International Journal of Biotechnology and Biochemistry ISSN 0973-2691 Volume 6 Number 3 (2010) pp. 419 426 Research India Publications http://www.ripublication.com/ijbb.htm Homo Sapiens DCC Gene: In Silico Analysis to Understand the Affect of Mutations in Colon Cancer Siddiqui M. Asif 1*, Amir Asad 1, Malik S. Anjali 1, Arya Arvind 1, Kapoor Neelesh 1 and Kumar Hirdesh 1 1 Department of Biotechnology, Meerut Institute of Engineering and Technology, N.H. 58, Delhi-Roorkee Highway, Baghpat Road Bypass Crossing, Meerut-250005, UP, India * Corresponding author E mail: - asifsiddiqui82@gmail.com Abstract Deleted in Colon Cancer (DCC) is a tumor suppressor gene, which is deleted in 70% of Colon cancers. Here, we investigate the protein product of DCC in much detail towards a better understanding how it leads to cancers. The protein sequence was retrieved through NCBI. Three mutations in protein sequence have been identified that are associated with colon cancer. Domain analysis shows that all the three mutations are present in functional domains. It was concluded from the results of physicochemical and structural analysis that the change of a single amino acid disrupts the structure of domain that leads to tumorigenesis. The analyses were performed using various in silico methods. Keywords: Deleted in Colon Cancer (DCC), NCBI, BLASTP, Multiple Sequence Alignment (MSA), PROTPRAM and SOPMA. Introduction Colon cancer is defined as any malignant neoplasm arising from the inner lining of the colonic epithelium, and is the third most common cancer and the third leading cause of cancer related deaths for both men and women in United States (American Cancer Society, 2008). The occurrence of colon cancer is mainly associated with the incidence of aberrant crypt foci (ACF), an earliest neoplastic lesion, which are clusters of mucosal cells with an enlarged and thicker layer of epithelia than the surrounding normal crypts that progress into polyps followed by adenomas and adenocarcinomas (Cappell, 2007). Epidemiological studies have suggested that colon cancer is a manifestation of a number of inherited cancer predisposition syndromes, including familial adenomatous polyposis, hereditary non-polyposis Colon cancer,
420 Siddiqui M. Asif et al and personal or family history of Colon cancer and/or polyps and inflammatory bowel disease (Rowley, 2005). Furthermore, other factors such as obesity, lack of exercise, smoking, alcohol consumption, diet rich in high fat, red and processed meats and inadequate intake of dietary fiber, fruits and vegetables are also associated with increased colon cancer risk (Cappell, 2007; Papapolychroniadis, 2004; Kim and Milner, 2007) and mutations due to loss of heterozygosity as well as chromosomal instability in oncogenes, or tumor suppressor genes have been implicated in 80% of colon cancer (Mutch, 2007). A tumor suppressor gene protects a cell to develop cancer. When that gene is mutated to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes. DCC is one such gene that is deleted in 70% of Colon cancers. DCC is functionally related to other dependence receptors such as p75ntr, the androgen receptor, RET, Ptc, UNC5H, and neogenin (Rabizadeh et al., 1993; Mehlen and Thibert, 2004). Such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied but inhibiting apoptosis in the presence of their respective ligands (Mehlen and Thibert, 2004). Recent studies found that the DCC gene is involved in the regulation of axonal development as a component of the Netrin-1 receptor (Fazeli et al., 1997). The DCC gene product was found to induce apoptosis activating caspase-3 and high levels of DCC expression were associated to an effective apoptotic process (Chen et al., 1999; Mehlen et al., 1998). Thus, DCC may function as tumor suppressor gene which controls programmed cell death. Mutations of the Deleted in Colon Cancer (DCC) with functional abnormalities in the encoded proteins may be involved in the development of variety of tumors. Therefore, the present investigation was carried out with the objectives to identify the DCC orthologous across different organisms, functional domains present in DCC, mutations that are responsible for carcinogenesis and to understand the evolutionary relationship of DCC. Methodology Searching for H. sapiens DCC gene The gene was searched using NCBI (http://www.ncbi.nlm.nih.gov) and its protein sequences was downloaded and stored in FASTA format. Homology searching Similarity search was performed using BLASTP against the NR databases with expect threshold as 1 and BLOSUM matrix 80. Identification of Mutations and Domain analysis The mutations responsible for colon cancer were identified using Uniprot Database (http://www.uniprot.org). The sequences of functional domains were retrieved and subjected to BLASTP to find out their occurrence in different model organisms.
Homo Sapiens DCC Gene: In Silico Analysis 421 Multiple sequence alignment (MSA) and evolutionary analysis Sequences of functional domains were subjected to T-Coffee (www.ebi.ac.uk/tcoffee) and MEGA4 (http://www.megasoftware.net) for multiple sequence alignment and phylogenetic analysis respectively. Physicochemical properties analysis and Secondary structure prediction The sequences of functional domains were further subjected to physicochemical properties analysis using PROTPRAM (http://www.expasy.ch/tools/protparam.html) and secondary structure prediction using SOPMA (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_sopma.html) with the aim to find out the affect of mutations on the properties and structure of domains. Results The DCC gene for H. sapiens was searched and its protein sequence (NP_005206) of length 1447 aa was retrieved from the protein database of NCBI and stored in FASTA format. Homology search results in a number of proteins similar to DCC provide that DCC is conserved across different organisms. The protein sequences having high score, identities and similarities and fewer gaps were retrieved from the results of the similarity search (Table 1). Three different mutations were identified from UNIPROT that are responsible for carcinoma and after obtaining the results of PROSITE domain search it was found that all the three mutations are present in three separate functional domains (Table 2). Sequences of all three functional domains were retrieved for all orthologous species and subjected to T-Coffee for MSA to find out the conservedness of mutated amino acid as well as similarities and differences among different species. On the basis of mol. wt., pi of domain, half-life, instability index, hydropathicity, alpha helix, extended strand, beta turn, random coil stability, analysis of domains were performed and it was observed that mutation of single amino acid has a great affect on the domain structure and stability (Table 3). Evolutionary tree obtained by MEGA4 can be divided in three separated clusters representing each domain suggesting independent evolution of domains (Fig. 4). Table 1: Similar proteins to H. sapiens DCC as obtained from BLASTP search. Organism Gene ID Protein Acc. No. Length of protein Expect Bit Score Identities P. troglodytes 455425 XP_512137 1447 0.0 3112 1445/1447 (99%) E. caballus 100054013 XP_001916613 1447 0.0 2975 1401/1447 (96%) M. musculus 13176 CAA59786 1447 0.0 2974 1396/1447 (96%) R. norvegicus 25311 NP_036973 1445 0.0 2968 1395/1447 (96%) C. familiaris 483976 XP_541094 1486 0.0 2932 1384/1435 (96%) M. domestica 100010164 XP_001363009 1448 0.0 2791 1312/1435 (91%) O. anatinus 100078379 XP_001509438 1546 0.0 2721 1272/1417 (89%)
422 Siddiqui M. Asif et al X. laevis 378529 AAI70145 1428 0.0 2512 1179/1448 (81%) B. taurus 537176 XP_617332 1208 0.0 2503 1168/1207 (96%) D. rerio 569360 NP_001030157 1421 0.0 2172 1029/1445 (71%) S. scrofa 100152102 XP_001926851 1112 0.0 1511 798/1156 (69%) G. gallus 395822 AAC59662 1443 0.0 1466 750/1461 (51%) Table 2: Domains/Motif along with their position, mutated position, length of mutation and description. Domain/Motif Ig-like domain Position Mutated position 154-214 168 Fibronectin type-iii domain 946-1041 1039 Neogenin_C domain 1073-1445 1375 Length Description 1 M T (Esophageal carcinoma) 1 F S (Colon cancer); somatic mutation. 1 P H (Colon carcinoma) Table 3: Analysis of physicochemical properties and secondary structure prediction of functional domains (N-Normal, M-Mutated) using PROTPRAM and SOPMA. Features IG-LIKE FN3 Neogenin_C N M N M N M Mol. Wt. 39813.1 39853.2 6612.6 6582.5 10966.6 10906.5 pi of Domain 7.76 7.77 5.55 5.55 6.51 6.51 Half-Life 1.9 hrs 1.9 hrs 30 hrs 30 hrs 1.9 hrs 1.9 hrs Instability index 76.06 76.18 44.10 38.14 20.07 22.08 Hydropathicity -0.466-0.471-0.077-0.120-0.197-0.234 Alpha helix 12.87% 16.09% 1.64% 0.00% 16.67% 11.46% Extended strand 7.77% 7.77% 42.62% 42.62% 33.33% 34.38% Beta turn 0.27% 0.27% 4.92% 4.92% 4.17% 4.17% Random coil 79.09% 75.87% 50.82% 52.46% 45.83% 50.00% Stability Stable unstable unstable stable stable stable
Homo Sapiens DCC Gene: In Silico Analysis 423 Discussion Similar to other cancers, Colon cancer is a multifactorial disease. Diet, life-style, ageing (Boyle and Leon, 2002), and genetic predisposition (mutation) are known to affect its development. Prediction and anlaysis on these mutated regions can be use in phramacogenomics for drug discovery process. In silico approaches are very quick to produce desirable results. These approaches easily help us to performed homology searching, domain prediction and also the prediction of mutated regions using various biological tools and databases. Integration and analysis of above predictions results in the important finding for the future drug development. Conclusion Sequences with high similarity are structurally conserved and are functionally related. With this approach homology searching was done and DCC orthologous were identified in twelve different organisms (Table 1). A total of three mutations were identified and further it was confirmed from PROSITE results that mutations responsible for carcinoma were lie within the functional domains of DCC gene (Table 2). MSA of domains shown that mutated amino acid M is conserved in IG LIKE domain except G. gallus (Fig. 1), mutated amino acid F is conserved in FN3 domain except D. rerio (Fig. 2) and mutated amino acid P shows a higher degree of variability across different organisms (Fig. 3). It was also observed from the results of MEGA4 (MSA tool) that the three domains were evolved separately as the tree is clearly separated in three different clusters representing each domain (Fig. 4).Table 3 shows the physico-chemical properties of predicted domains (using PROTPRAM and SOPMA) before and after mutation. From the results we concluded that the change of single amino acid will disrupt the confirmation of protein that may prevent it from its normal function (Table 3). Evolutionary tree suggests that the three domains were evolved separately. Figure 1: Multiple alignment of IG - LIKE domain using T-Coffee showing that Methionine (M) is conserved except G. gallus.
424 Siddiqui M. Asif et al Figure 2: Multiple alignment of FN3 domain using T-Coffee showing that Phenylalanine (F) is conserved except D. rerio. Figure 3: Multiple alignment of Neogenin C domain using T-Coffee showing variability of Proline (P).
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