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- Bimai, Ornella, et al.
(författare)
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Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding
- 2024
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- A small, nucleotide-binding domain, the ATP-cone, is found at the N- terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimertetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP- cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain ( GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 A away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
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| 2. |
- Bimai, Ornella, et al.
(författare)
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Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding
- 2024
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Ingår i: eLife. - 2050-084X. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer– tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
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| 5. |
- Schagatay, Felix, et al.
(författare)
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Serum concentration of extracellular cold-inducible RNA-binding protein is associated with respiratory failure in COVID-19
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Uncontrolled release of damage-associated molecular patterns (DAMPs) is suggested to be a major trigger for the dysregulated host immune response that leads to severe COVID-19. Cold-inducible RNA-binding protein (CIRP), is a newly identified DAMP that aggravates inflammation and tissue injury, and induces respiratory failure in sepsis. Whether CIRP contributes to the pathogenesis of respiratory failure in COVID-19 has not yet been explored.Aim: To investigate if the concentration of extracellular CIRP (eCIRP) in serum associates with respiratory failure and lung involvement by chest computed tomography (CT) in COVID-19.Methods: Herein we report a prospective observational study of patients with COVID-19 included at two University Hospitals in Sweden between April 2020 and May 2021. Serum from hospitalized patients in Örebro (N=97) were used to assess the association between eCIRP and the level of respiratory support and its correlation with pulmonary involvement on chest CT and inflammatory biomarkers. A cohort of hospitalized and non-hospitalized patients from Umeå (N=78) was used as an external validation cohort. The severity of disease was defined according to the highest degree of respiratory support; mild disease (no oxygen), non-severe hypoxemia (conventional oxygen or high-flow nasal oxygen, HFNO <50% FiO2), and severe hypoxemia (HFNO ≥50% FiO2, mechanical ventilation). Unadjusted and adjusted linear regression was used to evaluate peak eCIRP day 0-4 in respect to severity, age, sex, Charlson comorbidity score, symptom duration, and BMI.Results: Peak eCIRP concentrations were higher in patients with severe hypoxemia and were independently associated with the degree of respiratory support in both cohorts (Örebro; p=0.01, Umeå; p<0.01). The degree of pulmonary involvement measured by CT correlated with eCIRP, rs=0.30, p<0.01 (n=97).Conclusion: High serum levels of eCIRP are associated with acute respiratory failure in COVID-19. Experimental studies are needed to determine if treatments targeting eCIRP reduces the risk of acute respiratory failure in COVID-19.
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| 6. |
- Schaufelberger, Maria, 1954, et al.
(författare)
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Validity of heart failure diagnoses made in 2000-2012 in western Sweden
- 2020
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Ingår i: ESC Heart Failure. - : WILEY PERIODICALS, INC. - 2055-5822. ; 7:1, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- Aims The aim of this study is to validate a diagnosis of heart failure (HF) according to the European Society of Cardiology (ESC) guidelines among patients hospitalized at Sahlgrenska University Hospital, Gothenburg, Sweden, between 2000 and 2012. Methods and results In Sweden, it is mandatory to report all hospital discharge diagnoses to the Swedish national inpatient register. In total, 27 517 patients were diagnosed with HF at the Sahlgrenska University hospital between 2000 and 2012. Altogether, 1100 records with a primary (n = 550) or contributory (n = 550) diagnosis of HF were randomly selected. The diagnosis was validated according to the ESC guidelines from 1995, 2001, 2005, and 2008, and cases were divided into three groups: definite, probable, and miscoded. In total, 965 cases were validated, while 135 records were excluded for various reasons. Of the 965 records, the diagnosis was validated as definite in 601 (62.3%) and as probable in 310 (32.1%); only 54 (5.6%) of cases had been miscoded. Echocardiography, as an objective evidence of cardiac dysfunction, had been performed in 581 (96.7%) of the definite, 106 (34.2%) of the probable, and 31 (57.4%) of the miscoded cases. Among the probable cases, the main reason they had not been classified as a definitive diagnosis of HF was lack of examination by echocardiography (63.8%). Conclusions The overall validity of HF diagnosis at Sahlgrenska University Hospital is high. This may reflect a high diagnostic validity at the time of diagnosis in the national Swedish patient register, supporting the continued use of this register in epidemiological research.
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| 7. |
- Sele, Céleste, et al.
(författare)
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New insights into complex formation by SARS-CoV-2 nsp10 and nsp14
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - 1525-7770.
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Tidskriftsartikel (refereegranskat)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.
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