| 1. |
- Wehlin, Anna, 1994-
(författare)
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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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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 2. |
- Anandapadamanaban, Madhanagopal
(författare)
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Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 3. |
- Johansson-Åkhe, Isak, 1993-
(författare)
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Development and Application of Computational Models for Peptide-Protein Complexes
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein-protein interactions between a protein and a smaller protein fragment or a disordered segment of a protein are called peptide-protein interactions. Such interactions are commonplace in nature and vital for normal cell function in humans. For example, the onco-protein Myc con- tains a large disordered region with several segments involved in peptide-protein interactions as part of transcription regulation, and it is mis-regulated in the vast majority of all human can- cers. As such, understanding the structural details of peptide-protein interactions on an atomic level is a necessary endeavor for understanding disease pathways as well as facilitating targeted drug-design. While experimental methods for structure determination such as X-ray crystallography and NMR can determine the structure of many peptide-protein complexes, these methods are time- consuming and costly. Additionally, the disordered nature of peptides and a sometimes lower binding affinity than for protein-protein binding can lead to transient or weak (but still highly specific) interactions impossible to fully capture with experimental methods. This leads to the need for computational methods as support and complement. Such methods have classically used statistical potentials or simple template search approaches, but as the number of deposited structures in the protein databank (PDB) grows so does the potential for supervised machine learning. The papers in this thesis present the contributions of the author to the field of peptide-protein in- teraction complex prediction, mainly through use of machine learning models. The first papers apply a Random Forest classifier to detect similarities between binding interfaces deposited in the PDB and a peptide-protein pair being investigated to find the optimal templates for struc- ture prediction. In excess of producing predictions with good self-evaluation of performance, the development of the method also confirmed theories on the similarity of protein-protein, domain-domain, and peptide-protein interfaces. Two more method for peptide-protein docking are presented in later papers. One utilizes graph convolution neural networks to improve model selection from rigid-body-docking methods by including MSA profile information as a feature, which also lead to the discovery that while profile information such as position conservation does improve predictive performance, something also seen in the first papers, the most impor- tant features are the ones describing the structural details of the complex and the bonds between residues. The other uses a graph neural network as an additional scoring term to improve upon the already state-of-the-art performing local refinement method FlexPepDock, and is capable of refining even models generated by AlphaFold-multimer. Finally, two manuscripts focus on the application of computational approaches for research into the interactions of human cMyc with TBP and PPP1R10, respectively. In the first of these pa- pers, the template-based peptide-protein complex prediction methods developed in the earlier papers of the thesis are employed together with prior knowledge of the interaction to model the complex to a high degree of certainty not achievable by NMR alone. In the second of these papers, experimental data is used as a basis for computational modeling of the complex, and the modeled complex could act as a basis for further experiments characterizing the interaction.
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| 4. |
- Oanh, Ho Ngoc Hoang, 1986-
(författare)
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Regulation of the multi-functional protein YscU in assembly of the Yersinia type III secretion injectisome
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Yersinia pseudotuberculosis is a Gram-negative zoonotic pathogenic bacterium causing gastroenteritis in human and animals. It shares a conserved virulence plasmid encoding for a needle-like secretion machinery, or type III secretion system, which can be found in other pathogenic Gram-negative bacteria. The type III secretion system (T3SS) is a macromolecular assembly that enables pathogenic effector proteins (or Yersinia outer proteins, Yops) to be transported into eukaryotic host cells. This export machinery is assembled in a highly ordered stepwise mechanism. The activation of T3SS is also dependent on calcium concentration, temperature, and pH of the growth media as mimic factors for host cell’s contact. The T3SS-associated inner-membrane protein, YscU, of Yersinia is proposed to function as a substrate specificity switch protein and forms basal structure of T3SS. YscU has four α helical transmembrane domain and a soluble cytoplasmic domain YscUC which undergoes auto-proteolysis at a conserved N↑PTH motif. The auto-proteolysis process, which is required for the assembly of the injectisome and secretion of Yops, results in a 10-kDa C-terminal polypeptide fragment, denoted YscUCC and 6-kDa N-terminal fragment YscUCN. In this thesis, we showed that YscUC dissociation was important for Yops secretion and resulted in unfolded YscUCN and oligomeric YscUCC. By combination in vivo and in vitro methods, growth media conditions as calcium, temperature, and pH were indicated to control secretion by regulation of YscUC dissociation. The calcium-binding isotherm to YscUC was fit best with a one-site binding model resulting in Kd 800 µM, which is identical to calcium level that blocks secretion in vivo. YscU is also the key protein for the T3SS pH dependence, demonstrated by thermal unfolding profile and secondary structure of protein were altered between pH 7.4 and 6.0. In addition, bacterial inner membrane was proposed to assist the YscUCN folding, monitored by using lipid bilayer as a mimic environment in nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. This binding is important for Yops secretion and YscUC is anchored to bacterial membrane upon dissociation. The other substrate specificity switch protein YscP has function as a “molecular ruler” controlling length of the secretion needle. Previous genetic experiments have suggested that YscP and YscU interact physically, when mutation at defined residues on yscU (suppressor mutants) rescued Yops secretion in null-yscP mutant. In this research, direct binding of YscU and YscP was proved as weak but important interaction with Kd 430 mM by application of NMR and the binding interface of YscP was centred on the last helix of YscUC. Furthermore, we found that the YscP interaction could inhibit YscU auto-proteolysis. Studying the dissociation kinetic of suppressor YscUC variants at temperature 30 and 37oC provides strong support to a model where YscU is a temperature sensor for T3SS and YscUC dissociation is required for Yops secretion. Interestingly, the NPTH motif is conserved through most of YscU family members, meaning that role of dissociation may be conserved also in other bacterial injectisomes. To this end, the dissociation of YscU can be used as a therapeutic target in drug discovery. We attempted to identify the small-molecules that can hinder YscU dissociation. The small compound methyl(5-methyl-2-phenyl-1,3-thiazolidin-4-yl)acetate was found to be able to inhibit dissociation and to crystalize full YscUC, which has never been successfully done before. Finally, we found that the inner-rod protein YscI is binding to YscUC with a 1:1 stoichiometry as shown with pull-down assays and isothermal titration calorimetry. Taken together we have made several discoveries that expand the functional palette of YscU and all these functions were shown to have biological relevance with Yops secretion levels. In light of the strong sequence conservation between T3SS utilizing pathogenic bacteria the findings are likely to be general characters.
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| 5. |
- Pietras, Zuzanna, 1993-
(författare)
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Small angle scattering as a tool to study protein structure and interactions
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 6. |
- Pilstål, Robert, 1985-
(författare)
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On protein structure, function and modularity from an evolutionary perspective
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We are compounded entities, given life by a complex molecular machinery. When studying these molecules we have to make sense of a diverse set of dynamical nanostructures with wast and intricate patterns of interactions. Protein polymers is one of the major groups of building blocks of such nanostructures which fold up into more or less distinct three dimensional structures. Due to their shape, dynamics and chemical properties proteins are able to perform a plethora of specific functions essential to all known cellular lifeforms.The connection between protein sequence, translated into protein structure and in the continuation into protein function is well accepted but poorly understood. Malfunction in the process of protein folding is known to be implicated in natural aging, cancer and degenerative diseases such as Alzheimer's.Protein folds are described hierarchically by structural ontologies such as SCOP, CATH and Pfam all which has yet to succeed in deciphering the natural language of protein function. These paradigmatic views centered on protein structure fail to describe more mutable entities, such as intrinsically disordered proteins (IDPs) which lack a clear defined structure.As of 2012, about two thirds of cancer patients was predicted to survive past 5 years of diagnosis. Despite this, about a third do not survive and numerous of successfully treated patients suffer from secondary conditions due to chemotherapy, surgery and the like. In order to handle cancer more efficiently we have to better understand the underlying molecular mechanisms.Elusive to standard methods of investigation, IDPs have a central role in pathology; dysfunction in IDPs are key factors in cellular system failures such as cancer, as many IDPs are hub regulators for major cell functions. These IDPs carry short conserved functional boxes, that are not described by known ontologies, which suggests the existence of a smaller entity. In an investigation of a pair of such boxes of c-MYC, a plausible structural model of its interacting with Pin1 emerged, but such a model still leaves the observer with a puzzle of understanding the actual function of that interaction.If the protein is represented as a graph and modeled as the interaction patterns instead of as a structural entity, another picture emerges. As a graph, there is a parable from that of the boxes of IDPs, to that of sectors of allosterically connected residues and the theory of foldons and folding units. Such a description is also useful in deciphering the implications of specific mutations.In order to render a functional description feasible for both structured and disordered proteins, there is a need of a model separate from form and structure. Realized as protein primes, patterns of interaction, which has a specific function that can be defined as prime interactions and context. With function defined as interactions, it might be possible that the discussion of proteins and their mechanisms is thereby simplified to the point rendering protein structural determination merely supplementary to understanding protein function.
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| 7. |
- Andrésen, Cecilia
(författare)
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Protein Structure and Interaction in Health and Disease
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 8. |
- Caporaletti, Francesca, 1990-
(författare)
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MYC and MexR interactions with DNA : a Small Angle Scattering perspective
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 9. |
- Helander, Sara, 1981-
(författare)
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Structural biology of transcriptional regulation in the c-Myc network
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The oncogene c-‐Myc is overexpressed in many types of human cancers and regulation of c-‐Myc expression is crucial in a normal cell. The intrinsically disordered N-‐terminal transactivation domain interacts with a wide range of proteins regulating c-‐Myc activity. The highly conserved Myc box I region includes residues Thr58 and Ser62, which are involved in the phosphorylation events that control c-‐Myc degradation by ubiquitination. Aggressive cell growth, leading to tumor formation, occurs if activated c-‐ Myc is not degraded by ubiquitination. Such events may be triggered by defects in the regulated network of interactions involving Pin1 and phospho-‐dependent kinases.In this thesis, the properties of the intrinsically disordered unphosphorylated c-‐Myc1-‐88 and its interaction with Bin1 are studied by nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR). Furthermore, the interaction of Myc1-‐88 with Pin1 is analyzed in molecular detail, both for unphosphorylated and Ser62 phosphorylated c-‐Myc1-‐88, providing a first molecular description of a disordered but specific c-‐Myc complex. A detailed analysis of the dynamics and structural properties of the transcriptional activator TAF in complex with TBP, both by NMR spectroscopy and crystallography, provides insight into transcriptional regulation and how c-‐Myc could interact with TBP. Finally, the structure of a novel N-‐terminal domain motif in FKBP25, which we name the Basic Tilted Helix Bundle (BTHB) domain, and its binding to YY1, which also binds c-‐Myc, is described. By investigating the structural and dynamic properties of c-‐Myc and c-‐Myc-‐interacting proteins, this thesis thus provides further insight to the molecular basis for c-‐Myc functionality in transcriptional regulation.
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| 10. |
- Hennig, Janosch, 1977-
(författare)
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Structure-function studies on TRIM21/Ro52, a protein involved in autoimmune diseases
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Several members of the tripartite motif (TRIM) protein family are involvedin antiviral activity and immunity and have been linked to severaldiseases. TRIM21, the main object of this thesis, is involved in Sjögrensyndrome (SS) and systemic lupus erythematosus (SLE), where patientsoften have autoantibodies against different epitopes on TRIM21. Duringthe course of this study a role of TRIM21 in regulation of proinflammatorycytokines and autoimmunity emerged. The aim of this thesis is to providea better understanding of the structure-function relationship of TRIM21.A conformational epitope in the coiled-coil domain of TRIM21 has beencharacterized, whose autoantibodies cause congenital heart block. A widerange of biophysical methods were employed to establish a model of theprotein domain arrangement of TRIM21, and functional implications werederived. By sequence comparisons, TRIM proteins were classified into threesubgroups, sharing a conserved amphipathic helix in the region, linkingthe conserved N-terminal Zn2+-binding domains RING and B-box, calledthe RING-B-box linker (RBL). A structural dependence of this region on theRING has been observed and a model of the RING-RBL was derived frombioinformatics and proteolysis data. Anti-RING-RBL antibodies inhibit theE3 ligase activity of TRIM21 in ubiquitination. Interferon regulatory factors(IRFs), the substrate for TRIM21-dependent ubiquitination could thereforeretain their high cellular levels after stress-induced inflammation, increasingthe susceptibility to SS and SLE. According to NMR data, the antibodiesbind to the Zn2+-binding loop regions of the RING, which usually bind tothe E2 conjugating enzyme. Antibodies against the C-terminus of the RBLregion do not inhibit the E3 ligase activity.
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| 11. |
- Salomonsson, Johannes, 1991-
(författare)
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Structural, Dynamic and Functional Studies of USP14 : A Proteasome-Associated Deubiquitinating Enzyme Regulating Protein Degradation
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 12. |
- Wallenhammar, Amélie
(författare)
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Protein modularity : Structure and interactions by NMR and SPR
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein degradation is a fundamental cornerstone of regulation of protein levels in the human body. For proper function, numerous molecular processes rely on fine-tuned communication through specific interactions between proteins. Tagging proteins by a small protein ubiquitin, called ubiquitination, is mediated by set of ubiquitin-conjugating and transferring enzymes with ubiquitin as well as substrates. This process enables specific selection of substrate modifications, which is directly coupled to biological activity.In this work, the E3 ligase enzyme TRIM21, a member of the TRIM (TRIpartite Motif) protein family, and its interactions are explored. First, the three-dimensional solution structure of the TRIM21 B-box2 domain solved by nuclear magnetic resonance (NMR) spectroscopy is presented. The structure reveals how different surfaces of the B-box motif are employed for various modular interactions. Using NMR titration experiments, an exposed interaction surface is identified, described as a novel interaction patch where the B-box2 is likely to bind the RING domain. Taken together, this work establishes an extended understanding of the structural role of the B-box2 domain, and how this domain is related to flanking domains in multimodular TRIM proteins.Our crystal structure of TRIM21-UBE2E1 complex reveals the specific E2 recognition, which is conserved in other TRIMs. Additionally, solution scattering provides further information on the complexes and also confirms that TRIM21 RING1-91 is predominantly dimer. We also understand how the acceptor ubiquitin can be transferred to Lysine residue of TRIM211-91.Another highly regulated system is the RNA polymerase-II transcription, which is the first step for all protein synthesis. TATA-box Binding Protein (TBP) is an essential subunit for initiating the gene transcription, which is regulated by number of other transcription factors. Among these, the oncoprotein c-Myc regulates the transcription by directly mediating its interaction with TBP. The interplay of these interaction and competing binding of TBP and c-Myc is studied in this thesis.Conclusively, this thesis highlights TRIM molecular recognition and its interface with E2, and also provides the first glimpse of interaction between TBP and c-Myc.
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