Genome-wide and structural analyses of signalling proteins with special reference to protein kinases

Abstract

The work done in the dissertation involves genome?wide sequence analysis on the Plasmodium falciparum proteins with a broad objective of understanding further the functional diversity and evolution. P. falciparum is one of the most prevalent pathogens in the third world countries and worldwide efforts have concentrated on developing effective drugs against this pathogen to combat malaria. The genome of P. falciparum is atypical and consists of long stretches of low?complexity regions in the proteins encoded in the genome. The composition of the genome itself is atypical with an above?average percentage of asparagines (which is also present in most of the low?complexity regions) among other anomalies. Due to the atypical nature of P. falciparum proteins, conventional methods involving homology?based information transfer are not entirely successful and presently proper function annotation is not available for more than 60% of the proteome. Due to the lack of annotation and failure of conventional homology?based tools in detecting remote homologs, many enzymes that are expected in the organism based on experimental studies are not yet identified. It has been seen that close to 130 enzymatic steps involved in various pathways in plasmodium are not yet detected through any computational tool. A typical example is cytidine deaminase involved in de novo pyrimidine biosynthesis, for which experimental proof exists that it operates inside the organism. We have attempted to provide information about such "missing enzymes" in P. falciparum metabolic pathways (Chapter 2). The methodology involves recognition of pathway 'holes' and the required enzymatic function based on the information available on orthologs in other organisms. These orthologs are then used as queries and an in?house?developed powerful approach for remote homology detection has been employed to detect the "missing" enzymes. Using this approach we have successfully annotated 48 of the "missing" enzymes which includes 20 non?redundant enzymes. The accuracy of the method is assessed by a fold?recognition method and the results of fold recognition for all the 48 proteins are consistent with our annotation. P. falciparum is extensively studied and hence information about various aspects of its biology and physiology is available. More importantly, stage?specific gene expression of P. falciparum genes has been identified using the available microarray datasets which is a valuable input for the annotation of hitherto unknown functions (Chapter 3). We have applied a clustering?based approach for analysis of the presence of a pathway in P. falciparum using the microarray data. The methodology involves assessment of the expression of the enzymes required for the operation of a pathway in the cell. The main drawback of the method is in assuming that no transfer of metabolites takes place to/from the host cells. The assumption is made since it is difficult at present to correctly ascertain the metabolites transferred between the host cell and P. falciparum cells. Using our approach we have found that 4446 out of 5222 genes show stage?specific expression and these can be clustered into various groups based on which stages the genes are potentially expressed. The evolution of proteins in an atypical parasite like P. falciparum is an interesting case study. We chose small monomeric G?proteins and their exchange factors for the analysis of their evolution in P. falciparum. Since the G?proteins are involved in signal transduction, the interactions should be highly conserved as otherwise the regulation of physiological processes would be impaired. We chose the Arf sub?family of G?proteins as an example of small G?protein sub?family (Chapter 4). We detected 14 Arf?like sequences from P. falciparum genome and an exchange factor protein containing the typical Sec7 domain using the sensitive homology detection approach. We modeled the structure of an atypical Arf protein and its complex with its cognate substrate Sec7 and found that most of the core functional residues and the interface are largely conserved; however, there are flanking regions which are quite unique and found only in plasmodium.