Development of a generic, structural bioinformatics information management system and its application to variation in foot-and-mouth disease virus proteins

Structural biology forms the basis of all functions in an organism from how enzymes work to how a cell is assembled. In silico structural biology has been a rather isolated domain due to the perceived difficulty of working with the tools. This work focused on constructing a web-based Functional Genomics Information Management System (FunGIMS) that will provide biologists access to the most commonly used structural biology tools without the need to learn program or operating specific syntax. The system was designed using a Model-View-Controller architecture which is easy to maintain and expand. It is Python-based with various other technologies incorporated. The specific focus of this work was the Structural module which allows a user to work with protein structures. The database behind the system is based on a modified version of the Macromolecular Structure Database from the EBI. The Structural module provides functionality to explore protein structures at each level of complexity through an easy-to-use interface. The module also provides some analysis tools which allows the user to identify features on a protein sequence as well as to identify unknown protein sequences. Another vital functionality allows the users to build protein models. The user can choose between building models online on downloading a generated script. Similar script generation utilities are provided for mutation modeling and molecular dynamics. A search functionality was also provided which allows the user to search for a keyword in the database. The system was used on three examples in Foot-and-Mouth Disease Virus (FMDV). In the first case, several FMDV proteomes were reannotated and compared to elucidate any functional differences between them. The second case involved the modeling of two FMDV proteins involved in replication, 3C and 3D. Variation between the several different strains were mapped to the structures to understand how variation affects enzymes structure. The last example involved capsid protein stability differences between two subtypes. Models were built and molecular dynamics simulations were run to determine at which protein structure level stability was influenced by the differences between the subtypes. This work provides an important introductory tool for biologists to structural biology.Thesis (PhD)--University of Pretoria, 2009.Biochemistryunrestricte

Genetic heterogeneity in the leader and P1-coding regions of foot-and-mouth disease virus serotypes A and O in Africa

Genetic information regarding the leader (L) and complete capsid-coding (P1) region of FMD serotype A and O viruses prevalent on the African conti- nent is lacking.Here,we present the complete L-P1 sequences for eight serotype A and nine serotype O viruses recovered from FMDV outbreaks in East and West Africa over the last 33 years.Phylogenetic analysis of the P1 and capsid-coding regions revealed that the African isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus.However,similar analysis of the L region revealed random groupings of isolates from serotypes O and A.Comparisons between the phylogenetic trees derived from the structura lcoding regions and the L region pointed to a possibility of genetic recombination.The in- tertypic nucleotide and amino acid variation of all the isolates in this stud ysupported results from previous studies where the externally located 1D was the most variable whilst the internally located 1A was the most conserved,which likely reflects the selective pressures on these proteins.Amino acids identified previously as important for FMDV structure and functioning were found to be highly conserved.The information gained from this study will contribute to the construction of structurally designed FMDV vaccines in Africa.SA-UKcol-laboration initiative via the Department of Science and Technologyhttp://link.springer.com/journal/705hb201

In silico structural characterisation of Plasmodium falciparum dihydro-6-hydroxymethylpterin pyrophosphokinase dihydropteroate synthase (PPPK-DHPS)

Malaria kills nearly 1.5 million and affects more than 500 million people annually, mostly in sub-Saharan Africa. The malaria parasite has developed resistance against almost all of the known drugs used for treatment. This fact has resulted in a constant battle between developing new anti-malarials and the parasite evolving resistance. One of the main drug combinations, pyrimethamine/sulfadoxine, targets the dihydrofolate reductase (DHFR) and dehydropteroate synthase (DHPS) proteins in the folate synthesis pathway of human malaria parasite, Plasmodium falciparum. The folate synthesis pathway is absent from the human host and thus presents itself as an ideal target for parasite-specific drugs. The three dimensional atomic coordinates of a target protein can help in designing new, more effective drugs. Malarial proteins are notoriously difficult to crystallize and thus homology modelling was chosen as an alternative method to obtain a protein structure. DHPS and PPPK occur as a bifunctional protein in the folate metabolism pathway. In this study, homology modelling was used to do in silico modelling of P. falciparum DHPS and hydroxymethyldihydropteridine pyrophosphokinase (PPPK). For the P. falciparum DHPS model the crystal structures of M. tuberculosis and B. anthracis DHPS were used as templates and for the P. falciparum PPPK model, the crystal structure ofE. coli PPPK. Molecular dynamics was used to investigate loop movement in DHPS and PPPK as well as to reveal the effect of resistance-causing mutations on sulfadoxine binding in P. falsiparum DHPS. This study revealed that four of the five known sulfadoxine resistance-causing mutations in DHPS disrupt the interaction between sulfadoxine and DHPS. This translates to a reduced capacity for sulfadoxine to inhibit DHPS, and results in resistance. The simulations also showed that both DHPS and PPPK have extensive loop movements during catalysis. The loop movements in DHPS and PPPK may also play a role in determining the catalytic rate of the enzymes. The work presented here provides researchers with models of P. falsiparum DHPS and PPPK. These models can be used to design experiments to investigate resistance, design new drugs and probe the structure of the PPPK-DHPS bifunctional enzyme.Dissertation (MSc (Biochemistry))--University of Pretoria, 2005.Biochemistryunrestricte

Protein homology modelling and its use in South Africa

No abstract availabl

Insight into neutral and disease-associated human genetic variants through interpretable predictors

A variety of methods that predict human nonsynonymous single nucleotide polymorphisms (SNPs) to be neutral or disease-associated have been developed over the last decade. These methods are used for pinpointing disease-associated variants in the many variants obtained with next-generation sequencing technologies. The high performances of current sequence-based predictors indicate that sequence data contains valuable information about a variant being neutral or disease-associated. However, most predictors do not readily disclose this information, and so it remains unclear what sequence properties are most important. Here, we show how we can obtain insight into sequence characteristics of variants and their surroundings by interpreting predictors. We used an extensive range of features derived from the variant itself, its surrounding sequence, sequence conservation, and sequence annotation, and employed linear support vector machine classifiers to enable extracting feature importance from trained predictors. Our approach is useful for providing additional information about what features are most important for the predictions made. Furthermore, for large sets of known variants, it can provide insight into the mechanisms responsible for variants being disease-associated.Intelligent SystemsElectrical Engineering, Mathematics and Computer Scienc

Determination of common genetic variants within the non-structural proteins of foot-and-mouth disease viruses isolated in sub-Saharan Africa

The non-structural proteins of foot-and-mouth disease virus (FMDV) are responsible for RNA replication, proteolytic processing of the viral polyprotein precursor, folding and assembly of the structural proteins and modification of the cellular translation apparatus. Investigation of the amino acid heterogeneity of the non-structural proteins of seventy- nine FMDV isolates of SAT1, SAT2, SAT3, A and O serotypes revealed between 29 and 62% amino acid variability. The Leader protease (Lpro) and 3A proteins were the most variable whilst the RNA-dependent RNA polymerase (3Dpol) the most conserved. Phylogeny based on the non-structural protein-coding regions showed separate clusters for southern African viruses for both the Lpro and 3C protease (3Cpro) and sequences unique to this group of viruses, e.g. in the 2C and 3Cpro proteins. These groupings were unlike serotype groupings based on structural protein-coding regions. The amino acid substitutions and the nature of the naturally occurring substitutions provide insight into the functional domains and regions of the non-structural proteins that are critical for structure–function. The Lpro of southern African SAT type isolates differed from A, O and SAT isolates in northern Africa, particularly in the auto-processing region. Three-dimensional structures of the 3C protease (3Cpro) and 3Dpol showed that the observed variation does not affect the enzymatic active sites or substrate binding sites. Variation in the 3Cpro cleavage sites demonstrates broad substrate specificity.THRIP of the National Research Foundation of South Africahttp://www.elsevier.com/locate/vetmic2016-05-31hb201