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Unraveling the segmentation clock with bioinformatics tools and techniques.
Sourav Roy School of Informatics Indiana University Advisors: Dr. Predrag Radivojac & Dr. Santiago Schnell Capstone Instructor: Dr. Mehmet Dalkilic. 11/27/2018 Capstone Presentation /21/2006
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Background and Motivation Notch Signaling Pathway (NSP)
Introduction Background and Motivation Notch Signaling Pathway (NSP) Structure of NSP proteins (NSPPs) Potential Binding Sites in NSPPs Probable Protein-Protein Interactions Interaction Map & Boolean Model for the Pathway Overview Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Beginning of Life Sourav Roy School of Informatics Indiana University
Introduction Background Beginning of Life zygote Cleavage Sourav Roy School of Informatics Indiana University Blastula Start of Gastrulation 11/27/2018 Capstone Presentation /21/
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Vertebrate segmentation
Introduction Background Vertebrate segmentation Segmentation was nature’s answer to the development of complex organisms Presomitic mesoderm (PSM) or paraxial mesoderm gives rise to vertebrate equal- sized segments – somites Repeated formation of intersomitic boundaries is driven by a molecular oscillator (segmentation clock) Sourav Roy School of Informatics Indiana University 11/27/2018 Source: Cancer Research UK Website Capstone Presentation /21/
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Periodicity Sourav Roy School of Informatics Indiana University
Introduction Background Motivation Periodicity Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Why are we interested? Sourav Roy School of Informatics
Introduction Background Motivation Why are we interested? Sourav Roy School of Informatics Indiana University To date models of the pathway have been built on the basis of knockout experiments, but nobody knows whether there is a single interaction or a cascade for most of the cases 11/27/2018 Capstone Presentation /21/
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Notch Signaling Pathway
Introduction Background Motivation Notch Pathway Notch Signaling Pathway Sourav Roy School of Informatics Indiana University 11/27/2018 Source - Biocarta Capstone Presentation /21/
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Proteins in Mouse Notch Signaling Pathway
Introduction Background Motivation Notch Pathway Proteins in Mouse Notch Signaling Pathway Notch1 - Transmembrane receptor. Dll1 - Ligand. Dll3 - Ligand. Psen1 - Membrane bound protein with γ- secretase activity. Lfng Beta-1,3-N- acetylglucosaminyltransferase. RbpSuh DNA binding transcription. Mesp bHLH protein. Hes bHLH transcription repressor. Hes7 - bHLH transcription repressor. Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Flexibility, Rigidity and binding
Introduction Background Motivation Notch Pathway Structure of NSPPs Flexibility, Rigidity and binding Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Order and Disorder Sourav Roy School of Informatics Indiana University
Introduction Background Motivation Notch Pathway Structure of NSPPs Order and Disorder Ordered Regions Disordered Regions Sourav Roy School of Informatics Indiana University 11/27/2018 Source : Bachinsky and V.V. Solovyev Capstone Presentation /21/
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Disorder Prediction Sourav Roy School of Informatics
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder Prediction Disorder has a role to play in signal transduction, cell cycle regulation and transcriptional activity (Dunker et al. 2002; Iakoucheva et al. 2002; Ward et al. 2004) Swiss-Prot (June 2005, version) database was downloaded and all mouse proteins were extracted with the help of a Matlab code VL3 model (Obradovic et al., 2003) was used to calculate the disorder for each protein Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Notch Signaling Pathway Proteins Intrinsically Disordered?
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway Notch Signaling Pathway Proteins Intrinsically Disordered? Proteins %Disorder Average of 9 pathway proteins 56.4 Average of all 9448 Mouse Proteins in the Swiss Prot database on 05/10/2005 32.2 Sourav Roy School of Informatics Indiana University A Matlab code was used with VL3 within it to calculate the average disorder 11/27/2018 Capstone Presentation /21/
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Disorder Percentage in general
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway Disorder Percentage in general Sampling Method I: a) 10 / 30 / 60 / 90 / 100 / 300 / 600 / 900 /1000 random samples were taken from the set of 9448 proteins. b) Average Disorder and standard deviation calculated with the help of a Matlab code. c) t-test done – Fischer’s t-test. Sourav Roy School of Informatics Indiana University Steps a) and b) were done with the help of a Matlab code JAVA code was used for the t-test 11/27/2018 Capstone Presentation /21/
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t-test results 9 10 60 100 600 1000 Sourav Roy School of Informatics
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway t-test results # of Proteins Mean Sd P value Significance 9 56.4 25.2 0.0062 Yes 10 26.6 23.0 0.5029 No 60 31.6 26.5 0.8572 100 29.8 23.1 0.3628 600 30.6 25.7 0.1556 1000 32.1 26.1 0.9283 Sourav Roy School of Informatics Indiana University % Disorder # of Proteins # of Proteins 11/27/2018 Capstone Presentation /21/
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Sampling Method II Different sample sets 1,2,3,…,11 were taken.
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway Sampling Method II Different sample sets 1,2,3,…,11 were taken. Each of the sets had 9 random proteins from the entire set of 9448. The average of the 9 proteins in each set was calculated. The average of the averages and standard of the sample sets was calculated. Sourav Roy School of Informatics Indiana University A Matlab code was used for this method 11/27/2018 Capstone Presentation /21/
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Sampling Method II results
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway Introduction Background & Motivation Notch Pathway Ordered & Dis- ordered Proteins Disorder in the Pathway Sampling Method II results N # of Sample sets % Disorder Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Potential binding sites
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs Potential binding sites MoREs - short, interaction-prone loosely-structured or semi-structured regions in intrinsically disordered proteins α-MoRE a subclass of MoREs Source: Oldfield et al Biochemistry 2005 Ordered Regions Disordered Regions α-MoRE Sourav Roy School of Informatics Indiana University 11/27/2018 Source : Bachinsky and V.V. Solovyev Capstone Presentation /21/
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α-MoRE Prediction MoREs Sourav Roy School of Informatics
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs α-MoRE Prediction α-MoRE score Amino acid residue Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Disordered Regions and α-MoREs
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs Disordered Regions and α-MoREs Sourav Roy School of Informatics Indiana University Ordered regions, Disordered regions α-MoREs at p > α-MoREs at p >0.7 11/27/2018 Capstone Presentation /21/
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To check if the disordered regions and the α-MoREs are conserved
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs Evolution To check if the disordered regions and the α-MoREs are conserved PSI- BLAST of the pathway proteins Extraction of the aligned regions with the help of a PERL script Clustering of the aligned regions with the help of PERL script Multiple sequence alignment of the clustered sequences by ClustalW Parsing the Clustal report and calculating the entropy of each column with the help of a Matlab code Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Results for Hes7 Sourav Roy School of Informatics Indiana University
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs Evolution Results for Hes7 Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Prediction of possible protein-protein interactions
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs Evolution Probable PPI Prediction of possible protein-protein interactions ADVICE webtool was used for the prediction ADVICE is a web server providing Automated Detection and Validation of Interaction based on the Co-Evolutions between interacting proteins. It automated the steps needed to compute the similarities between proteins' evolutionary histories to detect co-evolved interacting proteins Step 1. Homolog Search. ADVICE detects orthologous sequences for pair(s) of protein sequences and retrieve the orthologous sequences if both appear in the same species Step 2. Distance Matrix Construction. ADVICE then constructs the distance matrix for both orthologous group of sequence. The distance matrices are derived from multiple sequences alignments using ClustalW Step 3. Linear correlation coefficient computation. The Pearson's Correlation Coefficient formula is used to calculate the similarities between the two distance matrices: The result r will fall into -1 to 1. Previous studies have indicated that interacting proteins share similarity in their evolutionary histories and have high r-value (>=0.8) Sourav Roy School of Informatics Indiana University 11/27/2018 Source: ADVICE website Capstone Presentation /21/
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Chances of interaction (r)
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the Pathway MoREs Evolution Probable PPI Predicted PPI Protein Pairs Chances of interaction (r) Notch1 & Dll1 89.50% Notch1 & Psen1 97.20% Notch1 & Lfng 98.90% Notch1 & RbpSuh 98.20% RbpSuh & Hes7 97.70% Hes1 & Lfng 97.80% Notch1 & Hes7 Lfng & Dll1 88.70% Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Interaction Map Sourav Roy School of Informatics Indiana University
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Interaction Map Notch Dll1 Dll Notch NICD Psen1 Rbp/Suh NICD Rbp/Suh Nucleus Sourav Roy School of Informatics Indiana University dll mesp2 Mesp2 lfng Lfng hes1/7 Hes1/7 Cell1 Cell2 11/27/2018 Capstone Presentation /21/
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Mesp2t ( Notch1t Psen1t RbpSuht ) Dll1/3t+1
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Boolean Model Boolean Logic Mesp2t ( Notch1t Psen1t RbpSuht ) Dll1/3t+1 Dll1/3t Lfngt Notch1t+1 {( Notch1t Psen1t RbpSuht ) Mesp2t } Hes1/7t Lfngt+1 Notch1t Psen1t RbpSuht Mesp2t+1 (Notch1t Psen1t RbpSuht) Hes1/7t Hes1/7t+1 Notch1t Psen1t RbpSuht+1 Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Expression of hes/her regulates the
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Boolean Model Expression of hes/her regulates the segmentation clock Sourav Roy School of Informatics Indiana University The molecular network constituting the segmentation clock in two adjacent cells. Notch pathway HER1/HER7- based oscillator, excluding the Notch pathway components Source: Pourquie & Goldbeter, Current Biology 2003 11/27/2018 Capstone Presentation /21/
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Oscillations within the pathway
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Boolean Model Oscillations within the pathway time Dll1 Notch1 Psen1 Lfng Mesp2 RbpSuh Hes7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Somitogenesis - controlled by segmentation clock.
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Boolean Model What was known? Somitogenesis - controlled by segmentation clock. Segmentation clock in turn is controlled by the expression of the hairy and enhancer of split family of genes. Expression of Hes family determined by the Notch signaling pathway. Review Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Information from this study:
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Boolean Model Review Information from this study: The NSPPs have more disordered regions than usual The MoREs seem to be either in the disordered regions or near to those – probable binding sites for binding partners These regions have low entropy and therefore seem to be conserved Interaction map on the basis of predicted ppi and genetic interactions Got the required oscillations from the Boolean model Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Review Sourav Roy School of Informatics Indiana University
Introduction Background Motivation Notch Pathway Structure of NSPPs Disorder in the pathway MoREs Evolution Probable PPI Interaction map Boolean Model Review Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/
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Thanks !!! Acknowledgements Dr. S. Schnell. Dr. P. Radivojac.
Dr. M. Dalkilic. Dr. H. Tang. Dr. S. Kim. Dr. C. Raphael. Junguk Hur. Systems Biology Group. School of Informatics. Everyone related to Bioinformatics at IUB. My family and friends. Thanks !!! Sourav Roy School of Informatics Indiana University 11/27/2018 Capstone Presentation /21/2006
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