Predicting protein function with hierarchical phylogenetic profiles: The Gene3D phylo-tuner method applied to eukaryotic Genomes

"Phylogenetic profiling'' is based on the hypothesis that during evolution functionally or physically interacting genes are likely to be inherited or eliminated in a codependent manner. Creating presence-absence profiles of orthologous genes is now a common and powerful way of identifying functionally associated genes. In this approach, correctly determining orthology, as a means of identifying functional equivalence between two genes, is a critical and nontrivial step and largely explains why previous work in this area has mainly focused on using presence-absence profiles in prokaryotic species. Here, we demonstrate that eukaryotic genomes have a high proportion of multigene families whose phylogenetic profile distributions are poor in presence-absence information content. This feature makes them prone to orthology mis-assignment and unsuited to standard profile-based prediction methods. Using CATH structural domain assignments from the Gene3D database for 13 complete eukaryotic genomes, we have developed a novel modification of the phylogenetic profiling method that uses genome copy number of each domain superfamily to predict functional relationships. In our approach, superfamilies are subclustered at ten levels of sequence identity from 30% to 100% - and phylogenetic profiles built at each level. All the profiles are compared using normalised Euclidean distances to identify those with correlated changes in their domain copy number. We demonstrate that two protein families will "auto-tune'' with strong co-evolutionary signals when their profiles are compared at the similarity levels that capture their functional relationship. Our method finds functional relationships that are not detectable by the conventional presence - absence profile comparisons, and it does not require a priori any fixed criteria to define orthologous genes

Predicting Protein Function with Hierarchical Phylogenetic Profiles: The Gene3D Phylo-Tuner Method Applied to Eukaryotic Genomes

“Phylogenetic profiling” is based on the hypothesis that during evolution functionally or physically interacting genes are likely to be inherited or eliminated in a codependent manner. Creating presence–absence profiles of orthologous genes is now a common and powerful way of identifying functionally associated genes. In this approach, correctly determining orthology, as a means of identifying functional equivalence between two genes, is a critical and nontrivial step and largely explains why previous work in this area has mainly focused on using presence–absence profiles in prokaryotic species. Here, we demonstrate that eukaryotic genomes have a high proportion of multigene families whose phylogenetic profile distributions are poor in presence–absence information content. This feature makes them prone to orthology mis-assignment and unsuited to standard profile-based prediction methods. Using CATH structural domain assignments from the Gene3D database for 13 complete eukaryotic genomes, we have developed a novel modification of the phylogenetic profiling method that uses genome copy number of each domain superfamily to predict functional relationships. In our approach, superfamilies are subclustered at ten levels of sequence identity—from 30% to 100%—and phylogenetic profiles built at each level. All the profiles are compared using normalised Euclidean distances to identify those with correlated changes in their domain copy number. We demonstrate that two protein families will “auto-tune” with strong co-evolutionary signals when their profiles are compared at the similarity levels that capture their functional relationship. Our method finds functional relationships that are not detectable by the conventional presence–absence profile comparisons, and it does not require a priori any fixed criteria to define orthologous genes

Proteasome Inhibitors: Harnessing Proteostasis to Combat Disease

The proteasome is the central component of the main cellular protein degradation pathway. During the past four decades, the critical function of the proteasome in numerous physiological processes has been revealed, and proteasome activity has been linked to various human diseases. The proteasome prevents the accumulation of misfolded proteins, controls the cell cycle, and regulates the immune response, to name a few important roles for this macromolecular “machine.” As a therapeutic target, proteasome inhibitors have been approved for the treatment of multiple myeloma and mantle cell lymphoma. However, inability to sufficiently inhibit proteasome activity at tolerated doses has hampered efforts to expand the scope of proteasome inhibitor-based therapies. With emerging new modalities in myeloma, it might seem challenging to develop additional proteasome-based therapies. However, the constant development of new applications for proteasome inhibitors and deeper insights into the intricacies of protein homeostasis suggest that proteasome inhibitors might have novel therapeutic applications. Herein, we summarize the latest advances in proteasome inhibitor development and discuss the future of proteasome inhibitors and other proteasome-based therapies in combating human diseases

Characterisation of the zinc fingers of Erythroid Kruppel-Like Factor

Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis

Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent

Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Identification and characterisation of somatic regulatory mutations in the breast cancer genome

Luminal breast cancer remains a major clinical challenge with over 2 million cases diagnosed annually. While prognosis is favourable in these patients, roughly 40% will relapse over the course of the next 20 years. Understanding the evolution of disseminated tumour cells at distal sites is critical to effectively treating these patients. While metastatic driver mutations, such as those in the Oestrogen Receptor (ESR1) gene, can be identified in many cases for a significant proportion of patients, clear drivers remain elusive. A limitation of previous genomics studies in metastatic breast cancer is their focus on the coding genome. Advances in our understanding have revealed the critical role of regulatory elements such as enhancers and promoters in transcriptional regulation. This effect is mediated through the functional and hierarchical organisation of chromatin within the nucleus, the key unit of chromatin organisation is the Topologically Associating Domains (TADs). TAD organisation is, in part, mediated by the CCCTC-Binding Factor (CTCF) protein which physically binds to DNA mediating the formation of loops and domains. Together promoters, enhancers, and CTCF-bound regions provide potential as sites for non-coding mutations to occur, drastically impacting gene regulation and tumour evolution. In this work we interrogate the contribution of regulatory element mutations in the evolution of metastatic breast cancer. This is done through two projects. First, a proof of principle study functionally characterising a clinically relevant CTCF binding site mutation. Second, through the design of an informed panel of regulatory regions utilised in a longitudinal targeted sequencing study in patient samples and a CRISPRi perturbation study in cell lines. Through these studies we provide evidence that the mutation of TAD boundary associated CTCF binding sites is unlikely to contribute to tumour evolution. We also fail to identify recurrence of non-coding drivers, though more patient specific mutations may contribute to metastatic evolution. Results obtained from the CRISPRi screen illustrate the functionality of the regulatory regions in the panel, identifying regulatory elements that confer fitness or vulnerabilities when specifically repressed. This study identifies that repression of several members of the NF-κB signalling pathway provides MCF7 cells with an advantage in adapting to oestrogen deprivation. This data underlines the importance of regulatory regions in the evolution of luminal breast cancers and indicates that non-genetic mechanisms may play a key role.Open Acces

Host-pathogen-drug interactions in the context of antibiotic resistance: How host xenobiotic metabolism can affect antibiotic efficacy in a Methicillin-Resistant Staphylococcus aureus infection

Our arsenal of weapons to fight against bacterial infections is weakening: bacteria are gaining resistance to the common antibiotics, while industries are struggling to develop new effective ones. To avoid triggering de-novo antibiotic resistance, we need the right antibiotic for the specific bacteria, at a dose adapted to the patient genetics. Genes driving the degradation of antibiotics have indeed known genetic variants that can dramatically affect the kinetics of antibiotic metabolism from one patient to another. This could lead to treatment failure, excessive side effects or emergence of resistance. I first investigated the clinical relevance of the vancomycin-rifampicin combination to treat Methicillin-Resistant Staphylococcus aureus infections (Chapter 3). I showed in various experimental settings that these two antibiotics may promote an environment prone for antibiotic resistance. Their interaction might be unstable in vitro because of environmental factors, one could wonder how the host environment might generate such instability. I then explored how interactions between antibiotics and host xenobiotic genetics could influence antibiotic concentrations, potentially triggering increased treatment failure, side-effects and antibiotic resistance in patients carrying particular variants. In silico, I estimated the effects of genetic variants of the Cytochrome P450 3A4 gene to its enzyme, and, as they are unequally distributed in the world, their global relevance (Chapter 4). In vivo, I focused on the Carboxylesterase 2 gene and I found two of its variants, rs11075646 and rs8192925, capable of significantly altering the degradation of various drugs, including rifampicin and mycophenolate mofetil. A clinical study was designed, to explore possible correlations between genotype for these variants and treatment response in patients (Chapter 5). Altogether, this body of work highlights the prescribing importance of considering not only the strain in bacterial infections, but also the genetics of the human host. This raises a need to make sure the right antibiotics are used in practices, at doses adapted to the patients. As part of personalised medicine, checking their genotype for these biomarkers could tailor their therapy, improving recovery while avoiding antibiotic resistance