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Title: Analyses of serine proteases and serine protease inhibitors : Evolutionary and structural insights
Other Titles: Analyse van serine proteasen en serine protease inhibitoren: evolutionaire en structurele inzichten
Authors: Kuchi, Srikeerthana
Issue Date: 28-Sep-2012
Abstract: Analyses of proteases and protease inhibitorsProteolytic enzymes (also known as proteases, proteinases) are enzymes capable of proteolysis (breakdown of proteins), by hydrolysis of the peptide bonds that link amino acids in a polypeptide chain. There are different types of proteases that are found to be important in various biological functions, such as in digestion, immunity, blood clotting etc,. whose deficiency leads to various important diseases such as, hemophilia B, C, neurogenerative disorders etc,.The analyses of proteases in this study consist of three parts, which include1. The sequence and structural analysis of proteases and their inhibitors.2. Development of a residue conservation method to identify members of enzyme families.3. Identification of immunity coding genes and serine protease coding genes in Daphnia magna.Proteolytic enzymes – Sequence analysisEvolutionary analysisSerine proteases are proteases which has at least one serine residue in the active site of the enzyme. The aim of this study is to exhibit the ancestral enzyme that had emerged into different classes and subclasses of serine protease.The serpin excluded list of 160 serine proteases of Homo sapiens have been obtained from KEGG database. Each of the selected proteins have been searched for absolute homologues (e-value lesser than or equal to 0.0001) from within all the human proteins using the Blastp program. 97 out of 160 proteins had absolute homologues only from within the serine proteases, which showed that there is high sequence similarity and homology, which suggest that, the classes and subclasses would have emerged from a single ancestral enzyme with a wide-spread cleavage activity. The multiple sequence alignment of the proteins has been performed with clustalW and the phylogenetic analysis with Phylip package. The resulted tree suggested that the different classes and subclasses had a common ancestor, from which they have emerged by different gene duplication events.Future work includes the evolutionary analysis of serine protease inhibitors (serpins) and also finding homologues (of the single ancestral enzyme which would be the result of a sequence clustering technique (described later)) from other organisms and building up the final ancestral enzyme which would have had a wide-spread cleavage activity.Structural analysisNeuroserpinA newly found enzyme, neuroserpin, a selective inhibitor of tPA(tissue-type plasminogen activator) has been taken for the structural analysis, since the unique structural patterns that differ from other serpinfamilies were thought to be involved in the function of the neuroserpin and distortions of these patterns affects the function of this serpin. The crystal structure of the human neuroserpin has been deposited in PDB as 3FGQ, which is found to have an important role to play in synaptic plasticity, memory and learning. In mouse models of cerebral infarction, neuroserpin acts as a neuro-protectant and protects the mouse against cortical damage.The aim of this study is to identify new members of the neuroserpin family in other organisms and also to identify the unique structural patterns and stabilizing interactions of the neuroserpin family. By comparative sequence analysis (tblastn and Pfam analysis), 13 new neuroserpins in different organisms have been identified. By comparative structural analysis, the unique structural patterns such as the omega (&###937;-) loop, crystal contacts that stabilize the RCL (reactive center loop) and a consensus sequence (Asn-Leu-Val) at the end of helix ‘F’ have been identified. Macaca fascicularis, a non-human primate has found to contain neuroserpin, that is 98% similar to that of the human neuroserpin.The importance of omega loops in human proteins have been analyzed by comparing and aligning the secondary structure elements of the omega loop containing proteins with their structural homologues, which might pave way for drug design strategies against the diseases, caused by the mutations of the omega loops. Functionally different proteolytic enzymes, serine racemases and the catalytic domain of the threonine deaminases have found to contain a similar fold, demonstrated by the superimposition of their secondary structures and comparative modeling.Residue conservationResidue conservation in a multiple sequence alignment is an indirect evidence for the importance of the residues of question in maintaining structure and function of a protein. In contrast, the conservation of the functionally important residues can be used as a tool to distinguish and identify different members of enzyme families, which differ generally in the active site residues. Using information-theoretic approach, a measure would be developed in order to distinguish and identify serine protease and serpin sub-families and an enhanced generalization of that method would be used to classify members of any enzyme family.Gene identification in Daphnia magnaBased on the 82 genes that are known to involve in the immunity of D. pulex, 20 new genes have been identified by comparative inter-genome analysis. Genes that are involved in immunity of D. magna have also been identified using these 82+20 genes, which also include the chitinases. These genes will be searched for polymorphism using re-sequencing techniques. Genes that code for the serine protease enzymes and their inhibitors will also be identified using the available data.Proteolytic enzymes are very important in various biological functions and analyses of these enzymes provide insights into structural, functional and evolutionary mechanisms of these enzymes and also might help in the drug design against diseases caused by the deficiency of these enzymes.Publications1. Poster : Evolution of serine proteases as different subclasses of the enzyme family in Homo sapiens. APBC’10, The Eighth Asia Pacific Bioinformatics Conference, Bangalore, India, 18-21 January 2010.2. Conference Paper : Comparative sequence and structural analyses of neuroserpin – the serine protease inhibitor family, In Proceedings of the International Symposium on Biocomputing (Calicut, Kerala, India, February 15 - 17, 2010). ISB '10., pp. no 1-7, ACM, New York. http://doi.acm.org/10.1145/1722024.1722031.3. Journal : Comparative sequence and structural analyses of neuroserpin: the serine protease inhibitor family, International Journal of Data Mining and Bioinformatics, 2010 (Accepted), In press.4. Conference Paper : Structural similarities between the catalytic domain of threonine deaminase and the mammalian serine racemases, International Conference on Advances in Computer Engineering, ACE 2010., ACE 2010, Bangalore, 20-21 June 2010, pages 371-373, IEEE Computer Society Digital Library (CSDL).5. Poster : Comparative inter-genome analysis using ESTs to identify new immune genes in Daphnia pulex, DGC’10, Daphnia Genomics Consortium 2010, Leuven, 26-30 March 2010.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Department of Biology - miscellaneous
Biology @ Kulak
Computer Science @ Kulak

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