38 research outputs found

    Exploring Humoral Immune Responses by Mass Spectrometry: Resolving Structures, Interactions, and Clonal Repertoires of Antibodies

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    In his thesis “Exploring Humoral Immune Responses by Mass Spectrometry”, Maurits den Boer uses mass spectrometry to shed new light on antibody responses. Antibodies play a crucial role in the immune protection against threats like bacteria, viruses, and cancers. When valuable antibodies are discovered, they can therefore be reproduced for use as a medicine. A better understanding of their structures, interactions, and repertoires is therefore key to finding novel treatments for many diseases. In the first part of his thesis, Maurits and coworkers used mass spectrometry to study antibody structures and interactions, leading to two major findings. They first uncovered a mechanism by which Staphylococcus aureus bacteria can evade antibody responses, and how this mechanism may be circumvented in future therapies. Second, he redefined the textbook structure of circulating IgM antibodies by showing that they are universally attached to an extra protein. This may have major implications for how these antibodies function, and their use as therapeutics. In a second line of research, Maurits focused on the development of innovative techniques for antibody repertoire analysis and discovery. Together with coworkers, he explored the use of electron-based fragmentation mass spectrometry, developing methods to obtain valuable pieces of antibody sequence information. Finally, he combined multiple layers of mass spectrometry analysis to discover and fully determine the sequence of a malignant patient antibody. Combined, this demonstrates the promise of mass spectrometry as a compelling new approach for therapeutic antibody discovery

    Development and Application of Software to Understand 3D Chromatin Structure and Gene Regulation

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    Nearly all cells contain the same 2 meters of DNA that must be systematically organized into their nucleus for timely access to genes in response to stimuli. Proteins and biomolecular condensates make this possible by dynamically shaping chromatin into 3D structures that connect regulators to their genes. Chromatin loops are structures that are partly responsible for forming these connections and can result in disease when disrupted or aberrantly formed. In this work, I describe three studies centered on using 3D chromatin structure to understand gene regulation. Using multi-omic data from a macrophage activation time course, we show that regulation temporally precedes gene expression and that chromatin loops play a key role in connecting enhancers to their target genes. In the next study, we investigated the role of biomolecular condensates in loop formation by mapping 3D chromatin structure in cell lines before and after disruption of NUP98-HOXA9 condensate formation. Differential analysis revealed evidence of CTCF-independent loop formation sensitive to condensate disruption. In the last study, we used 3D chromatin structure and multi-omic data in chondrocytes to link variant-gene pairs associated with Osteoarthritis (OA). Computational analysis suggests that a specific variant may disrupt transcription factor binding and misregulate inflammatory pathways in OA. To carry out these analyses I built computational pipelines and two R/Bioconductor packages to support the processing and analysis of genomic data. The nullranges package contains functions for performing covariate-matched subsampling to generate null-hypothesis genomic data and mitigate the effects of confounding. The mariner package is designed for working with large chromatin contact data. It extends existing Bioconductor tools to allow fast and efficient extraction and manipulation of chromatin interactions for better understanding 3D chromatin structure and its impact on gene regulation.Doctor of Philosoph

    Practical Applications of NMR to Solve Real-World Problems

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    Nuclear magnetic resonance spectroscopy (NMR) has developed from primarily a method of academic study into a recognized technology that has advanced measurement capabilities within many different industrial sectors. These sectors include areas such as national security, energy, forensics, life sciences, pharmaceuticals, etc. Despite this diversity, these applications have many shared technical challenges and regulatory burdens, yet interdisciplinary cross-talk is often limited. To facilitate the sharing of knowledge, this Special Issue presents technical articles from four different areas, including the oil industry, nanostructured systems and materials, metabolomics, and biologics. These areas use NMR or magnetic resonance imaging (MRI) technologies that range from low-field relaxometry to magnetic fields as high as 700 MHz. Each article represents a practical application of NMR. A few articles are focused on basic research concepts, which will likely have the cross-cutting effect of advancing multiple disciplinary areas

    Atherosclerosis: Methods and Protocols

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    This volume provides detailed, up-to-date methods used in research on Atherosclerosis. Chapters guide readers through an overview of the pathogenesis of atherosclerosis and model systems together with in vitro, ex vivo, in vivo and emerging methods in atherosclerosis research. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Atherosclerosis: Methods and Protocols serves as an invaluable resource for those engaging in research on atherosclerosis and cardiovascular disease, as well as for researchers who are new to t

    La spectrométrie de masse : un couteau suisse pour disséquer la structure et la fonction du spermatoprotéasome

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    Le protéasome est le complexe enzymatique protéolytique principal de la cellule. Son cœur catalytique (20S) est formé de quatre anneaux heptamériques. Son activité et sa spécificité de substrat peuvent être régulées par les complexes 19S, PA28alphaß, PA28gamma et PA200 ainsi que par des sous-unités 20S alternatives. La spermatogenèse est un processus de différenciation des cellules germinales mâles: les spermatogonies (SPG) se transforment en spermatocytes (SPC), en spermatides (SPT) puis en spermatozoïdes. Ce processus requière une protéolyse intense. Le spermatoprotéasome (spt20S) est spécifique des gamètes en développement et essentiel à la spermatogenèse. Il diffère du protéasome standard (std20S) par la sous-unité alpha4s qui remplace la sous-unité constitutive alpha4. Le spt20S joue un rôle important avec PA200 dans la progression de la méiose, mais les mécanismes qui le rendent différent du std20S restent inconnus. Nous avons établi des stratégies protéomiques complémentaires pour caractériser les complexes du protéasome immunopurifiés à partir de testicules. L'analyse Top-Down de protéasome purifié, nous a permis de montrer pour la première fois qu'alpha4s et alpha4 portent les mêmes MPTs. La protéomique Bottom-Up, nous a permis de comparer les immunopurifications (IPs) de protéasomes totaux avec celles obtenues avec un anticorps spécifique du stp20S que nous avons développé. Nous avons établi qu'alpha4 et alpha4s ne coexistent pas dans le même 20S, bien qu'ils soient presque également abondants dans les testicules. Nous avons également trouvé plus de 19S et de PA200 liés au spt20S qu'au std20S. Les autres protéines préférentiellement associées au spt20S incluent PI31 et Fbxo7 qui sont cruciales pour la fertilité et d'importants médiateurs du transport du protéasome et de l'ancrage des E3 ligases - deux processus qui semblent cruciaux pour la fonction du spt20S pendant la spermatogenèse. Nous avons ensuite obtenu des cellules germinales à différents stades de la différenciation et l'analyse protéomique des lysats ainsi que des IPs, nous a permis d'établir un interactome dynamique du protéasome tout au long de la spermatogenèse. Nous avons observé un changement total du std20S au spt20S entre les SPG pré-méiotiques et les SPC/SPT méiotiques et post-méiotiques. Un changement d'expression semble responsable, plutôt qu'une incorporation préférentielle d'alpha4s. En entrant dans la méiose, l'association de PA200 avec le protéasome a augmenté 7 fois, confirmant son importance dans le développement des gamètes. Bien que PA200 soit d'après la littérature le principal activateur du spt20S, nous montrons que le 19S est est en réalité majoritaire, lié à 60% des 20S dans les SPC - une activation du protéasome sans précédent. De nombreux partenaires du spt20S sont identifiés à la fois dans les cellules germinales et dans les testicules entiers, montrant la robustesse de nos méthodes. Ceux-ci incluent des protéines synaptonémales, de nombreuses protéines impliquées dans l'ubiquitylation, le cycle cellulaire et la progression méiotique ainsi que le transport cellulaire, en accord avec les fonctions du spt20S proposées dans la littérature. Le passage d'alpha4 à alpha4s semble crucial pour la méiose, mais quelles sont les bases moléculaires de cette transition ? L'échange hydrogène-deutérium nous a permis de montrer pour la première fois que les deux protéasomes présentent des interfaces d'interaction différentes : alpha4s contient des régions plus flexibles qu'alpha4. Cette découverte est confirmée par des pull-down in vitro montrant que le 19S se lie plus fortement au spt20S qu'au std20S, expliquant la hausse d'activité protéolytique pendant la méiose. L'activité trypsique du spt20S est plus élevée que celle du std20S in vitro, ce qui pourrait refléter la nécessité de dégradation des histones. Globalement, nos données révèlent un processus de régulation du spt20S qui est plus complexe que ce qui avait été suggéré précédemment et jettent les bases des différences structurales et fonctionnelles entre le spt20S et le std20S.The proteasome is the main enzymatic complex for targeted proteolysis in the cell. Its core complex (20S) consists of four stacked heptameric rings and requires activator complexes: 19S, PA28alphaß, PA28gamma and PA200, which regulate 20S activity and substrate specificity. Alternative 20S subunits exist to further modulate the proteasome activity. Spermatogenesis is a process of male germ cell differentiation, where spermatogonia (SPG) transform through spermatocyte (SPC), then spermatid (SPT) stages, to become spermatozoa. This process requires intense proteolysis. The spermatoproteasome (spt20S) is specific to the developing gametes and essential for spermatogenesis. It differs from the standard proteasome (std20S) by only one subunit - alpha4s, which replaces the constitutive alpha4 subunit. Together with PA200, the spt20S plays an important role in meiosis progression, however, the mechanisms that make it different compared to std20S remain unknown. We established complementary proteomic pipelines for characterisation of proteasome complexes in the testes, combining immunopurification (IP) and mass spectrometry (MS) analysis. Our Top-Down analysis of purified proteasome, showed for the first time that both alpha4 and alpha4s carry the same PTMs. Using Bottom-Up proteomics we compared the interactome of total proteasomes with that of the spt20S only, obtained using a specific antibody we developed for this purpose. We established that alpha4 and alpha4s do not co-exist in the same 20S, although they are almost equally abundant in the testes.  We also measured that 19S and PA200 regulators were bound in higher ratios to the spt20S compared to std20S. Among other preferentially-associated proteins of spt20S were PI31 and Fbxo7, both shown to be crucial for fertility. They mediate proteasome transport and docking of E3 ligases - both processes that could be crucial for spt20S function. We then obtained germ cells at different differentiation stages and performed a proteomics analysis of both lysates and IP-ed proteasome complexes, to establish a dynamic image of the proteasome throughout spermatogenesis. We observed a complete shift from std20S to spt20S between pre-meiotic SPG and meiotic and post-meiotic SPC and SPT cells. We explained this by a shift in expression, rather than preferential incorporation of alpha4s. Upon entering meiosis, the PA200 association with core proteasome increased 7-fold, marking its importance in gamete development. Although PA200 was represented in literature as the main spt20S interactor, we show that 19S was undoubtedly stoichiometrically dominant, occupying 60% of the existing 20S in SPCs - an unprecedented proteasome activation. Identified spt20S-interacting proteins largely correlated with previous interactome analysis on the whole testes, showing robustness of our methods. We identified synaptonemal proteins bound exclusively to spt20S and numerous proteins involved in ubiquitylation, cell cycle and meiotic progression as well as cellular transport, which fits the current model of spt20S role, proposed by earlier work. The shift from alpha4 to alpha4s in meiosis was shown to be crucial, but what is the molecular basis for this transition? The hydrogen-deuterium exchange experiment coupled to MS helped us to show for the first time that the two proteasomes exhibit different binding interfaces: alpha4s contains regions that are more flexible compared to alpha4. We further supported this finding with pull-down assays, which showed that 19S binds more strongly to the spt20S than to std20S, which would explain the increase in proteolytic activity required during meiosis. The spt20S showed a higher tryptic activity compared to the std20S in vitro, which might reflect a particular need for histone degradation. Altogether, our data reveal a more complex process of spt20S regulation than previously suggested and set the basis for structural and functional differences between the spt20S and std20S

    Design, Optimization and Syntheses of Small Molecules to Disrupt Protein-Protein Interactions

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    Protein-protein interactions (PPIs) are one of the basic mechanisms in cellular biology, but also involve in diseases if they are dysregulation. Disrupting aberrant PPI activities is useful in medicinal chemistry. One approach to inhibit PPIs is to design small molecule peptidomimetics bearing side-chain orientations similar to protein ligands, in which those mimics might displace or interfere the native PPIs. Previous research in our group developed Exploring Key Orientations (EKO) program that matches Cɑ-Cß coordinates of virtual small molecules to the side-chain vectors of proteins at PPI interfaces. Similar Cɑ-Cß orientations between mimics and protein ligands indicate that small molecules might be suitable to displace protein ligands, i.e. those compounds might interfere PPIs. We used EKO to deduce small molecules that might disrupt medicinally-relevant PPIs. Herein, EKO implicated our designed mimics, hydantoin-oxazoline, triazole-oxazole and triazole-oxazoline derivatives, might disrupt Nef•MHC-I•AP1 and NEDD8•NAE interactions, in which they are relevant to HIV-1 and cancer diseases respectively. After learning from these projects, we designed hydantoin-piperazine analogues to disrupt PCSK9•LDLR interaction that causes hypercholesterolemia disease. Although the firstgeneration hydantoin-piperazine derivatives did not show good PCSK9•LDLR inhibition, we modified chemotype structures by cooperating with a docking program, Glide, to improve inhibitory potencies. As a result, we successfully obtained lead compounds that significantly disrupt PCSK9•LDLR interaction with the measurable binding affinities. Besides these protein targets, we synthesized another minimalist mimic, oxazoline piperidine-2,4-dione, that has conformational biases toward helical and sheet-turn-sheet motifs. This structure potentially has favorable cellular- and oral-permeability calculated by QikProp. We are also interested in how to design molecules suitable for PPI inhibition. A concept of secondary structure mimicry is widely applied to design molecules that resemble a secondary structure at an PPI interface, hence possibly disrupt protein-protein interaction. However, there is no direct study to prove a correlation between secondary structure mimicry and interface mimicry. To respond this issue, we used EKO to match several new chemotypes on the ideal secondary structures and PPIs database, and then compared the frequencies of secondary structures that chemotypes matched at PPI interfaces to the ideal secondary structure biases of each chemotype. We found that, in general, good secondary structure mimics tend to match frequently at PPI interfaces; however, they mostly match on non-ideal secondary structure motifs

    The use of computer-aided drug design in small molecule drug discovery

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    Drug discovery is one of the most challenging research fields that contributes to the birth of novel drugs for therapeutic use. Due to the complexity and intricate nature of the research, lengthy processes are involved in identifying potential hit molecules for a therapeutic target. To shorten the time required to reach the hit-to-lead stage, computer-aided drug design (CADD) has been used to expedite the process and reduce laboratory expenses. Common strategies used within CADD involve structure-based drug design (SBDD) and ligand-based drug design (LBDD). Both strategies were used extensively in two projects showing the complementarity of each strategy throughout the process. In this work, two separate drug discovery projects are detailed: Design, synthesis and molecular docking study of novel tetrahydrocurcumin analogues as potential sarcoplasmic-endoplasmic reticulum calcium ATPases (SERCA) inhibitors – details the identification, synthesis and testing of potential hit candidate(s) targeting SERCA by using SBDD Filamenting temperature-sensitive mutant Z (FtsZ) as therapeutic target in ligand-based drug design – details the identification, synthesis and testing of potential hit molecule(s) targeting FtsZ In the first project, homology modelling and virtual compound library screening were utilised as the SBDD methods to identify potential hit molecules for testing in P-type calcium ATPases such as SERCA. Preliminary results have found compound 20, an analogue of tetrahydrocurcumin, to show some SERCA inhibitory effect at 300µM based on a SERCA-specific calcium signalling assay performed via fluorometric imaging plate reader. Molecular docking study has also reflected this outcome with desirable ligand-protein binding energies found for 20 when compared with other tested ligands. Pharmacophore screening was used as the main LBDD method in the second project to identify probable hit candidates targeting FtsZ. Potential ligands were synthesised, and tested for antibacterial effect in Bacillus Subtilis strain 168 (Bs168) and Streptococcus pneumoniae strain R6 (SpnR6) cells. One of the tetrahydrocurcumin analogues, compound 4, was found to have minimum inhibitory concentration (MIC) ≤ 10 µM in Bs168 cells and ≤ 2 µM in spnR6 cells. The IC50 values for 4 were 9.1 ± 0.01 µM and 1 ± 0.01 µM in Bs168 and SpnR6 cells respectively. The MIC of 4 was found to be very similar to the MIC of compound 1, a known hit compound targeting against Bs168 cells. On the other hand, the MIC of 4 was lower than the MIC (> 64 µg/mL) of a well-known FtsZ inhibitor, PC190723, against S. pneumoniae. Subsequent molecular docking analyses were completed to evaluate the ligand-protein binding energies to correlate against the testing results. Both compounds 20 and 4 possess some structural similarities and differences that may confer their different effects in these protein targets, which render both with potentials to become the next lead molecules for future development
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