel metal binding motifs as well as a structure solved by X-ray crystallography of one candidate found by using our program, illustrating the viability of the method.

References

1. Angamuthu, R., Byers, P., Lutz, M., Spek, A. L. & Bouwman, E. Electrocatalytic CO2 Conversion to Oxalate by a Copper Complex. Science 327, 313–315 (2010).
2. Johnson, L. S., Eddy, S. R. & Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics 11, 431 (2010).
3. Deorowicz, S., Debudaj-Grabysz, A. & Gudyś, A. FAMSA: Fast and accurate multiple sequence alignment of huge protein families. Sci. Rep. 6, 33964 (2016).
4. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).
5. Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022).
6. Kohl, M., Wiese, S. & Warscheid, B. Cytoscape: Software for Visualization and Analysis of Biological Networks. in Data Mining in Proteomics: From Standards to Applications (eds. Hamacher, M., Eisenacher, M. & Stephan, C.) 291–303 (Humana Press, 2011). doi:10.1007/978-1-60761-987-1_18.

Flore Kersten^1,2, Gabriele Cordara², Stefanie Schmieder³, Markus Künzler³, Ute Krengel²

¹ NCMM, University of Oslo, Norway
² Department of Chemistry, University of Oslo, Norway
³ Institute of Microbiology, Department of Biology, ETH Zürich, Switzerland

CCTX2 is a 89 kDa chimerolectin produced in the vegetative mycelium of the fungus Coprinopsis cinerea (grey shag) and acting as defence protein against fungivorous nematodes [1]. CCTX2 contains four ricin B chain-like lectin domains and a fifth domain, responsible of the cytotoxic activity. The fifth domain displays a “RDQ motif”, a signature sequence found in Poly-ADP-Ribose Polymerases (PARPs). Accordingly, preliminary in vitro experiments suggest a NAD-dependent self-ribosylating activity of CCTX2 [2]. However, the lack of significant sequence conservation with other PARPs outside of the RDQ-motif and the unknown fold of the fifth domain prevent the unambiguous identification of its enzymatic activity, or the full understanding of the reaction mechanism. We have determined the structure of CCTX2 using single particle cryo-EM. Our results shows the first high-resolution structure of CCTX2 as well as complementary data on a cleaved version of the protein, possibly revealing a biologically relevant intermediate. The structure of the fifth domain does not match any of the folds found the in the PDB database and may constitute a completely new family of PARPs.

References

1. Plaza, D.F., Schmieder, S.S., Lipzen, A., Lindquist, E., and Künzler, M. (2015). Identification of a novel nematotoxic protein by challenging the model mushroom Coprinopsis cinerea with a fungivorous nematode. G3 (Bethesda) 6, 87-98.
2. Schmieder, S.S. (2015). The spatiotemporal regulation and mechanism of action of fungal nematotoxic defense effectors. PhD thesis, doi: 10.3929/ethz-a-010607472.

Jan Benedict Spannenkrebs¹, Marianna Karava², Johannes Kabisch¹

¹ Department of Biotechnology and Food Science, Norwegian University of Science and Technology, Trondheim
² Institute of Molecular Biotechnology, Graz University of Technology, Austria

Although enzymes nowadays are often used to reduce the demand a certain process has in terms of energy, resources and water, the current large-scale production of enzymes, especially purified ones remains a significant consumer of said limited commodities¹.

The Gram-positive bacterium Bacillus subtilis is widely used in biotechnology due to the ease of genome manipulation, due to being a good secreter of enzymes and many processes with B. subtilis having obtained a generally recognized as safe (GRAS) status from the FDA. In case of worsening environmental conditions, Bacillus subtilis cells can sporulate, safeguarding their DNA in a spore highly resistant to environmental stress.

The possibility of displaying enzymes on these bacterial endospores, is a method to combine enzyme production and immobilization into a single process². This display usually involves the creation of a genetically linked fusion protein, which consists of a protein of interest, connected via a linker protein to an anchoring protein native to the spore. This technique offers the added advantage of using the sporulation process itself for protein production, compared to e.g. adsorption based methods of displaying proteins on the spore surface³.

The formed spores can be subdivided into three sections, namely the core, the inner & outer coat and outermost layer, the crust. Each layer incorporates a distinct set of proteins which can be used as anchoring proteins.

Their physical properties compared to dissolved enzymes, leads to them being readily purified by centrifugation and repeated washing steps⁴.

After having successfully employed spore display to immobilize a photodecarboxylase5 and sucrose phosphorylase, one of our current research goals is to utilize spore display to produce diagnostics enzymes currently in high demand due to the Covid-19 epidemic. We believe that our system can be especially helpful in low ressource environments such as rural Africa, because it allows for an easy, de-skilled production of said enzymes right at the point of care.

References

1. Becker, M., Lütz, S. & Rosenthal, K. Environmental Assessment of Enzyme Production and Purification. Molecules 26, Enzyme Microb. Technol. 49, 66–71 (2021)
2. Guoyan, Z. et al. Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications. J. Microbiol. Biotechnol. 29, 179–190 (2019)
3. Cho, E.-A., Kim, E.-J. & Pan, J.-G. Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores. Enzyme Microb. Technol. 49, 66–71 (2011)
4. Tavares, M. B. et al. Bacillus subtilis endospores at high purity and recovery yields: optimization of growth conditions and purification method. Curr. Microbiol. 66, 279–285 (2013)
5. Karava, M., Gockel, P., Kabisch, J. & Kabisch, J. Bacillus subtilis spore surface display of photodecarboxylase for the transformation of lipids to hydrocarbons. bioRxiv doi:10.1101/2020.08.30.27382, (2020)

Katja S. Håheim¹ and Magne O. Sydnes¹

¹ Faculty of Science and Technology, Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger

Indoloquinolines are an important class of bioactive compounds whose structural motifs are found in natural products, pharmaceuticals, agrochemicals and drug candidates^{[1, 2]}. The most notable pharmacological properties found in indoloquinolines are antimalarial ^[3], antiproliferative ^[4] and antibacterial ^[5].

Building on strategies from our previous synthetic endeavors to create indoloquinolines ^[6,7] it became apparent that it would be feasible to synthesize both indolo[2,3-b]quinolines (i.e. neocryptolepines) 2 and indolo[3,2-c]quinolines 3 from a common starting material 1. Neocryptolepines 2 were prepared by ­N-alkylation of haloquinolines 1 followed by a tandem Suzuki-Miyaura cross-coupling reaction and intramolecular cyclization. To furnish indolo[3,2-c]quinolines 3, a lengthier approach was necessary, the key synthetic strategies being a standard Suzuki-Miyaura cross-coupling reaction followed by diazotization-azidation and finally photochemical cyclization. The viability of the two pathways were evaluated by conducting a large scope and limitations study, which revealed a broad tolerance to both EDGs (e.g. OMe and Me) and EWGs (e.g. CN, CF₃ and F), however, nitro-functionalizations were overall poorly tolerated.

References

1. Zhu, J.-K.; Gao, J.-M.; Yang, C.-J.; Shang, X.-F.; Zhao, Z.-M.; Lawoe, R. K.; Zhou, R.; Sun, Y.; Yin, X.-D.; Liu, Y.-Q. J. Agric. Food Chem., 2020, 68, 2306-2315.
2. Symington, S. B., Zhang, A., Karstens, W., Van Houten, J., Clark, J. M. Pestic. Biochem. Phys., 1999, 65, 181-193.
3. Sofowora, A. Medicinal Plants and Traditional Medicine in Africa; John Wiley & Sons: Chichester, UK, 1982; pp. 183-256.
4. Lu, C.-M., Chen, Y.-L., Chen, H.-L., Chen, C. A., Lu, P.-J., Yang, C. N., Tzeng, C.-C. Bioorg. Med. Chem., 2010, 18, 1948-1957.
5. Paulo, A., Duarte, A., Gomes, E. T. J. Enthnopharmacol., 1994, 44, 127-130.
6. Håheim, K. S., Helgeland, I. T. U., Lindbäck, E., Sydnes, M. O. Tetrahedron, 2019, 75, 2924-2957.
7. Håheim, K. S., Linbäck, E., Tan, K. N., Albrigtsen, M., Helgeland, I. T. U., Lauga, C., Matringe, T., Kennedy, E. K., Andersen, J. H., Avery, V. M., Sydnes, M. O. Molecules, 2021, 26, 3268-3290.

Maria Wilhelmsen Hoff¹, Lindsey Martinsen², Sietske S. Grijseels³, Terje Vasskog³, J. Johannes Eksteen² and Peik Haugen³

¹ UiT Norges arktiske universitet, ² Amicoat, ³ UiT Norges arktiske universitet

Sustainable industrial food production and reduction of waste are recognized by the European Agency (EU) as essential parts of the future bioeconomy. Sustainable production entails the entire process from primary production to processing of a high-quality product, waste reduction and waste management^1,2 Several recent EU-funded projects have directly target upcycling of biological waste as important measures. Bioconversion by fermenting microorganisms have been explored as potential methods for increased value of underutilized materials^3,4,5. Accumulation and storage of polymers by microorganisms can be exploited to recover nutrients from low value materials, with polyhydroxyalkanoates representing a well-known case^4,5,6,7. Traditionally, the properties of these microbial polymers have been studied as candidates for biodegradable plastic. Multiple potential usages are emerging due to their compositional flexibility and biocompatibility. However, there are several challenges, including expensive production methods and variable product quality, which must be overcome before sustainable and economically viable PHA production is a reality^4,5,6,7. Some of these challenges could be overcome by use of Arctic marine bacteria. Arctic marine bacteria have been found to produce PHA naturally⁸, and their growth at lower temperatures could reduce the environmental footprint and production costs⁹. The resulting biomass could be implemented as part of fish feed. This is highly relevant, as aquaculture have been singled out as a key food production industry to meet the nutritional needs of a growing population. Here, the use of plant-based sources can be reduced and bring the aquaculture industry closer to circular food production^1,2,3,4,10.

References:

1. European Commission (2020). A farm to Fork Strategy for a fair, healthy and environmentally-friendly food system. EUR-Lex – 52020DC0381 – EN – EUR-Lex (europa.eu)
2. European Commission (2018). A sustainable Bioeconomy for Europe: Strengthening the connection between economy, society and the environment. EUR-Lex – 52018DC0673 – EN – EUR-Lex (europa.eu)
3. European Commission (2020). Bioeconomy. Bioeconomy | Research and Innovation (europa.eu)
4. European Commission (2021). Upcycling innovation to extract value from biowaste. Upcycling innovation to extract value from biowaste | Research and Innovation (europa.eu)
5. European commission (2021). Extracting value from waste to deliver high-end products. Available from: Extracting value from waste to deliver high-end products | Research and Innovation (europa.eu)
6. Tan et al. 2021, Trends in biotechnology, 39(9):953–963.
7. Pérez-Rivero et al. 2019, Biochemical Engineering Journal 150:107283.
8. Christensen et al. 2021, Microbial Cell Factories 20:225.
9. Söderberg et al. 2019, Microbial Cell Factories 18:197.
10. Hua et al. 2019, One Earth, 3:316-329.

Md Jalal Uddin¹ and Christian Lentz¹

¹ Research Group for Host-Microbe Interactions (HMI), Department of Medical Biology (IMB), UiT—The Arctic University of Norway, Tromsø, Norway

Staphylococcus aureus is a major human pathogen and a leading cause of bacterial infections worldwide. Many patients suffer from S. aureus infections that are often chronic and are never fully cured by antibiotics due to the ability of S. aureus to persist in biofilms or other protected niches. Therefore, developing novel diagnostic strategies and treatment options for life-threatening S. aureus infections is an urgent priority. Chemical probes, so-called activity-based protein profiling (ABPP) are a powerful technique for deciphering the specific functional enzymes in bacterial systems. Activity-based probes (ABPs) are functionalized enzyme inhibitors that can rapidly and irreversibly bind with their target enzymes by covalently modifying the active site of catalytically active enzymes via specific chemical reaction. This study aimed to identify novel (functional) enzymatic activities in S. aureus during biofilm formation.

Competitive ABPP was used to identify serine hydrolases/functional enzymes and inhibitors activity in live S. aureus by treating intact cells with carmofur/5-fluorouracil and fluorescent carmofur-derived ABP (G11) followed by analysis of labeled protein by SDS–PAGE analysis. LC-MS/MS was used to identify target enzymes using biotin-tagged G11 & transposon mutant strains for target validation. MIC & time-kill assay of carmofur and 5-fluorouracil was performed by the broth microdilution method.

The carmofur-derived ABP (G11) showed broad activity in targeting functional enzymes on biofilm-promoting growth conditions. The G11 labeled the secreted serine hydrolase/lipase and other fluorophosphate-binding serine hydrolases (fphB, fphE, and fphF). Most bands labeled by the G11 can be competed out by pretreatment with the unlabelled parent inhibitors carmofur and active drug, 5-fluorouracil and both drugs showed the same antibacterial activity (MIC-5μM) & time-dependent killing against S. aureus USA 300.

The G11 could be used for targeting & inhibiting a group of hydrolytic enzymes whose functions are likely to be important for various aspects of cellular physiology and host-pathogen interactions.

Keywords:

activity-based probes, serine hydrolases, Staphylococcus aureus

References:

1. P. N. Reddy, K. Srirama, V. R. Dirisala, An update on clinical burden, diagnostic tools, and therapeutic options of Staphylococcus aureus. Infect Dis (Auckl) 10, 1179916117703999 (2017).
2. H. Deng, Q. Lei, Y. Wu, Y. He, W. Li, Activity-based protein profiling: recent Advances in medicinal chemistry. European Journal of Medicinal Chemistry 191, 112151 (2020).

Monika Moreń¹, Helene Grøtting¹, Erling Berge Monsen¹ and Kåre Bredeli Jørgensen¹

¹ Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Stavanger

Many PAHs have been studied in terms of their toxicity and influence on the environment though, even more of them have not been analyzed thoroughly enough. One of the PAHs that needs to be examined more extensively is retene (7-isopropyl-1-methyl-phenantrene) (Scheme). Recent research revealed it is genotoxic¹ and cardiotoxic² to fish. Further studies showed retene’s cytotoxicity to human lung cell line³ and suggest it might be one of the factors that increases the risk of carcinogenesis⁴.

Collected data indicate that exposure to retene might pose a serious threat to people and the environment. Further thorough studies of retene’s toxic properties are crucial for developing standardized methods essential for assessing possible health risks. However, the lack of an efficient method of synthesis of retene or extracting it from natural sources preclude it. Therefore, we propose a simple and efficient method of synthesis of 7-isopropyl-1-methyl-phenantrene based on eliminative photocyclization (Scheme).⁵

References:

1. Maria, V. L.; Correia, A. C.; Santos, M. A. Anguilla Anguilla L. Ecotoxicology and Environmental Safety 2005, 61 (2), 230.
2. Vehniäinen, E.; Haverinen, J.; Vornanen, M. Environ Toxicol Chem 2019, 38 (10), 2145.
3. de Oliveira Alves, N.; Vessoni, A. T.; Quinet, A.; Fortunato, R. S.; Kajitani, G. S.; Peixoto, M. S.; Hacon, S. de S.; Artaxo, P.; Saldiva, P.; Menck, C. F. M.; Batistuzzo de Medeiros, S. R. Sci Rep 2017, 7 (1), 10937.
4. Peixoto, M. S.; da Silva Junior, F. C.; de Oliveira Galvão, M. F.; Roubicek, D. A.; de Oliveira Alves, N.; Batistuzzo de Medeiros, S. R. Chemosphere 2019, 231, 518.
5. Mallory, F. B.; Rudolph, M. J.; Oh, S. M. J. Org. Chem. 1989, 54 (19), 4619.

Oda C. Krokengen¹, Arne Raasakka¹, Petri Kursula ^1,2

¹ Department of Biomedicine, University of Bergen, Bergen, Norway
² Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu, Finland

Myelin protein zero (P0) constitutes 50% of the total protein in the peripheral nervous system and is a key protein in stacking the multilayered proteolipid membrane of the myelin sheath. It functions as a homophilic adhesion molecule, and mutations in P0 are estimated to account for 5% of all cases of the neurological disease Charcot-Marie-Tooth, effecting 1 in 2500 [1]. P0 is in the immunoglobulin (Ig) superfamily and is a transmembrane protein consisting of a N-terminal extracellular Ig-like domain, one transmembrane helix and a C-terminal cytoplasmic tail. The cytoplasmic tail of P0 (P0ct) is unfolded in aqueous solution but gains a-helical secondary structure when interacting with lipids. Mutations in the tail result in decreased adhesiveness, which is important for P0 function within the myelin sheath [2-4]. The mechanisms of this complex system of vital proteins and multilayered lipid membranes are still poorly understood.  

To shed further light on structure-function relationships in P0, further characterization of the cytoplasmic tail of P0 and its interaction with lipids was carried out with use of two membrane mimetic models: liposomes and bicelles. Several structural analytical methods were utilised, including small-angle X-ray diffraction (SAXD), electron microscopy and synchrotron radiation circular dichroism spectroscopy (SRCD).

Negative staining of protein-lipid complexes in various ratios revealed that P0ct induces different types of aggregation depending on the lipid model. The distinct organization increased with lower protein-to-lipid ratio, suggesting a concentration-dependent lipid arrangement. Diffraction data gave several new Bragg peaks when using bicelles, while only one peak is appearing with liposomes [2,3,5]. This suggests an improved lattice structure formed by P0ct when it interacts with bicelles that can potentially be explained by the slower kinetic rates found with stopped-flow SRCD. The gradual binding to bicelles can be favouring the higher order arrangement found in SAXD. Further examination using different membrane model systems can potentially give us more complete understanding of myelin formation at the molecular level.  

References:

1. Reilly, M.M., S.M. Murphy, and M. Laurá, Charcot-Marie-Tooth disease. J Peripher Nerv Syst, 2011. 16(1): p. 1-14.
2. Raasakka, A., et al., Molecular structure and function of myelin protein P0 in membrane stacking. Scientific Reports, 2019. 9(1): p. 642.
3. Krokengen, O.C., A. Raasakka, and P. Kursula, Manuscript in preparation.
4. Gao, Y., W. Li, and M.T. Filbin, Acylation of myelin Po protein is required for adhesion. Journal of Neuroscience Research, 2000. 60(6): p. 704-713.
5. Raasakka, A., et al., Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail. PLOS ONE, 2019. 14(6): p. e0216833.

Selina Cannon Homaei¹, Elaheh Mahootchi¹, Jarl Underhaug² and Jan Haavik^1,3

¹ Department of Biomedicine, University of Bergen, Norway,
² Department of Chemistry, University of Bergen, Norway,
3 Division of Psychiatry, Haukeland University Hospital, Bergen, Norway

b-alanine is synthesized through multiple pathways and serves as a precursor for carnosine (β-alanyl-L-histidine) and related dipeptides. Carnosine is highly abundant in excitatory tissues such as the olfactory bulb, whole brain, and skeletal muscle¹. Several lines of evidence indicate that it has multiple biochemical functions such as antioxidation, neurotransmission and modulation, and pH buffering¹. In addition, endogenous carnosine levels are known to vary with age, gender, and diet. For instance, the age-related decrease of carnosine may play a vital role in the development of diseases related to oxidative stress. Based on this, carnosine and related histidine-containing peptides (HCDs) are attractive therapeutic targets for several diseases. Likewise, b-alanine supplementation is widely popular in sports nutrition to increase endogenous carnosine and boost its pH buffering capacity in muscles during high-intensity exercise¹. Still, the biosynthetic routes and functional potential of these peptides are debated.

We generated the first Glutamate Decarboxylase Like 1 (GADL1)  animal model and described a novel biosynthetic pathway for b-alanine production. This shows that GADL1 decarboxylates aspartate to b-alanine, consequently altering metabolite levels of b-alanine and carnosine peptides in a tissue-specific manner^2,3. We are the first to show that carnosine depletion alters the levels of antioxidant enzymes in an animal model, without giving large pharmacological doses of β-alanine or carnosine^2,3. The mice also displayed changes in both energy and lipid metabolism. The established GADL1 mouse model serves as a new tool to study β-alanine synthesis, carnosine peptide biology, and their relationship to oxidative stress and diseases. This Ph.D. project aims to further investigate these aspects.

References:

1. Boldyrev, A. A., et al. (2013). “Physiology and pathophysiology of carnosine.” Physiol Rev 93(4): 1803-1845.
2. Winge, I., et al. (2015). “Mammalian CSAD and GADL1 have distinct biochemical properties and patterns of brain expression.” Neurochem Int 90: 173-184.
3. Mahootchi, E., et al. (2020). “GADL1 is a multifunctional decarboxylase with tissue-specific roles in beta-alanine and carnosine production.” Sci Adv 6(29): eabb3713.

Yomkippur Perez¹, Erik Agner² and Magne Olav Sydnes³

^{1 Polypure AS and University of Stavanger (UiS), 2 Polypure AS, 3 UiS}

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer, killing about 95% of patients within 5 years of diagnosis. Despite extensive efforts, traditional therapies such as chemotherapy and radiotherapy have not shown significant improvement in overall survival rates. Surgery is only applicable for those in the early stages of the disease however, most patients have locally advanced or metastatic PDAC at the time of diagnosis. In recent years, immunotherapy-based strategies have shown promising results in treating various types of cancers and managing minimal residual disease after pancreatic resection. Our project is part of a multidisciplinary group that will develop novel immunotherapy approaches for the treatment of PDAC. In this study, a multi-component nanovaccine comprising of tumor antigens, immunomodulators, and imaging contrast agents will be developed and delivered through nanocarriers. Additional surface functionalization with various PEG derivatives will also be done. Finally, the efficacy of the multi-component nanovaccine will be tested in vivo in mice and porcine models.

References:

1. O’Kane, G. M.; Ladak, F.; Gallinger, S. Advances in the Management of Pancreatic Ductal Adenocarcinoma. Canadian Medical Association Journal 2021, 193.
2. Sturm, N.; Ettrich, T. J.; Perkhofer, L. The Impact of Biomarkers in Pancreatic Ductal Adenocarcinoma on Diagnosis, Surveillance and Therapy. Cancers 2022, 14, 217. 
3. The illustration was created using BioRender.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 861190