| 1. |
- Collins, Ruairi, et al.
(författare)
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Biochemical discrimination between selenium and sulfur 1 : a single residue provides selenium specificity to human selenocysteine lyase
- 2012
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Ingår i: PLoS One. - Stockholm : Karolinska Institutet, Dept of Medical Biochemistry and Biophysics. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.
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| 2. |
- Davey, Norman E., et al.
(författare)
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Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome
- 2017
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 284:3, s. 485-498
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Tidskriftsartikel (refereegranskat)abstract
- The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.
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| 3. |
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| 4. |
- Gustafsson, Nina M. S., et al.
(författare)
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Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anticancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-gamma H2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.
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| 5. |
- Lehtiö, Lari, et al.
(författare)
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Structural basis for inhibitor specificity in human poly(ADP-ribose) polymerase-3.
- 2009
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Ingår i: Journal of medicinal chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 52:9, s. 3108-11
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Tidskriftsartikel (refereegranskat)abstract
- Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors.
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| 6. |
- Lindgren, Joel, et al.
(författare)
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N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
- 2010
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 19:12, s. 2319-2329
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)
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| 7. |
- Lindgren, Joel, et al.
(författare)
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N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
- 2010
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 19:12, s. 2319-2329
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)
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| 8. |
- Markova, Natalia
(författare)
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Sorption Microcalorimetry: Method Development and Applications
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A double twin isothermal microcalorimeter has been designed and its performance has been tested experimentally. The calorimeter makes it possible to continuously and isothermally scan a solid sample in a broad range of relative vapor pressures at near-equilibrium conditions. The method is suited for measurements with water vapor as well as organic vapors and has been tested at temperatures between 25 and 40ºC. With the technique it is possible to simultaneously and independently in one experiment obtain the sorption isotherm of a sample along with the corresponding differential enthalpies of sorption. The method provides a rather unique combination of information on both partial molar enthalpy and chemical potential change and thus also the partial molar entropy change for the vapor in the sorption process. This is sufficient to characterize the sorption process thermodynamically. It is demonstrated that the method can be applied to the study of a wide range of physicochemical phenomena associated with the uptake of vapor by a substance/material including capillary condensation, hydrate and solvate formation, crystallization and lyotropic phase transitions. The calorimetric results agree well with data from other established techniques such as desiccator storage, sorption microbalance, osmotic stress, solution calorimetry and DSC. The advantage of the present technique is that the sorption data are continuous and cover the whole range of water activities; the isotherms are supported by differential enthalpies of sorption obtained simultaneously for the same sample. It is shown that with the present instrument the critical vapor pressures of hydrate formation and phase boundaries of the lyotropic phase transitions can be determined along with corresponding differential enthalpies of sorption. It is demonstrated that the results of the sorption calorimetric measurements, supported by the data obtained with more specific techniques, can help in understanding of the phase behavior of substances that can self–associate upon hydration.
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| 9. |
- Rozman Grinberg, Inna, et al.
(författare)
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Novel ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit
- 2018
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 A crystal structure. We also present evidence for an (MnMnIV)-Mn-III metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.
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| 10. |
- Wadsö, Lars, et al.
(författare)
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A method to simultaneously determine sorption isotherms and sorption enthalpies with a double twin microcalorimeter
- 2002
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Ingår i: Review of Scientific Instruments. - : AIP Publishing. - 1089-7623 .- 0034-6748. ; 73:7, s. 2743-2754
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Tidskriftsartikel (refereegranskat)abstract
- Sorption of vapors of water, ethanol, and other liquids on solids like pharmaceuticals, textiles and food stuffs are of both practical and theoretical importance. In this article we present a technique to simultaneously measure sorption isotherms and sorption enthalpies. The sample is contained in one end of a sorption vessel. In the other end a vaporizable liquid is introduced to start the measurement. Mass transfer from the liquid to the sample is by vapor diffusion and the rate of mass transfer is calculated from the measured thermal power of vaporization. Simultaneously, the thermal power of sorption is measured and from this one may calculate the differential enthalpy of sorption. The thermal power measurements are made by inserting the sorption vessel in an isothermal double twin microcalorimeter.
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