| 1. |
- Begum, Afshan, et al.
(author)
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Transthyretin Binding Mode Dichotomy of Fluorescent trans-Stilbene Ligands
- 2023
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 14:5, s. 820-828
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Journal article (peer-reviewed)abstract
- The orientations of ligands bound to the transthyretin (TTR) thyroxine (T4) binding site are difficult to predict. Conflicting binding modes of resveratrol have been reported. We previously reported two resveratrol based trans-stilbene fluorescent ligands, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (SB-11) and (E)-4-(2-(naphthalen-2-yl)vinyl)-benzene-1,2-diol (SB-14), that bind native and misfolded protofibrillar TTR. The binding orientations of these two analogous ligands to native tetrameric TTR were predicted to be opposite. Herein we report the crystal structures of these TTR:ligand complexes. Opposite binding modes were verified but were different than predicted. The reverse binding mode (SB14) placing the naphthalene moiety toward the opening of the binding pocket renders the fluorescent ligand pH sensitive due to changes in Lys15 amine protonation. Conversely, the forward binding mode (SB-11) placing the naphthalene inward mediates a stabilizing conformational change, allowing intersubunit H-bonding between Ser117 of different monomers across the dimer interface. Our structures of TTR complexes answer important questions in ligand design and interpretation of trans-stilbene binding modes to the TTR T4 binding site.
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| 2. |
- Malinski, Bartosz, et al.
(author)
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Novel pathological variants of NHP2 affect N-terminal domain flexibility, protein stability, H/ACA Ribonucleoprotein (RNP) complex formation and telomerase activity
- 2023
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In: Human Molecular Genetics. - : OXFORD UNIV PRESS. - 0964-6906 .- 1460-2083. ; 32:19, s. 2901-2912
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Journal article (peer-reviewed)abstract
- Telomere biology disorders (TBDs) are characterized by short telomeres, premature aging, bone marrow failure and cancer predisposition. Germline mutations in NHP2, encoding for one component of the telomerase cofactor H/ACA RNA binding complex together with Dyskerin, NOP10 and GAR1, have been previously reported in rare cases of TBDs. Here, we report two novel NHP2 variants (NHP2-A39T and NHP2-T44M) identified in a compound heterozygous patient affected by premature aging, bone marrow failure/myelodysplastic syndrome and gastric cancer. Although still able to support cell viability, both variants reduce the levels of hTR, the telomerase RNA component, and telomerase activity, expanding the panel of NHP2 pathological variants. Furthermore, both variants fail to be incorporated in the H/ACA RNA binding complex when in competition with wild-type endogenous NHP2, and the lack of incorporation causes their drastic proteasomal degradation. By RoseTTAFold prediction followed by molecular dynamics simulations, we reveal a dramatic distortion of residues 33-41, which normally position on top of the NHP2 core, as the main defect of NHP2-A39T, and high flexibility and the misplacement of the N-terminal region (residues 1-24) in NHP2-T44M and, to a lower degree, in NHP2-A39T. Because deletion of amino acids 2-24 causes a reduction in NHP2 levels only in the presence of wild-type NHP2, while deletion of amino acids 2-38 completely disrupts NHP2 stability, we propose that the two variants are mis-incorporated into the H/ACA binding complex due to the altered dynamics of the first 23 amino acids and/or the distortion of the residues 25-41 loop.
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| 3. |
- Minh, Nghia Nguyen Thi, et al.
(author)
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Binding of a Pyrene-Based Fluorescent Amyloid Ligand to Transthyretin : A Combined Crystallographic and Molecular Dynamics Study
- 2023
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 127:30, s. 6628-6635
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Journal article (peer-reviewed)abstract
- Misfolding and aggregation of transthyretin (TTR) causeseveralamyloid diseases. Besides being an amyloidogenic protein, TTR hasan affinity for bicyclic small-molecule ligands in its thyroxine (T4)binding site. One class of TTR ligands are trans-stilbenes. The trans-stilbenescaffold is also widely applied for amyloid fibril-specific ligandsused as fluorescence probes and as positron emission tomography tracersfor amyloid detection and diagnosis of amyloidosis. We have shownthat native tetrameric TTR binds to amyloid ligands based on the trans-stilbenescaffold providing a platform for the determination of high-resolutionstructures of these important molecules bound to protein. In thisstudy, we provide spectroscopic evidence of binding and X-ray crystallographicstructure data on tetrameric TTR complex with the fluorescent salicylicacid-based pyrene amyloid ligand (Py1SA), an analogue of the Congored analogue X-34. The ambiguous electron density from the X-ray diffraction,however, did not permit Py1SA placement with enough confidence likelydue to partial ligand occupancy. Instead, the preferred orientationof the Py1SA ligand in the binding pocket was determined by moleculardynamics and umbrella sampling approaches. We find a distinct preferencefor the binding modes with the salicylic acid group pointing intothe pocket and the pyrene moiety outward to the opening of the T4binding site. Our work provides insight into TTR binding mode preferencefor trans-stilbene salicylic acid derivatives as well as a frameworkfor determining structures of TTR-ligand complexes.
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| 4. |
- Pietras, Zuzanna, 1993-
(author)
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Small angle scattering as a tool to study protein structure and interactions
- 2022
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Doctoral thesis (other academic/artistic)abstract
- This thesis uses small angle X-ray and neutron scattering (SAXS/SANS) to gain structural and functional insight into the molecular regulation of critical life processes in prokaryotic and eukaryotic species. The presented studies highlight the strength of combining low-resolution structure determination with biophysical and in silico modelling methods to extensively characterize proteins and their interactions. DNA-binding: MexR protein belongs to the family of bacterial transcription regulators and control the expression of multidrug efflux pumps in Pseudomonas Aeruginosa by binding to a DNA region of the operator. SAXS/SANS data supported by MD (Molecular Dynamics) simulations demonstrated that the MexR dimer in solution undergoes a DNA-binding conformational selection mechanism. To gain a better understanding about the system, a low-resolution structural model was resolved in order to assess protein binding to the entire operator region comprising of two closely located DNA recognition sites. The study demonstrates that the use of scattering techniques to investigate similar systems is straightforward and provides knowledge of relevance for clinical understanding and future drug design. Viral host factors: Picornaviruses represent a large family of small RNA viruses that are responsible for a range of diseases in humans and animals. Recently a non-essential human phospholipase PLAAT3 was identified as a key host factor for some picornaviruses. Several picornaviruses representing different branches of the picornaviral phylogenetic tree contain a type of 2A protein in their genome that share a conserved H-box/NC motif with PLAAT3. To understand the role of these 2A proteins in the viral life cycle and to map their plasticity, high resolution techniques were complemented with SAXS to evaluate the structural rearrangements and flexibility. Ubiquitination: In eukaryotes, ubiquitination is a fundamental posttranslational modification, where a small protein ubiquitin is covalently attached to a target protein via sophisticated multienzyme process. SANS can be used to study this mechanism in solution by modular deuteration of ubiquitin complexes. To explore this possibility further, an E2 conjugating enzyme was attached to a deuterated ubiquitin via an isopeptide bond, and a neutron contrast variation experiment was performed. To investigate the flexibility of the E2~Ub conjugate, a multi-state modelling approach was employed to sample its conformational landscape. SANS methods in protein science: A final methods paper outlines and details the experimental requirements, procedures and pre-studies that need to be considered to optimise a successful experimental approach for SANS with contrast variation on biomolecular complexes and assemblies in solution.
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| 5. |
- Salomonsson, Johannes, 1991-
(author)
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Structural, Dynamic and Functional Studies of USP14 : A Proteasome-Associated Deubiquitinating Enzyme Regulating Protein Degradation
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Ubiquitin-Specific Protease 14 (USP14) is a proteasome-associated deubiquitinating enzyme (DUB) involved in regulation of protein degradation and recycling of ubiquitin. USP14 consists of a Ubl domain connected to a USP domain via a flexible linker. The Ubl domain is believed to be responsible for initial association to Rpn1 on the 19S proteasome while the USP domain binds ubiquitin and cleaves the isopeptide bond in ubiquitin chains. Due to its prevalence in various cancers, USP14 has been identified as a promising target for drug development and has been shown to decrease tumor growth and dissemination in zebrafish when inhibited by small molecules.The structure of the USP14 USP domain has been characterized by X-ray crystallography in its free state as well as when bound to ubiquitin or catalytic inhibitors. Additionally, structures of full-length USP14 in complex with the proteasome and ubiquitin have been solved by cryo- EM. However, by these methods, only stable structural elements are visible, limiting the understanding of the dynamic properties of USP14.In this thesis, I have used a wide range of biophysical analysis methods together with nuclear magnetic resonance (NMR) to study the structural, functional, and dynamic properties of USP14. In collaboration with pharmacologists, I have applied biophysical analysis and modelling to the analysis of a library of USP14 binders to elucidate binding affinities and specificities. By combining NMR with small-angle X-ray scattering, I have described the dynamic interaction between the USP14 Ubl domain and its catalytic USP domain and its predisposition toward proteasome binding. Deuteration of USP14 and full NMR analysis of the dynamic properties of the USP domain in the presence and absence of the Ubl domain provided extended understanding of how dynamic networks could be involved in regulating the catalytic activities of USP14. Finally, NMR analysis of USP14 non-Ub-binding mutants jointly with functional analysis has extended our understanding on how USP14 regulates the activities of the proteasome. Taken together, the studies presented here contribute to the investigation of novel therapeutic pathways designed to improve cancer treatment and patient care.
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| 6. |
- Sengupta, Anirban, et al.
(author)
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Intranasal Coronavirus SARS-CoV-2 Immunization with Lipid Adjuvants Provides Systemic and Mucosal Immune Response against SARS-CoV-2 S1 Spike and Nucleocapsid Protein
- 2022
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In: Vaccines. - Basel, Switzerland : MDPI. - 2076-393X. ; 10:4
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Journal article (peer-reviewed)abstract
- In this preclinical two-dose mucosal immunization study, using a combination of S1 spike and nucleocapsid proteins with cationic (N3)/or anionic (L3) lipids were investigated using an intranasal delivery route. The study showed that nasal administration of low amounts of antigens/adjuvants induced a primary and secondary immune response in systemic IgG, mIL-5, and IFN-gamma secreting T lymphocytes, as well as humoral IgA in nasal and intestinal mucosal compartments. It is believed that recipients will benefit from receiving a combination of viral antigens in promoting a border immune response against present and evolving contagious viruses. Lipid adjuvants demonstrated an enhanced response in the vaccine effect. This was seen in the significant immunogenicity effect when using the cationic lipid N3. Unlike L3, which showed a recognizable effect when administrated at a slightly higher concentration. Moreover, the findings of the study proved the efficiency of an intranasally mucosal immunization strategy, which can be less painful and more effective in enhancing the respiratory tract immunity against respiratory infectious diseases.
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| 7. |
- Wehlin, Anna, et al.
(author)
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Crystal structure of the phospholipase A and acyltransferase 4 (PLAAT4) catalytic domain
- 2022
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In: Journal of Structural Biology. - : Academic Press Inc - Elsevier Science. - 1047-8477 .- 1095-8657. ; 214:4
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Journal article (peer-reviewed)abstract
- Phospholipase A and Acyltransferase 4 (PLAAT4) is a class II tumor suppressor, that also plays a role as a restrictor of intracellular Toxoplasma gondii infection through restriction of parasitic vacuole size. The catalytic N-terminal domain (NTD) interacts with the C-terminal domain (CTD), which is important for sub-cellular tar-geting and enzymatic function. The dynamics of the NTD main (L1) loop and the L2(B6) loop adjacent to the active site, have been shown to be important regulators of enzymatic activity. Here, we present the crystal structure of PLAAT4 NTD, determined from severely intergrown crystals using automated, laser-based crystal harvesting and data reduction technologies. The structure showed the L1 loop in two distinct conformations, highlighting a complex network of interactions likely influencing its conformational flexibility. Ensemble refinement of the crystal structure recapitulates the major correlated motions observed in solution by NMR. Our analysis offers useful insights on millisecond dynamics based on the crystal structure, complementing NMR studies which preclude structural information at this time scale.
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| 8. |
- Wehlin, Anna, 1994-
(author)
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Structural and Functional Studies on Evolutionary Repurposing of H-box/NC-proteins : From Host Factor to Virus Protein
- 2023
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Doctoral thesis (other academic/artistic)abstract
- The Picornaviridae are a large family of biomedically important viruses causing diseases such as the common cold, hepatitis A and polio in humans and foot-and-mouth disease in cattle. These diseases have great impact on people’s everyday life and cause economical losses all around the world. To date, no antiviral treatments are available. In attempts to identify potential drug targets for novel antiviral therapies, a human protein was identified as a common host factor for several enteroviruses, a genus within the picornavirus family. This host factor, PLAAT3, facilitates genome transfer from the virus particle into the cytoplasm early in the viral lifecycle prior to virus clearance by autophagy. PLAAT3 is part of a human phospholipid-modifying enzyme family of five members, PLAAT1-5, which all have a conserved H-box/NC-motif forming the active site of these enzymes as well as a hydrophobic C-terminal region that is critical for enzymatic function. This H-box/ NC-motif is also found in the 2A locus of some picornaviruses, suggesting that these viruses might have acquired the protein through horizontal gene transfer to become independent of the human host factor.This thesis focuses on understanding the structural mechanism allowing picornavirus infection. Therefore, two members of the PLAAT-family were studied together with viral 2A proteins sharing the H-box/NC-motif.PLAAT3 was studied with the aim to elucidate its molecular mechanism underpinning its role as a host factor enabling genome transfer. PLAAT3 is composed of a globular N-terminal domain (NTD), whose structure has previously been determined, followed by a 30 amino acid long hydrophobic region (CTR). The catalytic site is located within the NTD, but the hydrophobic CTR is essential both for the catalytic activity as well as cellular localization of PLAAT3.PLAAT4 shares 50% sequence identity with PLAAT3 and exhibits a similar structure with a globular NTD followed by a hydrophobic tail. However, PLAAT4 shows a different activity pattern and displays enzymatic activity even in the absence of the CTR. By comparing the structural properties of PLAAT3 and PLAAT4 more can be understood of the structural characteristics enabling biological functions.The viral 2A proteins studied in this thesis originate from different picornavirus genera but all share the conserved H-box/NC-motif with the PLAAT-family. By investigating the structure and function of representative 2AH/NC proteins from different branches of the phylogenetic tree we aim to identify different steps of evolutionary repurposing to help us understand their role(s) in the viral lifecycle and determine the molecular mechanism allowing them to by-pass PLAAT3 as a host factor.
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