STRUCTURAL AND FUNCTIONAL ANALYSIS OF AF9-MLL ONCOGENIC FUSION PROTEIN USING HOMOLOGY MODELING AND SIMULATION BASED APPROACH Original Article MEDHA DAVE 1 , ADITI DAGA 2 , RAKESH RAWAL 3* 1 Department of Bioinformatics, Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar, Gujarat, India, 2 Department of Microbiology, MVM Science Collage, Saurashtra University, Rajkot, Gujarat, India, 3 Received: 26 Aug 2015 Revised and Accepted: 27 Oct 2015 Department of Cancer Biology, The Gujarat Cancer and Research institute, Ahmedabad, Gujarat, India. Email: rakeshmrawal@gmail.com ABSTRACT Objective: AF9-MLL has been implicated in the pathogenesis of AML, New Therapeutic regimens are prerequisite for this category of hematological malignancy due to the poor prognosis. The experimental 3D structure of AF9-MLL is not available. Therefore, present study aims in developing the homology model and evaluating the best model through Energy Minimization and MD simulation. The structure further analyzed for functional Annotation. Methods: To the best of our knowledge, our study is novel in terms of predicting homology based 3D model of AF9-MLL leukemogenic fusion protein, facilitated by I-TASSER. The 3D modeled structure was subsequently optimized with MD simulation for 2 ns. Further stereo-chemical analysis and verification of the best structure so obtained were undertaken by different computational programs including PROCHECK, PROVE, Verify3D and ERRAT. Results: Homology model predicted from I-TASSER and refined by YASARA showed results with 86.5% residues in the most favorable region, 14.7% in the allowed region, 0.8% in the generously allowed region and 0.3% in the disallowed region. The RMSD between the modeled and the refined structure was found to be 2.37 Å. The results of ERRAT, Verify_3D, Prove and ProSA confirmed that the simulated model and energy minimized model is very good then the predicted raw model. The final structure was successfully submitted in Protein Model Database (PMDB) under ID: PM0080061. Conclusion: In this study, homology model was developed and Validated for MLL-AF9 using bio-informatics tools. These analyses validated that the simulated model is best, robust as well as reliable enough to be used for future study and the functional analysis shows the presence of CXXC domain. Eventually, these molecular and structural studies result in advancement of newer therapies. Keywords: MLL, Fusion Protein, Molecular modeling, Simulation, Structure Prediction. INTRODUCTION Chromosomal anomalies are regarded as one of the major hallmark of neoplastic cells, and the continual occurrence of chromosomal instability has been manifested in human neoplasia. Amongst these, recurrent reciprocal chromosomal translocations between non- homologous chromosomes are exclusively found to be implicated in the etiology of numerous hematological malignancies [1]. Balanced chromosomal rearrangements are crucial cellular mechanism, which lead to malignant transformation of normal cell via formation of chimeric fusion protein. 5–6% cases of Acute Myeloid Leukemia (AML) and 5–10% of acute lymphoblastic leukemia’s (ALLs) cases are observed with the presence of chromosomal translocations involving the long arm (q23) of chromosome 11 [2]. Remarkably, the occurrence of 11q23 rearrangements is appreciably higher in pediatric AML and infant ALL. The Mixed-Lineage Leukemia (MLL) gene encodes the complex transcription factor that leads to the formation of unique hybrid genes, whose protein products are believed as critical elements in initiation of leukemogenesis. This multi exonic segment contains cluster of translocation breakpoints around exon 8 and various translocations partner genes combine with MLL gene yielding specific fusion protein responsible for development of a specific subtype of leukemia [3-8]. Till date, there have been more than 50 fusion gene partners reported for MLL. Amongst all MLL translocations, around 50% of infant AML cases comprises of t(9,11)(p22,q23) rearrangement. AF9 gene also known as LTG9 or MLLT3 is located at short arm p22 of chromosome 9 [9-11]. From several experimental studies, it was evident that leukemogenesis is caused by formation of MLL-AF9 fusion protein but still the mechanism of these partner genes is anonymous. In contrast, few other in-vitro and in-vivo analysis revealed that MLL-AF9 alters myeloid progenitor cells and suppresses specific HOX gene e. g. mice with knock-in AF9-MLL fusion gene demonstrated anomalous proliferation of hematopoietic cell and developed AML identical to patient with t (9; 11) translocation [12-14]. Also, MLL and AF9 wild protein forms participate indispensably during hematopoiesis/embryogenesis process and are elements of protein complexes resulting in target gene transcriptional initiation (MLL) and elongation (AF9). Therefore it is hypothesized that MLL-AF9 fusion combines these characteristics, resulting in increased activation of target genes which may be interrupt hematopoietic cell differentiation and ultimately leads to leukemogenesis [15-19]. As the occurrence of 11q23 translocations is associated with extremely poor prognosis, novel therapeutic strategies are needed to be explored for this category of hematological malignancy. In spite of tremendous interest concerned with designing of target specific drug like molecules against this fusion protein. However, blocked by the unavailability of pertinent structural data. Additionally, structural & functional analysis of this chimeric gene (AF9-MLL) is required to be profoundly studied to get better insight into the causal mechanism leading to leukemogenesis. To resolve these problems, development of three dimensional molecular structure of AF9-MLL fusion protein is of prime importance with aim to discover newer alternative drug like compounds that precisely targets MLL-AF9 positive AML. The fig. 1 shows the reciprocal chromosomal translocation between chromosome 9 and 11. Due to these translocation two genes fused and codes for oncogenic Fusion protein. To the best of our knowledge, our study is novel in terms of predicting the homology based 3D model of AF9-MLL leukemogenic fusion protein, undertaken by I-TASSER. The 3D model structure was subsequently optimized with MD simulation and further stereo-chemical validation studies and functional analysis of the best structure so obtained were executed by means of different computational International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 7, Issue 12, 2015 Innovare Academic Sciences