6 results on '"Knecht W"'
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2. A mild skeletal phenotype with overlapping features of Miller syndrome and functional characterisation of two new variants of human dihydroorotate dehydrogenase.
- Author
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Mero IL, Orozco Rodriguez JM, Bjørgo K, Hankin RA, Krupinska E, Kulseth MA, Rossow MA, and Knecht W
- Abstract
Dihydroorotate dehydrogenase (DHODH) catalyzes the fourth enzymatic reaction of the pyrimidine biosynthesis pathway. Miller syndrome, also known as postaxial acrofacial dysostosis, is caused by biallelic pathogenic variants in DHODH . We present a patient with a relatively mild skeletal phenotype carrying a novel variant of unknown significance in DHODH : c.829G > A, p.(D277N), in combination with a known variant, c.403C > T, p.(R135C). We functionally characterized the DHODH variant D277N in comparison to a very recently reported, but functionally uncharacterized variant P43L, that was found in a patient with more pronounced Miller syndrome features. Because both cases share the same DHODH variant R135C, we aimed to study the effect on enzyme activity of the two variants D277N and P43L to determine pathogenicity and possibly a genotype-phenotype relationship on the R135C background. We found a significant reduction in enzyme activity for both variants. The variant P43L showed a more pronounced loss of function in all assays compatible with other pathogenic variants reported in Miller, whereas the D277N variant showed milder changes that could reflect the mild phenotypic features in our patient. Yet due to a lack of a known threshold of residual enzyme activity to determine pathogenicity, this needs to be confirmed in further studies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.) more...
- Published
- 2024
- Full Text
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3. New insights into complex formation by SARS-CoV-2 nsp10 and nsp14.
- Author
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Sele C, Krupinska E, Andersson Rasmussen A, Ekström S, Hultgren L, Lou J, Kozielski F, Fisher SZ, and Knecht W
- Subjects
- Protein Binding, Exoribonucleases metabolism, Exoribonucleases chemistry, Humans, Methyltransferases metabolism, Methyltransferases chemistry, Protein Domains, Models, Molecular, Viral Regulatory and Accessory Proteins, Viral Nonstructural Proteins metabolism, Viral Nonstructural Proteins chemistry, Viral Nonstructural Proteins genetics, SARS-CoV-2
- Abstract
SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone. more...
- Published
- 2024
- Full Text
- View/download PDF
4. Oligomeric State of β-Coronavirus Non-Structural Protein 10 Stimulators Studied by Small Angle X-ray Scattering.
- Author
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Knecht W, Fisher SZ, Lou J, Sele C, Ma S, Rasmussen AA, Pinotsis N, and Kozielski F
- Subjects
- Humans, SARS-CoV-2, Scattering, Small Angle, X-Ray Diffraction, X-Rays, COVID-19, Middle East Respiratory Syndrome Coronavirus
- Abstract
The β-coronavirus family, encompassing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS), and Middle East Respiratory Syndrome Coronavirus (MERS), has triggered pandemics within the last two decades. With the possibility of future pandemics, studying the coronavirus family members is necessary to improve knowledge and treatment. These viruses possess 16 non-structural proteins, many of which play crucial roles in viral replication and in other vital functions. One such vital protein is non-structural protein 10 (nsp10), acting as a pivotal stimulator of nsp14 and nsp16, thereby influencing RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to unraveling its multifunctional roles. Our study involves the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. To elucidate their oligomeric state, we employed a combination of Multi-detection Size exclusion chromatography (Multi-detection SEC) with multi-angle static light scattering (MALS) and small angle X-ray scattering (SAXS) techniques. Our findings reveal that full-length nsp10s primarily exist as monomers in solution, while truncated versions tend to oligomerise. SAXS experiments reveal a globular shape for nsp10, a trait conserved in all three coronaviruses, although MERS nsp10, diverges most from SARS and SARS-CoV-2 nsp10s. In summary, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 exhibit a globular and predominantly monomeric state in solution. more...
- Published
- 2023
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5. Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).
- Author
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Sullivan HM, Krupinska E, Rasmussen AA, Orozco Rodriguez JM, and Knecht W
- Subjects
- Recombinant Proteins metabolism, Baculoviridae genetics, Genetic Vectors
- Abstract
The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) more...
- Published
- 2023
- Full Text
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6. New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition.
- Author
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Orozco Rodriguez JM, Wacklin-Knecht HP, Clifton LA, Bogojevic O, Leung A, Fragneto G, and Knecht W
- Subjects
- Cardiolipins metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Phosphatidylcholines metabolism, Protein Conformation, Protein Domains, Dihydroorotate Dehydrogenase chemistry, Dihydroorotate Dehydrogenase metabolism, Escherichia coli enzymology, Lipid Bilayers metabolism, Ubiquinone metabolism
- Abstract
The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH ( Hs Δ29DHODH) and the DHODH from Escherichia coli ( Ec DHODH) with ubiquinone (Q
10 ) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10 . Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10 . We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that Ec DHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than Hs Δ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10 , as well as lipid composition, on enzyme binding. more...- Published
- 2022
- Full Text
- View/download PDF
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