An exploration of phosphorylases for the synthesis of carbohydrate polymers

Phosphorylases are interesting enzymes with regard to both their role in metabolism and their use in the in vitro synthesis of carbohydrates. The disaccharide phosphorylases have attracted attention because of their strict stereo- and regiospecificity and their tractability. The polymerising phosphorylases have received less attention due to heterogeneous product formation, requiring more complex analyses. In this work three polymerising carbohydrate phosphorylases have been studied. The plant α-1,4-glucan phosphorylase PHS2 is closely related to the well characterised mammalian glycogen phosphorylase. We present the first crystal structures of the plant enzyme which reveals a unique surface binding site. PHS2 allowed the production of novel starch like surface, both in two and three dimensions, which show some of the same properties as a native starch granule. This can now be used to study starch-active enzymes on an insoluble glucan surface which is analogous to the native starch granule. The bacterial β-1,4-glucan phosphorylase CDP is involved in degradation of cellulose. In the reverse direction this enzyme allows the rapid synthesis of cellulose polymers in solution and also allows the synthesis of hemicellulose-like materials. The substrate specificity can in part be probed in the crystal structure presented here, which represents the first structure of a polymerising, inverting phosphorylase. Together these data provide the foundation for further work with this enzyme in the synthesis of plant cell wall related glycans. The third enzyme studied was the β-1,3-glucan from the unsequenced alga Euglena gracilis, which was used for the facile enzymatic synthesis of β-glucosyl glycerols. In order to identify the sequence of this enzyme we obtained de novo transcriptome sequencing data from this alga, which has revealed unexpected metabolic diversity. Aside from complex carbohydrate metabolism, there are also many surprising features, including novel enzyme architectures, antioxidants only previously noted in human parasites and complex natural product synthases

Phylogenomic and structure-function relationship studies of proteins involved in EBV associated oncogenesis

This study covers the investigation of evolutionary and structure-function relationship aspects of several cancer related proteins. One part of the study deals with the investigation of a critical protein of Epstein-Barr Virus (EBV) the Nuclear Antigen 1 (EBNA1), and its interactions with different host proteins. One of these host proteins is a member of a large gene family, encoding ubiquitin specific proteases (USP), known as USP7. The second section of the thesis deals with the molecular evolution of the USP gene family. Another set of cellular proteins deregulated during EBV associated oncogenesis are members of the glycoside hydrolase (GH18) family. Their phylogenetic relationships and protein structures were investigated in the third section of this thesis. EBNA1 is the only EBV protein that consistently expressed in all latent forms of the EBV infections. The protein is involved in the genome maintenance and a substantial body of evidence suggests that it has a role in EBV associated oncogenesis. In this study, full length molecular models of the EBNA1 protein were generated using the programmes, I-TASSER, MOE and Modeller. The best models were selected on the basis of plausibility in structural and thermodynamical parameters and from this models of EBNA1 homologues of primates lymphocryptoviruses (LCVs) were generated. The C-terminal DNA binding and homodimerisation domain was predicted to be structurally similar between different LCV EBNA1 homologues, indicative of functional conservation. The central glycine alanine repeat (GAr) domain was predicted to be primarily composed of α helices, while almost all of the protein interaction region was found to be unstructured, irrespective of the prediction approach used and sequence origin. Predicted USP7 and Casein kinase 2 (CK2) binding sites and GAr were observed in the EBNA1 homologues of Old World primate LCVs, but not in the marmoset homologue suggesting the co-evolution of both these sites. Dimer conformations of the EBNA1 monomer models were constructed using SymmDock, where the C-terminal tail was predicted to wrap around the proline rich loop of another monomer, possibly contributing to dimer stability. This feature could be exploited in therapeutic design, hence an inhibitor peptide was designed and a preliminary evaluation was conducted to explore its ability to inhibit EBNA1 function in cell survival. The peptide array libraries of EBNA1 were used to investigate the binding regions and critical contact points between EBNA1 and partner proteins. Human EBP2 and USP7 proteins were expressed in bacteria and probed on the EBNA1 array. The data confirm the previously known binding region for EBNA1-EBP2 and EBNA1-USP7 interactions. In addition further information was gained regarding the critical contact residues and the potential role of phosphorylation of serine residues of EBNA1 in its binding with EBP2 and USP7. The human genome encodes nearly 100 USPs which contribute to regulate the turnover of cellular proteins. These homologues are divided into 16 paralogous groups, all sharing a characteristic peptidase C19 domain. Evolutionary relationships between these homologues were explored by datamining and the phylogenetic reconstruction of peptidase C19 domain sequences. The data reveal an ancient relationship between the genes, with expansion occurring throughout the course of evolution, but particularly at the base of the vertebrates, at the time of the two whole genome duplications. A comparison between the phylogenetic architecture and protein interaction networks suggests the parallel emergence of many molecular pathways and the associated USPs. The GH18 gene family includes chitinases and related non catalytic proteins. Most mammals encode at least three chitinases (CHIT1, CHIA/AMCase and CTBS), as well as several homologues encoding catalytically inactive chitinase-like proteins or chilectins. Phylogenomic analysis shows that the family has undergone extensive expansion, initiating with a duplication event at the root of the vertebrate tree, resulting in the origin of the ancestors of CHIT1 and CHIA. Two further duplications of ancestral CHIA predate the divergence of bony fishes, one leading to a newly identified paralogous group (we have termed CHIO). In tetrapods, additional CHIA duplications predate and postdate the amphibian/mammalian split and relics of some exist as pseudogenes in the human genome. Homology modelling of structurally unresolved GH18 homologues in mouse and human was conducted using Modeller and I-TASSER. All resolved and predicted structures share a TIM barrel (β/α)8 and α+β domain. A central ligand binding cavity was also found in all GH18 homologues. The variation in size and shape of different paralogous proteins, indicate the difference in their ligands specificity and in turn potential functions

The Regulation of Surface Responsive Genes in Blumeria graminis f. sp. hordei

Powdery mildew of barley is caused by the ascomycete pathogen Blumeria graminis f. sp. hordei (Bgh). Bgh is economically important throughout the world, causing crop losses varying between 5 to 20 % and in extreme cases as much as 60 %. Bgh is an obligate biotroph, relying on its host for growth and reproduction. This characteristic has hindered attempts to carry out biochemical and molecular biological analysis. Previous work had highlighted differential gene expression during Bgh development on surfaces other than the host. Consequently, this thesis had three aims. The first attempted to elucidate the nature of this gene expression. Work listed within includes studies of Bgh morphological development on the host barley, wheat, cellulose membrane, and glass. Additional studies included the assessment of gene expression, via RT-qPCR, on glass surfaces enhanced with 1-hexacosonal (a synthetic C26 aldehyde known to spur Bgh development), 16-hydroxyhexadecanoic acid (a cutin monomer found within the barley leaf), as well as surfaces of differing hydrophobicity. Results collected reenforce the surface-dependent nature of gene regulation, and highlight how gene expression is determined by the integration of multiple signal inputs. The second aim of this thesis was the transformation of Bgh utilising Agrobacterium tumefaciens. Efforts are discussed as are approaches for future work aimed at transforming this fungus. The final aim of the thesis aimed to lay foundations for work involving the assessment of 5‟-regulatory regions of genes showing clustered, and differential, expression on alternate surfaces. Utilising the phytopathogenic model fungus Magnaporthe oryzae (the causal agent of rice blast disease), 22 promoter regions were tested for their ability to drive GFP in this pathogen. 2 regions (for genes encoding a H4 histone and an aconitase) along with promoter regions selected for their conservation, were able to do so

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin