| 1. |
- Selvaraj, Karthik, et al.
(author)
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Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system
- 2019
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In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Journal article (peer-reviewed)abstract
- A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (similar to 20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.
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| 2. |
- 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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| 3. |
- Ahmed, Niaz, et al.
(author)
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Consensus statements and recommendations from the ESO-Karolinska Stroke Update Conference, Stockholm 11-13 November 2018.
- 2019
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In: European Stroke Journal. - : SAGE Publications. - 2396-9873 .- 2396-9881. ; 4:4, s. 307-317
-
Journal article (peer-reviewed)abstract
- The purpose of the European Stroke Organisation-Karolinska Stroke Update Conference is to provide updates on recent stroke therapy research and to give an opportunity for the participants to discuss how these results may be implemented into clinical routine. The meeting started 22 years ago as Karolinska Stroke Update, but since 2014 it is a joint conference with European Stroke Organisation. Importantly, it provides a platform for discussion on the European Stroke Organisation guidelines process and on recommendations to the European Stroke Organisation guidelines committee on specific topics. By this, it adds a direct influence from stroke professionals otherwise not involved in committees and work groups on the guideline procedure. The discussions at the conference may also inspire new guidelines when motivated. The topics raised at the meeting are selected by the scientific programme committee mainly based on recent important scientific publications. This year's European Stroke Organisation-Karolinska Stroke Update Meeting was held in Stockholm on 11-13 November 2018. There were 11 scientific sessions discussed in the meeting including two short sessions. Each session except the short sessions produced a consensus statement (Full version with background, issues, conclusions and references are published as web-material and at www.eso-karolinska.org and http://eso-stroke.org) and recommendations which were prepared by a writing committee consisting of session chair(s), scientific secretary and speakers. These statements were presented to the 250 participants of the meeting. In the open meeting, general participants commented on the consensus statement and recommendations and the final document were adjusted based on the discussion from the general participants Recommendations (grade of evidence) were graded according to the 1998 Karolinska Stroke Update meeting with regard to the strength of evidence. Grade A Evidence: Strong support from randomised controlled trials and statistical reviews (at least one randomised controlled trial plus one statistical review). Grade B Evidence: Support from randomised controlled trials and statistical reviews (one randomised controlled trial or one statistical review). Grade C Evidence: No reasonable support from randomised controlled trials, recommendations based on small randomised and/or non-randomised controlled trials evidence.
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| 4. |
- Anandapadamanaban, Madhanagopal, et al.
(author)
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E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2
- 2019
-
In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 294:30, s. 11404-11419
-
Journal article (peer-reviewed)abstract
- The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-angstrom crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called linchpin residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.
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| 5. |
- Anandapadamanaban, Madhanagopal
(author)
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Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
- 2015
-
Doctoral thesis (other academic/artistic)abstract
- Virtually every aspect of the cellular processes in eukaryotes requires that the interactions between protein molecules are well coordinated in different regulatory pathways. Any protein dysfunction involved in these regulatory pathways might lead to various pathological conditions. Understanding the structural and functional peculiarities of these proteins molecular machineries will help in formulating structure-based drug design.The first regulatory process studied here is the RNA polymerase-II mediated transcription of the eukaryotic protein-coding genes to produce mRNAs. This process requires the formation of the ‘transcription initiation’ by the assembly of Pre-Initiation Complex (PIC) formation at a core promoter region. Regulation at this initiation level is a key mechanism for the control of gene expression that governs cellular growth and differentiation. The transcription Factor IID (TFIID) is a conserved multiprotein general transcription factor with an essential role in nucleating the PIC formation, composed of TATA Binding Protein (TBP) and about 14 TBP Associated Factors (TAFs). The here presented crystal structure (1.97Å) of TBP bound to TAND1 and TAND2 domains from TAF1 reveals a detailed molecular pattern of interactions involving both transcriptionally activating and repressing regions in TBP, thereby uncovering central principles for anchoring of TBP-binding motifs. Together with NMR and cellular analysis, this work provides the structural basis of competitive binding with TFIIA to modulate TBP in promoter recognition.In eukaryotes, another fundamental mechanism in the regulation of cellular physiology is the posttranslational modification of substrate proteins by ubiquitin, termed ‘ubiquitination’. Important actors in this mechanism are the ubiquitin-ligases (E3s) that culminate the transfer of ubiquitin to the substrate and govern the specificity of this system. One E3 ligase in particular, TRIM21, defines a subgroup of the Tripartite Motif (TRIM) family, which belongs to the major RING-type of E3 ubiquitin ligases, and plays an important role in pathogenesis of autoimmunity by mediating ubiquitination of transcription factors. The crystal structure (2.86Å) of the RING domain from TRIM21 in complex with UBE2E1, an E2 conjugating enzyme, together with the NMR and SAXS analysis as well as biochemical functional analysis, reveals the molecular basis for the dynamic binding interfaces. The TRIM21 mode of ubiquitin recognition and activation for catalytic transfer of ubiquitin can be modeled onto the entire TRIM family.Finally, we explored the concepts of conformational selection in proteins as a possible key component for protein-mediated transcriptional regulation. In this framework, MexR, a bacterial repressor of the MexAB-OprM efflux pump, and its mutant Arg21Trp were studied as an example for proteins presenting different conformations. The residue Arg21Trp mutation is clinically identified to cause of Multi-Drug Resistant (MDR) by attenuated DNA binding, and leads to the overexpression of the MexAB-OprM efflux pump. With the crystal structure (2.19Å) of MexR mutant Arg21Trp, in combination with MD-simulations and SAXS for both wild-type and mutant, we could unravel the atomic details of the wild-type conformations consisting in subsets of populations of DNA bound and unbound forms. Remarkably, the mutant Arg21Trp stabilize the DNA unbound state and shifts MexR in a pre-existing equilibrium, from a repressed to a derepressed state.Taken together, these studies substantially broaden our knowledge at a molecular level in protein interactions that are involved in transcriptional regulation and ubiquitination, studied by a carefully selected combination of complementary structural methods spanning different resolutions and time scales.
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| 6. |
- Anandapadamanaban, Madhanagopal, et al.
(author)
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Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
-
Other publication (other academic/artistic)abstract
- TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM211- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.
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| 7. |
- Anandapadmanaban, Madhanagopal, et al.
(author)
-
High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation
- 2013
-
In: Nature Structural & Molecular Biology. - : NATURE PUBLISHING GROUP, 75 VARICK ST, 9TH FLR, NEW YORK, NY 10013-1917 USA. - 1545-9993 .- 1545-9985. ; 20:8, s. 1008-
-
Journal article (peer-reviewed)abstract
- The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.
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| 8. |
- Anandapadmanaban, Madhanagopal, et al.
(author)
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Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression
- 2016
-
In: Structure. - : CELL PRESS. - 0969-2126 .- 1878-4186. ; 24:8, s. 1311-1321
-
Journal article (peer-reviewed)abstract
- MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.
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| 9. |
- Andrésen, Cecilia, et al.
(author)
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Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
- 2010
-
In: Protein Science. - : Cold Spring Harbor Laboratory Press. - 0961-8368 .- 1469-896X. ; 19:4, s. 680-692
-
Journal article (peer-reviewed)abstract
- The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.
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| 10. |
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| 11. |
- Andrésen, Cecilia, et al.
(author)
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Molecular characterization of the interaction between the disordered c-Myc transactivation domain and the TATA-binding protein (TBP)
-
Other publication (other academic/artistic)abstract
- The proto-oncogene c-myc affects the occurrence, expansion, and evolution of numerous aggressive human cancers, and is often associated with the late-stage and/or poor prognostic disease. Regulation of target gene activity by c-Myc occurs through protein interactions with the c-Myc transactivation domain (TAD) which, in addition to binding the TATA-binding protein (TBP) also recruits a wide variety of co-activators and suppressor proteins. Here, we present a molecular model, based on NMR, X-ray crystallography and SPR measurements, which describes how the c-Myc TAD binds to TBP. Our model contributes to the understanding of how c-Myc can regulate individual genes as well as entire gene programs.
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| 12. |
- Andrésen, Cecilia
(author)
-
Protein Structure and Interaction in Health and Disease
- 2011
-
Doctoral thesis (other academic/artistic)abstract
- This thesis focuses on protein structure, dynamics and interaction and their relation to human disease. In particular, the biophysical and structural properties of both well-ordered and partially disordered proteins are studied using a range of biophysical techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. Pseudomonas aeruginosa is a human pathogen due to its multidrug resistance (MDR) caused by overexpression of efflux pump systems. This thesis describes how MDR mutations within the MexR repressor of the MexAB-OprM system reduce the DNA affinity by altering its stability with maintained structure. The oncogenic protein c-Myc is involved in many essential biological functions such as cell proliferation, differentiation and apoptosis and is also highly associated with several forms of human cancers, and where the N-terminal domain is regulated by a plethora of protein interactions. In this thesis the intrinsically disordered N-terminal part of c-Myc and its interactions with the proteins Bin1 and TBP are described. Myc binds Bin1 with maintained disorder in a multivalent manner, which may explain why the onco-protein can interact with such a wide range of binding partners. A similarly dynamic interaction is observed for Myc with the TATA-binding protein (TBP). The essential human multidomain glutaredoxin Grx3 is associated with several biological functions such as redox signaling, proliferation and signal transduction. We have solved the structure and analyzed the dynamic properties in the ps-ns and ms time scale for the two N-terminal domains, providing a platform for further analysis of the Grx3 protein and its interactions. Taken together, this thesis emphasizes the importance of joint structural, biophysical and dynamic studies to better understand protein function in health and disease.
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| 13. |
- Andrésen, Cecilia, et al.
(author)
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Structural and dynamic analysis of human glutaredoxin 3
-
Other publication (other academic/artistic)abstract
- Human glutaredoxin (Grx3) is an essential protein associated with biological functions including embryonic development and immune response, and is involved in human disease such as lung, colon cancer and cardiovascular disorder. Grx3 can harbour a [2Fe-2S]2+ cluster and is most likely involved in oxidative stress response. Grx3 consists of an N-terminal thioredoxin-like domain and two additional monothiol glutaredoxin domains, and is thus classified as a multidomain monothiol glutaredoxin. The Grx3 thioredoxin domain lacks both a characteristic active-site and catalytic activity, but is still essential in the yeast homologue and presumably functions together with its monothiol glutaredoxin domains. We have characterised the structures of the two Nterminal domains in Grx3, which have thioredoxin and glutaredoxin folds. We have analysed their dynamic and structural interdependence by analysing NMR relaxation data together with chemical shift changes between isolated and covalently linked domains. We find that although the two domains show interdomain mobility around a semi-flexible linker, there are indications for a preferred interaction surface between the two domains. Millisecond internal dynamics in a suggested ligand binding site in the isolated thioredoxin domain is dampened in the domain pair, suggesting that the two domains mutually affect each other on a profound level. Our results present a platform for further detailed studies of multidomain thioredoxin-glutaredoxin containing proteins, and their function in human cells.
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| 14. |
- Andrésen, Cecilia, et al.
(author)
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Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding
- 2012
-
In: Nucleic Acids Research. - : Oxford University Press (OUP): Policy C / Oxford University Press. - 0305-1048 .- 1362-4962. ; 40:13, s. 6353-6366
-
Journal article (peer-reviewed)abstract
- The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.
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| 15. |
- Andrésen, Cecilia, et al.
(author)
-
Transient structure and intrinsic disorder in the c-Myc transactivation domain and its effects on ligand binding
-
Other publication (other academic/artistic)abstract
- The crucial role of c-Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of c-Myc function. The transactivation domain of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of c-Myc-1-88, where hierarchical phosphorylation of T58 and S62 regulates activation and destruction of the c-Myc protein. By NMR chemical shift analysis, relaxation measurements and NOE analysis, we show that both the MBI region (residues 45-65) and residues 22-33 are transiently structured regions, conserved also in other members of the Myc family. Binding of Bin1-SH3 to c-Myc-1-88 as assayed by NMR and SPR revealed primary binding to the S62 region, but also a dynamically disordered and multivalent complex in which intrinsic disorder of c-Myc-1-88 was retained while releasing transient intramolecular interactions. Our findings describe a novel mode of regulatory recognition of c-Myc that is in agreement with the increasingly recognized capability of intrinsically disordered regions to efficiently mediate transient interactions with a wide range of targets, with important implications towards understanding the unique multifaceted biological functions of c-Myc.
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| 16. |
- Astori, Audrey, et al.
(author)
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ARID1a Associates with Lymphoid-Restricted Transcription Factors and Has an Essential Role in T Cell Development
- 2020
-
In: Journal of Immunology. - : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 205:5, s. 1419-1432
-
Journal article (peer-reviewed)abstract
- Maturation of lymphoid cells is controlled by the action of stage and lineage-restricted transcription factors working in concert with the general transcription and chromatin remodeling machinery to regulate gene expression. To better understand this functional interplay, we used Biotin Identification in human embryonic kidney cells to identify proximity interaction partners for GATA3, TCF7 (TCF1), SPI1, HLF, IKZF1, PAX5, ID1, and ID2. The proximity interaction partners shared among the lineagerestricted transcription factors included ARID1a, a BRG1-associated factor complex component. CUT&RUN analysis revealed that ARID1a shared binding with TCF7 and GATA3 at a substantial number of putative regulatory elements in mouse T cell progenitors. In support of an important function for ARID1a in lymphocyte development, deletion of Aridla in early lymphoid progenitors in mice resulted in a pronounced developmental arrest in early T cell development with a reduction of CD4(+)CD8(+) cells and a 20-fold reduction in thymic cellularity. Exploring gene expression patterns in DN3 cells from Wt and Aridla-deficient mice suggested that the developmental block resided in the DN3a to DN3b transition, indicating a deficiency in beta-selection. Our work highlights the critical importance of functional interactions between stage and lineage-restricted factors and the basic transcription machinery during lymphocyte differentiation.
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| 17. |
- Brauner, A., et al.
(author)
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Open-ended assignments and student responsibility
- 2007
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In: Biochemistry and molecular biology education. - : Wiley. - 1470-8175 .- 1539-3429. ; 35:3, s. 187-192
-
Journal article (peer-reviewed)abstract
- An inquiry-based laboratory course was created in an effort to increase student responsibility in learning and to improve teaching in areas related to molecular medicine. Authentic medical cases with both scientific and clinical aspects formed the basis of a project-oriented course that also included student laboratory work focused on the disease-related proteins. Students used basic biochemical techniques to develop and test hypotheses relating their results to the clinical findings. The course also included patient demonstrations to personalize students' knowledge of case presentations, lectures on basic biochemical principles relevant to the molecular basis of the cases, and seminars by invited guests with expertise in translational medicine. Students developed proposals for future research as part of the final examination. An inquiry matrix was used to evaluate the degree of learning responsibility taken during the course. By allowing for openness in how to explore the case including choice of methods and interpretation of unexpected results, students gained confidence in their ability to solve problems, formulate and test hypotheses, and collaborate with both clinical and non-clinical professionals.
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| 18. |
- Bromley Milton, Maria, et al.
(author)
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Is Pain Intensity Really That Important to Assess in Chronic Pain Patients? A Study Based on the Swedish Quality Registry for Pain Rehabilitation (SQRP)
- 2013
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In: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 8:6
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Journal article (peer-reviewed)abstract
- BackgroundIncorporating the patient's view on care and treatment has become increasingly important for health care. Patients describe the variety of consequences of their chronic pain conditions as significant pain intensity, depression, and anxiety. We hypothesised that intensities of common symptoms in chronic pain conditions carry important information that can be used to identify clinically relevant subgroups. This study has three aims: 1) to determine the importance of different symptoms with respect to participation and ill-health; 2) to identify subgroups based on data concerning important symptoms; and 3) to determine the secondary consequences for the identified subgroups with respect to participation and health factors.Methods and SubjectsThis study is based on a cohort of patients referred to a multidisciplinary pain centre at a university hospital (n = 4645, participation rate 88%) in Sweden. The patients answered a number of questionnaires concerning symptoms, participation, and health aspects as a part of the Swedish Quality Registry for Pain Rehabilitation (SQRP).ResultsCommon symptoms (such as pain intensity, depression, and anxiety) in patients with chronic pain showed great variability across subjects and 60% of the cohort had normal values with respect to depressive and anxiety symptoms. Pain intensity more than psychological symptoms showed stronger relationships with participation and health. It was possible to identify subgroups based on pain intensity, depression, and anxiety. With respect to participation and health, high depressive symptomatology had greater negative consequences than high anxiety.ConclusionsCommon symptoms (such as pain intensity and depressive and anxiety symptoms) in chronic pain conditions carry important information that can be used to identify clinically relevant subgroups.
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| 19. |
- Caporaletti, Francesca, 1990-
(author)
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MYC and MexR interactions with DNA : a Small Angle Scattering perspective
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Protein-DNA complexes govern transcription, that is, the cellular mechanism that converts the information stored in the DNA into proteins. These complexes need to be highly dynamic to respond to external factors that regulate their functions in agreement with what the cells need at that time. Macromolecular X-ray crystallography is very useful for structural studies of large molecular assemblies, but its general application is limited by the difficulties in crystallising highly dynamic and transient complexes. Furthermore, crystal lattices constrain the macromolecular conformation and do not entirely reveal the conformational ensemble adopted by protein-DNA complexes in the solution.Small-Angle X-Ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS) are two complementary techniques known jointly as Small-angle Scattering (SAS). SAS is a powerful tool for analysing the shape and changes of molecules in solution in their native state. It is beneficial if the variability of conformation or disorder complements high-resolution methods such as NMR or crystallography. With SANS, we can explore non-crystallisable protein-DNA complexes in solution without restrictions of artificially symmetrised DNA and limitations of a protein sequence. Neutrons are well-suited probes for studying protein-DNA complexes for the capability of the neutrons to scatter common atoms in biomolecules differentially and can thereby distinguish between hydrogen and deuterium. Together with varying the solvent deuterium ratio, the contrast variation approach can reveal shapes of distinct components within a macromolecular complex.The goal of this thesis is to explore unchartered territories of regulatory protein-DNA interactions by studying such complexes by SAS, with a specific focus on the flexibility of the complexes. In my study of the MexR-DNA complex, I try to elucidate the molecular mechanism by which the MexR repressor regulates the expression of the MexAB-OPrM efflux pump through DNA binding. This pump is one of the multidrug-resistant tools of the pathogen Pseudomonas Aeruginosa (P. Aer.). It can extrude antibacterial drugs from the bacteria enabling them to survive in hostile environments. In the second project, I strive to explore the MYC:MAX:DNA complex. This heterodimer assembly functions as a central hub in cellular growth control by regulating many biological functions, including proliferation, apoptosis, differentiation and transformation. Overexpression or deregulation of MYC is observed in up to 70% of human aggressive cancer forms, including prostate and breast cancers. By combining SAS with biophysical methods, the work presented in this thesis reveals novel information on the shape and dynamics of biomolecular assemblies critical to health and disease.This thesis comprises five chapters, each dealing with a different aspect of the work in those years. The first chapter introduces the reader to the motivations of this research, and it will give the reader a brief state of the art of the two projects. In the second chapter, I will give you all the theoretical instruments to understand better all the methods used in this thesis, I write first to provide an overview regarding the proteins and their capability to bind other macromolecules. I then will exploit the basics of the small-angle technique, focusing on the neutron contrast variation: the fundamental technique used throughout this thesis and the ab-initio modelling.In the third chapter, Methods, I will discuss the SAS measurements and the requirements for the experiments themselves, the procedure for the data reduction and the data processing and analysis to obtain the structural information.The fourth chapter is a summary of the results of the submitted papers and my contributions:Small-angle X-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding modelResolving the DNA interaction of the MexR antibiotic resistance regulatory proteinUpgraded D22 SEC-SANS set-up dedicated to the biology communitySAS studies on the regulation of MYC303:MAX:DNA and MAX:MAX:DNA binding in cancer.
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| 20. |
- Caporaletti, Francesca, 1990-, et al.
(author)
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Small-angle X-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model.
- 2023
-
In: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 122:2, s. 408-418
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Journal article (peer-reviewed)abstract
- In this work, we used Small-angle X-ray and neutron scattering (SAS) to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa (P.aeruginosa) transcriptional regulator MexR, a member of the MarR family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux-proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterisation of both free and DNA-bound MarR proteins. Here, we show how combined neutron and X-ray small-angle scattering (SAS) of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.
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| 21. |
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| 22. |
- Cider, Åsa, 1960, et al.
(author)
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Aquatic Exercise Is Effective in Improving Exercise Performance in Patients with Heart Failure and Type 2 Diabetes Mellitus
- 2012
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In: Evidence-Based Complementary and Alternative Medicine. - : Hindawi Limited. - 1741-427X .- 1741-4288. ; 2012
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Journal article (peer-reviewed)abstract
- Background. Peak oxygen uptake (VO2peak) and muscle function are more decreased in patients with a combination of chronic heart failure (CHF) and type 2 diabetes mellitus (2DM) compared to patients with only one of the conditions. Further, patients with 2DM have peripheral complications that hamper many types of conventional exercises. Aim. To evaluate the efficacy and applicability of eight-week aquatic exercise in patients with the combination of CHF and 2DM. Methods. Twenty patients (four women) with both CHF and 2DM (age 67.4 +/- 7.1, NYHA II-III) were randomly assigned to either aquatic exercise or a control group. The patients exercised for 45 minutes 3 times/week in 33-34 degrees C, swimming pool. Results. The training programme was well tolerated. Work rate (+11.7 +/- 6.6 versus -6.4 +/- 8.1watt, P < 0.001) and VO2peak (+2.1 +/- 0.8 versus -0.9 +/- 1.4 mL.kg(-1) . min(-1), P < 0.001) and walking capacity (P = 0.01) increased significantly in the training group. Muscle function was also significantly improved and Hba1c decreased significantly (P < 0.01) during training, while fasting glucose, insulin, c-peptide, and lipids were unchanged. Training also increased vitality measured by SF-36 significantly (P = 0.05). Conclusion. Aquatic exercise could be used to improve exercise capacity and muscle function in patients with the combination of CHF and 2DM.
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| 23. |
- Cider, Åsa, 1960, et al.
(author)
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Cardiorespiratory effects of warm water immersion in elderly patients with chronic heart failure
- 2005
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In: Clin Physiol Funct Imaging. ; 25:6, s. 313-7
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Journal article (peer-reviewed)abstract
- BACKGROUND: Hydrotherapy might be included in the rehabilitation of patients with chronic heart failure (CHF), but little is known about the acute cardiorespiratory reaction in warm water. The aim of this study was to assess the acute cardiorespiratory effect of immersion in warm water, in a clinical setting, in elderly patients with CHF compared with healthy age and sex matched persons. METHODS: Twelve patients (three females) with CHF, NYHA II-III, age 64 +/- 6 years, and 12 healthy subjects were studied. Cardiorespiratory changes, on land and in a temperature-controlled swimming pool (33-34 degrees C) were assessed during rest and exercise, in a sitting position, using continuous gas analyses. RESULTS: There were no significant differences, land versus water, in carbon dioxide production, total ventilation, respiratory frequency, respiratory exchange ratio, heart rate or blood pressure in either of the groups. A significant difference was found in oxygen uptake, at rest, land versus water in patients with CHF in comparison with healthy subjects (-0.2 +/- 0.4 versus +0.3 +/- 0.6 ml kg(-1) min(-1), P < 0.01). Oxygen kinetics (tau) increased significantly (P = 0.01) in both groups during exercise in water. CONCLUSION: Hydrotherapy was well tolerated and the vast majority of the cardiorespiratory responses, during warm water immersion in a clinical setting, are similar in patients with CHF compared with healthy subjects. However, further larger studies, are needed to better understand the physiological reactions during hydrotherapy.
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| 24. |
- Cider, Åsa, 1960, et al.
(author)
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Hydrotherapy--a new approach to improve function in the older patient with chronic heart failure
- 2003
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In: Eur J Heart Fail. ; 5:4, s. 527-35
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Journal article (peer-reviewed)abstract
- AIMS: Hydrotherapy, i.e. exercise in warm water, as a rehabilitation program has been considered potentially dangerous in patients with chronic heart failure (CHF) due to the increased venous return caused by the hydrostatic pressure. However, hydrotherapy has advantages compared to conventional training. We studied the applicability of an exercise programme in a temperature-controlled swimming pool, with specific reference to exercise capacity, muscle function, quality of life and safety. METHODS AND RESULTS: Twenty-five patients with CHF (NYHA II-III, age 72.1+/-6.1) were randomised into either 8 weeks of hydrotherapy (n=15), or into a control group (n=10). The training program was well tolerated with no adverse events. Patients in the hydrotherapy group improved their maximal exercise capacity (+6.5 vs.-5.9 W, P=0.001), isometric endurance in knee extension (+4 vs.-9 s, P=0.01) together with an improvement in the performance of heel-lift (+4 vs. -3 n.o., P=<0.01), shoulder abduction (+12 vs. -8 s, P=0.01) and shoulder flexion (+6 vs. +4, P=0.01) in comparison to patients in the control group. CONCLUSION: Physical training in warm water was well tolerated and seems to improve exercise capacity as well as muscle function in small muscle groups in patients with CHF. This new approach broadens the variety of training regimes for older patients with CHF.
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| 25. |
- Csizmok, Veronika, et al.
(author)
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Multivalent Interactions with Fbw7 and Pin1 Facilitate Recognition of c-Jun by the SCFFbw7 Ubiquitin Ligase
- 2018
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In: Structure. - : CELL PRESS. - 0969-2126 .- 1878-4186. ; 26:1, s. 28-
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Journal article (peer-reviewed)abstract
- Many regulatory proteins, including the transcription factor c-Jun, are highly enriched in disordered protein regions that govern growth, division, survival, differentiation, and response to signals. The stability of c-Jun is controlled by poorly understood regulatory interactions of its disordered region with both the E3 ubiquitin ligase SCFFbw7 and prolyl cis-trans isomerase Pin1. We use nuclear magnetic resonance and fluorescence studies of c-Jun to demonstrate that multisite c-Jun phosphorylation is required for high-affinity interaction with Fbw7. We show that the Pin1 WW and PPIase domains interact in a dynamic complex with multiply phosphorylated c-Jun. Importantly, Pin1 isomerizes a pSer-Pro peptide bond at the c-Jun N terminus that affects binding to Fbw7 and thus modulates the ubiquitin-mediated degradation of c-Jun. Our findings support the general principle that multiple weak binding motifs within disordered regions can synergize to yield high-affinity interactions and provide rapidly evolvable means to build and fine-tune regulatory events.
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