Disseksjon av vitellogenins molekylære egenskaper : et protein med funksjoner i grenseflaten mellom sosial adferd og aldring

Vitellogenin is a central honey bee (Apis mellifera) life-history regulator. This thesis represents an initiative to study this protein, which affects aging and task-division of the bees, at the molecular level. I have used, among others, molecular modeling, nuclear magnetic resonance spectroscopy and surface plasmon resonance techniques to understand the structure and properties of vitellogenin. In addition to the three experimental papers of this thesis, vitellogenin is discussed from the molecular viewpoint in two invited publications (Papers II-III). The studies have resulted in more detailed understanding of the structural organization of the protein and its modifications: the novel findings include the cleavage of honey bee vitellogenin at a phosphorylated polyserine linker site, and the ability of the protein to bind to membranes and to interact with Escherichia coli. Thus, this vitellogenin study exceeds its primary molecular scope, and provides a new perspective on the protein as a membrane-active factor in bee physiology. Since little is known of the detailed molecular properties of insect vitellogenins in general and honey bee vitellogenin in particular, the thesis also contributes towards expanding vitellogenin molecular biology beyond the more studied vertebrate vitellogenins.Vitellogenin er et sentralt protein i reguleringen av livsutviklingen hos honningbie (Apis mellifera). Dette proteinet påvirker aldring og oppgavefordeling hos bier, og avhandlingen er en studie av proteinet på molekylært nivå. Jeg har blant annet brukt molekylær modellering, kjernemagnetisk resonans spektroskopi og en teknikk for å måle overflateplasmonresonans (surface plasmon resonance) for å forstå strukturen og egenskapene til vitellogenin. I tillegg til de tre eksperimentelle artiklene i denne avhandlingen er vitellogenin diskutert fra et molekylært synspunkt i to inviterte publikasjoner (Papers II-III). Studiene har resultert i en økt forståelse av den strukturelle oppbygning av proteinet og dets modifikasjoner. Blant viktige funn i avhandlingen kan det nevnes at vitellogenin blir spaltet på et fosforylert polyserin-linker sted, at det binder seg til membraner og at det interagerer med Escherichia coli. Denne studiens omfang dekker dermed mer enn det primært molekylære, og gir nye perspektiv på proteiner som en membran-aktiv faktor i biefysiologi. Detaljert kjennskap om de molekylære egenskapene til vitellogenin hos insekter, og hos honningbie spesielt, er mangelfull, og denne avhandlingen gir derfor et bidrag til å utvide kunnskapen om molekylærbiologien til vitellogenin utover de mer studerte vitellogeninene hos vertebrater

A New Method for Ligand-supported Homology Modelling of Protein Binding Sites: Development and Application to the neurokinin-1 receptor

In this thesis, a novel strategy (MOBILE (Modelling Binding Sites Including Ligand Information Explicitly)) was developed that models protein binding-sites simultaneously considering information about the binding mode of bioactive ligands during the homology modelling process. As a result, protein binding-site models of higher accuracy and relevance can be generated. Starting with the (crystal) structure of one or more template proteins, in the first step several preliminary homology models of the target protein are generated using the homology modelling program MODELLER. Ligands are then placed into these preliminary models using different strategies depending on the amount of experimental information about the binding mode of the ligands. (1.) If a ligand is known to bind to the target protein and the crystal structure of the protein-ligand complex with the related template protein is available, it can be assumed that the ligand binding modes are similar in the target and template protein. Accordingly, ligands are then transferred among these structures keeping their orientation as a restraint for the subsequent modelling process. (2.) If no complex crystal structure with the template is available, the ligand(s) can be placed into the template protein structure by docking, and the resulting orientation can then be used to restrain the following protein modelling process. Alternatively, (3.) in cases where knowledge about the binding mode cannot be inferred by the template protein, ligand docking is performed into an ensemble of homology models. The ligands are placed into a crude binding-site representation via docking into averaged property fields derived from knowledge-based potentials. Once the ligands are placed, a new set of homology models is generated. However, in this step, ligand information is considered as additional restraint in terms of the knowledge-based DrugScore protein-ligand atom pair potentials. Consulting a large ensemble of produced models exhibiting di erent side-chain rotamers for the binding-site residues, a composite picture is assembled considering the individually best scored rotamers with respect to the ligand. After a local force-field optimisation, the obtained binding-site models can be used for structure-based drug design

Ligand selectivity: binding at the protein-protein interface of Keap1 and NEMO

This dissertation comprises identifying the structural determinants of binding selectivity as demonstrated in three systems. The first system involves the structure determination of Keap1-small molecule fragment complexes to locate binding surfaces. The second system involves the structural determination of a NEMO/IKKbeta complex to serve as a platform for future fragment binding validation studies. The third system involves the structural investigation of a bacterial phosphoglycosyltransferase found in Campylobacter concisus to find the active site. Keap1 binding of Nrf2 is a regulatory mechanism to inhibit the transcription factor activity of Nrf2 to upregulate Nucleoporin p62 (p62). Nucleoporin p62 is a regulator of tau protein aggregates in Alzheimer's disease. The determination of binding hot spots in the Keap1 active site could serve as a starting point for the development of inhibitors as a treatment method for Alzheimer’s disease. To achieve this, I have developed a crystal form of Keap1 that allows for fragment-based study of binding in the active site via small molecule fragment screening and X-ray crystallography. Analysis of collected data has resulted in the solution of four structures, one containing a peptide fragment and three containing small molecule fragments that occupy a region of binding within the Keap1 active site, demonstrating the utility of the crystal form and affording information on binding hot spots. Nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) is a transcription factor and has been linked to cancer, inflammation, and immune dysfunction. The enzyme complex IκB kinase (IKK) is a regulator of NF-κB and consists of three subunits: IKK-α, IKK-β, and NEMO. If NEMO activity is abrogated, IKK is unable to activate NF-κB, making it a promising therapeutic target. My research has found crystallization conditions and performed trials of phase determination on an N terminal IKKβ-binding construct of NEMO containing previously uncharacterized regions of this protein. Glycosylation is a commonly occurring post-translational modification that affects a number of processes including protein folding, trafficking, cell-cell interactions and host immune response. The phosphoglycosyl transferase PglC is an essential part of the Campylobacter glycosylation pathway and a possible antibacterial target. My research determined the crystallization conditions and has developed complexes and protein constructs for phase determination of this single-pass transmembrane protein and will in the future provide a platform for structure-based inhibition of this protein

Identification and characterisation of proteases in Mycobacterium tuberculosis

Virulence determinants of M. tuberculosis remain largely unknown. Of key interest has been the ability of the bacterium to survive intracellularly within its host cell, the macrophage, and its ability to cause extensive tissue necrosis. Exported proteases are commonly associated with virulence in bacterial pathogens, yet their role in Mycobacterium tuberculosis has virtually not been studied. Preliminary experiments showed M. tuberculosis culture filtrates contained a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca²⁺, features typical of some serine proteases, notably subtilisins, and possibly metalloproteases. Purification attempts were unsuccessful. A family of five genes that encode putative, secreted, serine proteases has recently been described in M. tuberculosis. These proteases share 36-47% sequence identity and are all encoded with putative signal peptides, suggesting that they are translocated across the cytoplasmic membrane. One member, mycP1, was selected for further study. The gene product, mycosin-1, was 30-35% identical to bacterial subtilisin-like serine proteases and contained the classic catalytic triad and oxyanion hole. Mycosin-1 also contained a typical signal peptide, a likely propeptide, and a Cterminal hydrophobic sequence with a high transmembrane potential. Topology analyses predicted mycosin-1 to be a type I ectoprotein. Consistent with this, expression of mycosin-1 in M. tuberculosis and in Mycobacterium smegmatis transformed with mycP1 (M. smegmatis-P1) was limited strictly to the cell envelope, as seen by Western blotting, and immunogold electron microscopy. Only full-length, 50-kDa mycosin-1 was observed by Western blotting in broth-grown M. tuberculosis and M. smegmatis-P1 lysates, whereas a 40-kDa species was detected in 6-week M. tuberculosis culture filtrates. A similar 40-kDa immunoreactive band was also observed in lysates of macrophages infected with M. tuberculosis, consistent with robust transcription of the mycP 1 gene during growth in macrophages. Since putative mature mycosin-1 has a molecular weight of 38.6 kDa, the 40-kDa protein may represent activated mycosin-1 after propeptide cleavage. In conclusion, mycosin-1 is an exported, cell envelopeassociated subtilisin homolog that is expressed during growth of M. tuberculosis in vitro and in macrophages

Identification and characterization of the activated defence response in the commercially important Agarophyte, Gracilaria Gracilis, following exposure to disease elicitors

To our knowledge, this study represents the first analysis of gene expression using cDNA microarrays in the red macroalga G. gracilis. Western hybridization analysis was used to establish whether the observed changes in gene expression following exposure to disease elicitors positively correlated to changes at the protein level

Investigating The Grey Field Slug

High-throughput sequencing was used to analyse cDNA generated from tissues of the grey field slug, Deroceras reticulatum, a significant invertebrate pest of agricultural and horticultural crops. Almost no sequence data is available for this organism. In this project, we performed de novo transcriptome sequencing to produce sequence dataset for the Deroceras reticulatum. A total of 132,597 and 161,419 sequencing reads between 50-600bp from the digestive gland and neural tissue were obtained through Roche 454 pyrosequencing. These reads were assembled into contiguous sequences and annotated using sequence homology search tools. Multiple sequence assemblies and annotation data was amalgamated into a biological database using BioSQL. Analysis of the dataset with predictions of probable protein function were made based on annotation data. InterPro (IPR) terms generated with InterProScan software were mapped to read counts and used to identify more frequently sequenced gene families. Digestive hydrolases were major transcripts in the digestive gland, with cysteine proteinases and cellulases being the most abundant functional classes. A Cathepsin L homologue is likely to be responsible for the proteinase activity of the digestive gland which was previously detected by biochemical analysis. Cathepsin L and several other predicted proteins were used to design RNAi experiments to assess potential for crop pest defence strategy. Further work on protein expression of a native tumour necrosis factor (TNF) ligand homologue was also conducted as an exemplar study

Protein structure prediction and structure-based protein function annotation

Nature tends to modify rather than invent function of protein molecules, and the log of the modifications is encrypted in the gene sequence. Analysis of these modification events in evolutionarily related genes is important for assigning function to hypothetical genes and their products surging in databases, and to improve our understanding of the bioverse. However, random mutations occurring during evolution chisel the sequence to an extent that both decrypting these codes and identifying evolutionary relatives from sequence alone becomes difficult. Thankfully, even after many changes at the sequence level, the protein three-dimensional structures are often conserved and hence protein structural similarity usually provide more clues on evolution of functionally related proteins. In this dissertation, I study the design of three bioinformatics modules that form a new hierarchical approach for structure prediction and function annotation of proteins based on sequence-to-structure-to-function paradigm. First, we design an online platform for structure prediction of protein molecules using multiple threading alignments and iterative structural assembly simulations (I-TASSER). I review the components of this module and have added features that provide function annotation to the protein sequences and help to combine experimental and biological data for improving the structure modeling accuracy. The online service of the system has been supporting more than 20,000 biologists from over 100 countries. Next, we design a new comparative approach (COFACTOR) to identify the location of ligand binding sites on these modeled protein structures and spot the functional residue constellations using an innovative global-to-local structural alignment procedure and functional sites in known protein structures. Based on both large-scale benchmarking and blind tests (CASP), the method demonstrates significant advantages over the state-of-the- art methods of the field in recognizing ligand-binding residues for both metal and non- metal ligands. The major advantage of the method is the optimal combination of the local and global protein structural alignments, which helps to recognize functionally conserved structural motifs among proteins that have taken different evolutionary paths. We further extend the COFACTOR global-to-local approach to annotate the gene- ontology and enzyme classifications of protein molecules. Here, we added two new components to COFACTOR. First, we developed a new global structural match algorithm that allows performing better structural search. Second, a sensitive technique was proposed for constructing local 3D-signature motifs of template proteins that lack known functional sites, which allows us to perform query-template local structural similarity comparisons with all template proteins. A scoring scheme that combines the confidence score of structure prediction with global-local similarity score is used for assigning a confidence score to each of the predicted function. Large scale benchmarking shows that the predicted functions have remarkably improved precision and recall rates and also higher prediction coverage than the state-of-art sequence based methods. To explore the applicability of the method for real-world cases, we applied the method to a subset of ORFs from Chlamydia trachomatis and the functional annotations provided new testable hypothesis for improving the understanding of this phylogenetically distinct bacterium

Computational Approaches to Drug Profiling and Drug-Protein Interactions

Despite substantial increases in R&D spending within the pharmaceutical industry, denovo drug design has become a time-consuming endeavour. High attrition rates led to a long period of stagnation in drug approvals. Due to the extreme costs associated with introducing a drug to the market, locating and understanding the reasons for clinical failure is key to future productivity. As part of this PhD, three main contributions were made in this respect. First, the web platform, LigNFam enables users to interactively explore similarity relationships between ‘drug like’ molecules and the proteins they bind. Secondly, two deep-learning-based binding site comparison tools were developed, competing with the state-of-the-art over benchmark datasets. The models have the ability to predict offtarget interactions and potential candidates for target-based drug repurposing. Finally, the open-source ScaffoldGraph software was presented for the analysis of hierarchical scaffold relationships and has already been used in multiple projects, including integration into a virtual screening pipeline to increase the tractability of ultra-large screening experiments. Together, and with existing tools, the contributions made will aid in the understanding of drug-protein relationships, particularly in the fields of off-target prediction and drug repurposing, helping to design better drugs faster

Structural, functional and genetic analyses of novel candidate genes for tuber quality traits in potato (Solanum tuberosum L.)

The development of diagnostic markers by exploiting the natural allelic variation of candidate genes is a powerful tool in tetraploid potato cultivars for precision breeding regarding complex agronomic traits. Multiple genetic and environmental factors are the basis for important quality traits like starch and processing characteristics. The latter can rely on the balance between tuber starch and sugars that affect potato chip quality due to enzymatic reactions at low temperature (cold-sweetening). The major aim was to screen for superior natural alleles in an interesting set of candidate genes. A candidate gene approach for starch and chip quality traits mainly targets functional candidates involved in carbohydrate metabolism. First, an α-glucan water dikinase (GWD) and a phosphoglucan water dikinase (PWD) were analyzed which trigger the initiation of starch breakdown in Solanum tuberosum. Second, as a post-transcriptional regulatory factor of vacuolar invertase, a putative invertase inhibitor was studied for its contribution to tuber quality traits and its functional role. In order to identify new candidates which do not operate in the starch pathway, in silico homology studies with well characterized genes on the released Solanum phureja genome and “-omics” approaches were used as an experimental basis. A hitherto uncharacterized 6th invertase and a leucine aminopeptidase (LAP) were obtained as third and fourth candidate genes. In all candidate genes, SNPs (single nucleotide polymorphisms) having an effect on starch and chip quality traits were identified. The functional candidates GWD and PWD showed moderate association with all tested tuber quality traits and explained between 4 % and 7 % of the phenotypic variance. The 6th invertase (Inv6) showed high associations and explained up to 10 % and 11 % of the phenotypic variation of chip quality and starch quality, respectively. The role of Inv6 remains to be elucidated, as semi-quantitative expression studies showed rather low expression in tubers and no strong increase upon cold treatment. Interestingly, LAP showed the highest association with tuber quality traits and explained up to 17 % of starch variance and 10 % of variance of chip quality after cold-storage. LAP has been selected as a candidate gene based on a proteomics analysis showing strong differences at protein level between good and bad processing genotypes. Finally, superior alleles of the putative invertase inhibitor were identified which explained 10 % of the phenotypic variance of starch quality traits, whereas no association was detected for chip quality traits. Functional analysis revealed a tuber-specific expression pattern. In vitro inhibition assays of heterologously expressed invertase and inhibitor did not confirm an inhibition of invertase activity. Although natural DNA variants in the different candidate genes showed associations with starch and chip quality traits, their functional role remains to be identified. However, the candidate gene approach is a powerful tool to identify diagnostic molecular markers within functional genes which will facilitate marker-assisted selection and increase breeding efficiency