The evolution of protein kinase specificity

All research conducted at EMBL-EBI under the supervision of Dr. Pedro Beltrao. Work on the PhD project was paused temporarily in the Spring of 2017 for me to undertake a 3-month internship at EMBO Press (in Heidelberg).Protein phosphorylation represents one of the most important post-translational modifica- tions (PTMs) for cell signalling, and is is catalysed by a group of enzymes called protein kinases. Through this activity they serve as key regulators of almost all cellular processes. This is achieved at any time by a network of different kinases that are transiently active. The fidelity of cell systems control therefore requires that each kinase targets only a restricted set of substrates. This specificity is achieved partly by contextual factors that separate kinases spatially and temporally, but also by sequence features that are encoded in the kinase domain itself. For this thesis I focus on elements of kinase specificity that are encoded in the the active site of the enzyme. During these investigations I have tried to address three main questions: 1) How is specificity for residues surrounding the phosphorylation site determined in the kinase? 2) How did these specificities evolve? and 3) To what extent does kinase evolution correlate with the evolution of its substrates? First, I developed a sequence-based method for the automated detection of kinase speci- ficity determining residues (SDRs). The putative determinants were then rationalised using available structural data, and in two specific cases were validated experimentally. I also used mutation data from The Cancer Genome Atlas (TCGA) to demonstrate that kinase SDRs are often targeted during cancer. Second, a global analysis of SDR evolution was performed for kinases following gene duplication and speciation, revealing that SDRs often diverge between paralogues but not between orthologues. This global analysis is followed by a detailed case study of G-protein coupled receptor kinase (GRKs) evolution using ancestral sequence reconstructions. Third, I inferred global substrate preferences in a taxonomically broad range of species using phosphoproteome data. I then related the evolution of substrate motif sequences to that of their cognate effector kinases where possible. The results strongly suggest that many of the motifs emerged in a universal eukaryotic ancestor. I finish by summarising the major findings of this doctoral research, which to my knowl- edge represents the most comprehensive analysis to date of protein kinase specificity and its evolution.BBSR

Elucidating the molecular genetics of host and nonhost resistance in barley to stripe rust

Plants have a remarkable ability to resist the majority of pathogenic microbes they encounter. As such, they are described as nonhosts. Nonhost resistance is often conceptualised as a qualitative separation from host resistance. Classification into these two states is generally facile, as they fail to fully describe the range of states that exist in the transition from host to nonhost. This poses a problem when studying pathosystems that cannot be classified into either of these categories due to their intermediate status relative to the two extremes. Therefore, the terms intermediate host and intermediate nonhost have been proposed to describe pathosystems in the evolutionary transition between host and nonhost status. At present, a significant amount of research exists into the molecular genetics of host and nonhost pathosystems but very little is known about intermediate systems. The work in this Ph. D. thesis focuses on the interaction of barley with Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, as an intermediate host pathosystem. The first research chapter describes the development of two microscopic phenotypic assays used to quantify P. striformis f. sp. tritici in barley leaves challenged with the pathogen. These assays are then used to screen a large panel of barley accessions to define the intermediate host status of barley relative to a host pathosystem. Subsequently, these assays play a key role in determining that the genetic architecture of resistance in barley is underpinned by three major effect resistance loci: Rpst1, Rpst2, and Rpst3. Using a combination of classical map-based genetics and contemporary genomics information I identify a candidate NLR gene underlying Rpst2 resistance on chromosome 7HL. Furthermore, I show that distinct genes condition host and nonhost resistance in barley by mapping the host resistance gene, rps2 to chromosome 2HL

The Evolution of Function in the Rab family of Small GTPases

Dissertation presented to obtain the PhD degree in Computational Biology.The question how protein function evolves is a fundamental problem with profound implications for both functional end evolutionary studies on proteins. Here, we review some of the work that has addressed or contributed to this question. We identify and comment on three different levels relevant for the evolution of protein function. First, biochemistry. This is the focus of our discussion, as protein function itself commonly receives least attention in studies on protein evolution.(...

Population Genomics of Cercospora beticola

Fungal plant pathogens pose a serious threat to global food safety and security, and can result in significant yield loss. Fungal plant pathogens have evolved with their hosts during the history of crop domestication. While some fungal phytopathogens of modern crops have maintained the ability to infect the wild relatives of these crops, many have evolved host-specificity due to the evolutionary arms race. Co-evolution between plants and their pathogens spans many generations. Therefore, we have employed the pathosystem of the fungal pathogen Cercospora beticola and its hosts Beta vulgaris ssp. (domesticated beet) and B. maritima (sea beet). This pathosystem is exceptional as sugar beet has a relatively short domestication history of ~300 years, compared to several thousand year history of other modern crops. Investigating the effect crop domestication has on fungal evolution in such a short time frame may provide insight into the early processes underlying the evolution of host-specificity. The availability of whole genome sequencing data for entire populations of fungal plant pathogens has enabled detailed analyses of genomic variation within and among field populations. Using population genomic data, we are able to detect population structure of a phytopathogenic fungus, identify regions that are highly differentiated between isolates, and predict the evolutionary trajectory of disease epidemics. The primary focus of this thesis was to describe the population genomics of the fungus Cercospora beticola, and determine the influence of host domestication on recent evolution and population structure of the fungus. Chapter 1 addressed the challenge of assembling and analysing population genomic data of species with structural variation, as is the case for many pathogenic fungi. We compared and contrasted two variant calling methods used in population genomics. We show that the commonly used method of variant calling, reference mapping-based approaches, as well as more recently adapted multiple genome alignment-based methods perform equally well at high sequencing depths in species with variable amounts of repetitive content. However, we also found that reference mapping-based approaches are reliable at average and high sequencing depths, regardless of repetitive content. In Chapter 2, we analyse the population genetic structure of C. beticola with the aim of comparing the genetic variation in populations of domesticated and wild beet species. Specifically, we make use of population genomics tools to elucidate whether C. beticola isolates from wild and domesticated hosts show strong signals of host specialisation. Sugar beet is comparatively novel crop, and provides insight into the early specialisation processes pathogens of domesticated plants. We collected isolates from wild and domesticated beet from Europe and North America and show that there are not clear populations of C. beticola isolates that infect wild or domesticated beet. We show that C. beticola isolates are likely a global population, with substantial admixture between individuals from all hosts and locations. While there is admixture between individuals from all locations, isolates from sea beet in the UK showed more differentiation from the isolates from other locations suggesting some barriers to gene flow and distinct population histories of the sea beet isolates. We investigated regions where the isolates from the UK are different from isolates from mainland Europe and North America, and showed that there are likely phenotypic differences between isolates from Croatian sea beet and the English sea beet isolates. We illustrate a region where the isolates from Croatia contained a premature stop codon in a gene involved in the production of an aflatoxin in high frequency, while it was present at a low frequency the isolates from English sea beet. Thus, we show that while C. beticola may not show strong signatures of host specialisation yet, there are some differences between isolates from different locations indicating the potential for future population divergence. In Chapter 3, we compare and contrast C. beticola to four other Cercospora species to elucidate differences and similarities in genome content and synteny within the genus. We show that C. beticola has a higher number of genes encoding proteins that are involved host-pathogen interaction. We also note that the other Cercospora species that has a broad host range included in this, C. cf. flagellaris, has a similar repertoire of genes. We also show that these two species share substantial synteny. We postulate that they most recent common ancestor of these two species likely had a plastic genome that underwent several translocation events. Taken together, we show that the Cercospora genus is shaped by its interactions with its environment and the various hosts. We show that C. beticola has not yet shown strong association with either host or location

Population genomics of Cercospora beticola

Fungal plant pathogens pose a serious threat to global food safety and security, and can result in significant yield loss. Fungal plant pathogens have evolved with their hosts during the history of crop domestication. While some fungal phytopathogens of modern crops have maintained the ability to infect the wild relatives of these crops, many have evolved host-specificity due to the evolutionary arms race. Co-evolution between plants and their pathogens spans many generations. Therefore, we have employed the pathosystem of the fungal pathogen Cercospora beticola and its hosts Beta vulgaris ssp. (domesticated beet) and B. maritima (sea beet). This pathosystem is exceptional as sugar beet has a relatively short domestication history of ~300 years, compared to several thousand year history of other modern crops. Investigating the effect crop domestication has on fungal evolution in such a short time frame may provide insight into the early processes underlying the evolution of host-specificity. The availability of whole genome sequencing data for entire populations of fungal plant pathogens has enabled detailed analyses of genomic variation within and among field populations. Using population genomic data, we are able to detect population structure of a phytopathogenic fungus, identify regions that are highly differentiated between isolates, and predict the evolutionary trajectory of disease epidemics. The primary focus of this thesis was to describe the population genomics of the fungus Cercospora beticola, and determine the influence of host domestication on recent evolution and population structure of the fungus. Chapter 1 addressed the challenge of assembling and analysing population genomic data of species with structural variation, as is the case for many pathogenic fungi. We compared and contrasted two variant calling methods used in population genomics. We show that the commonly used method of variant calling, reference mapping-based approaches, as well as more recently adapted multiple genome alignment-based methods perform equally well at high sequencing depths in species with variable amounts of repetitive content. However, we also found that reference mapping-based approaches are reliable at average and high sequencing depths, regardless of repetitive content. In Chapter 2, we analyse the population genetic structure of C. beticola with the aim of comparing the genetic variation in populations of domesticated and wild beet species. Specifically, we make use of population genomics tools to elucidate whether C. beticola isolates from wild and domesticated hosts show strong signals of host specialisation. Sugar beet is comparatively novel crop, and provides insight into the early specialisation processes pathogens of domesticated plants. We collected isolates from wild and domesticated beet from Europe and North America and show that there are not clear populations of C. beticola isolates that infect wild or domesticated beet. We show that C. beticola isolates are likely a global population, with substantial admixture between individuals from all hosts and locations. While there is admixture between individuals from all locations, isolates from sea beet in the UK showed more differentiation from the isolates from other locations suggesting some barriers to gene flow and distinct population histories of the sea beet isolates. We investigated regions where the isolates from the UK are different from isolates from mainland Europe and North America, and showed that there are likely phenotypic differences between isolates from Croatian sea beet and the English sea beet isolates. We illustrate a region where the isolates from Croatia contained a premature stop codon in a gene involved in the production of an aflatoxin in high frequency, while it was present at a low frequency the isolates from English sea beet. Thus, we show that while C. beticola may not show strong signatures of host specialisation yet, there are some differences between isolates from different locations indicating the potential for future population divergence. In Chapter 3, we compare and contrast C. beticola to four other Cercospora species to elucidate differences and similarities in genome content and synteny within the genus. We show that C. beticola has a higher number of genes encoding proteins that are involved host-pathogen interaction. We also note that the other Cercospora species that has a broad host range included in this, C. cf. flagellaris, has a similar repertoire of genes. We also show that these two species share substantial synteny. We postulate that they most recent common ancestor of these two species likely had a plastic genome that underwent several translocation events. Taken together, we show that the Cercospora genus is shaped by its interactions with its environment and the various hosts. We show that C. beticola has not yet shown strong association with either host or location

The evolution of aggregative multicellularity and cell-cell communication in the Dictyostelia

AbstractAggregative multicellularity, resulting in formation of a spore-bearing fruiting body, evolved at least six times independently amongst both eukaryotes and prokaryotes. Amongst eukaryotes, this form of multicellularity is mainly studied in the social amoeba Dictyostelium discoideum. In this review, we summarise trends in the evolution of cell-type specialisation and behavioural complexity in the four major groups of Dictyostelia. We describe the cell–cell communication systems that control the developmental programme of D. discoideum, highlighting the central role of cAMP in the regulation of cell movement and cell differentiation. Comparative genomic studies showed that the proteins involved in cAMP signalling are deeply conserved across Dictyostelia and their unicellular amoebozoan ancestors. Comparative functional analysis revealed that cAMP signalling in D. discoideum originated from a second messenger role in amoebozoan encystation. We highlight some molecular changes in cAMP signalling genes that were responsible for the novel roles of cAMP in multicellular development

Transcription, signaling receptor activity, oxidative phosphorylation, and fatty acid metabolism mediate the presence of closely related species in distinct intertidal and cold-seep habitats

Bathyal cold seeps are isolated extreme deep-sea environments characterized by low species diversity while biomass can be high. The Hakon Mosby mud volcano (Barents Sea, 1,280 m) is a rather stable chemosynthetic driven habitat characterized by prominent surface bacterial mats with high sulfide concentrations and low oxygen levels. Here, the nematode Halomonhystera hermesithrives in high abundances (11,000 individuals 10 cm(-2)). Halomonhystera hermesi is a member of the intertidal Halomonhystera disjuncta species complex that includes five cryptic species (GD 1-5). GD1-5's common habitat is characterized by strong environmental fluctuations. Here, we compared the transcriptomes of H. hermesi and GD1, H. hermesi's closest relative. Genes encoding proteins involved in oxidative phosphorylation are more strongly expressed in H. hermesi than in GD1, and many genes were only observed in H. hermesi while being completely absent in GD1. Both observations could in part be attributed to high sulfide concentrations and low oxygen levels. Additionally, fatty acid elongation was also prominent in H. hermesi confirming the importance of highly unsaturated fatty acids in this species. Significant higher amounts of transcription factors and genes involved in signaling receptor activity were observed in GD1 (many of which were completely absent in H. hermesi), allowing fast signaling and transcriptional reprogramming which can mediate survival in dynamic intertidal environments. GC content was approximately 8% higher in H. hermesi coding unigenes resulting in differential codon usage between both species and a higher proportion of amino acids with GC-rich codons in H. hermesi. In general our results showed that most pathways were active in both environments and that only three genes are under natural selection. This indicates that also plasticity should be taken in consideration in the evolutionary history of Halomonhystera species. Such plasticity, as well as possible preadaptation to low oxygen and high sulfide levels might have played an important role in the establishment of a cold-seep Halomonhystera population

Lineage-specific proteins essential for endocytosis in trypanosomes

Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites

Inositol phosphate pathway evolution and synthesis in Dictyostelium discoideum

Inositol phosphates (InsPs) are polar water-soluble derivatives of the six- carbon cyclitol inositol. They are synthesized, through phosphorylation reactions, by kinases of four distinct families: IPK, IP5-2K, ITPK1 and PPIP5K, which are thought to be present across all eukaryotes. A pleiotropy of functions has been ascribed to InsPs, from nutrient storage as phytate (InsP6) in plant seeds to the regulation of energy metabolism for the highly phosphorylated inositol pyrophosphates (PP-InsPs). PP-InsPs were first identified and structurally described in the slime mould D. discoideum, in part due to the high concentrations of these molecules in amoeba. However, the amoeba knockout strains for the homologous enzymes synthesising PP-InsPs in humans (and yeast), IP6K (kcs1) and PPIP5K (vip1), do not present clear phenotypes. The work presented in this thesis expanded our biochemical understanding of these kinases by identifying additional isomers of inositol pyrophosphates and IpkA as the main source of InsP8 in the social amoeba. These findings shed light into the evolution of the inositol phosphate pathway and suggested an increased complexity of isomers and enzymes. Inositol phosphate kinase functions were identified in all domains of the tree of life as well as certain viruses, increasing our understanding of the origin and diversification of the inositol phosphate pathway