Updated: Jan 23
N.A. Mahalakshmi Srinivasan firstname.lastname@example.org
Lets find out the key regulated nodes in the Human Glycolysis Pathway, using KEGG PATHWAY! Any biochemical pathway have their own set of nodes (biomolecules) and the interactions between them represented as edges. Nodes get regulated and regulate other nodes. Using one of the vital biochemical processes of a cell - Glycolysis, we shall find out the most regulated nodes from the pathway. Glycolysis is the process that describes the breakdown of glucose (sugar) or glycogen (stored carbohydrate) and releases energy. While a pathway requires all its nodes and edges to complete the process, this study will help us understand the kind of regulations into which each of the node is involved. Though a node could play a significant role in regulation, it doesn’t stop it from being regulated by other nodes in the pathway. The scope of this project is to get information about highly regulated nodes of the biochemical pathway - Glycolysis.
We shall be able to analyse using KEGG and R. KEGG stands for “Kyoto Encyclopedia of Genes and Genomes”. It has been developed and maintained by Kaneshisa Laboratories, since 1995. KEGG is a database resource for understanding high level functions and utilities of the biological system such as single cell, organism or ecosystem, using molecular data.
With KEGG, we can get an understanding of:
• The bimolecular pathways, • Functional hierarchies of the biomolecules, • Explore subset of pathways or functional units,• Functional orthologs,
• Gain access to: Biological genomes which includes metagenomes, Genes, Proteins, Small molecules, Glycans, Enzymatic reactions, Perturbed states of the molecular network system when in a diseased state to investigate the “network variation maps”, Structurally and functionally related group of drugs.
The Architecture of KEGG website comprises of:
Top page, which is the KEGG homepage,
KEGG2 page is the main entry point to the KEGG web service, DB (database)
DB (database) contents,
Software tools (at the KEGG main site excluding those at the GenomeNet
GenomeNet is the mirror site of KEGG website. The GenomeNet service has been developed by the Kanehisa Laboratory in Kyoto University as part of the research projects.
Dataset for this project shall be the KEGG pathway for Human Glycolysis: hsa: 00010. During this analysis, we will see how KEGG PATHWAYS can be searched, visualised using R and obtain the answer for question in hand by probing this KEGG PATHWAY.
TASK I: SEARCH FOR KEGG PATHWAY OF INTEREST AND VISUALISE IN R
#KEGGgraph package: It parses the regularly updated KGML (KEGG XML) files into graph models maintaining all essential pathway attributes. The package offers functionalities including parsing, graph operation, visualization. #Rgraphviz package: Interfaces R with the graphviz (Graph Visualization) library for plotting R graph objects from the graph package.
#igraph package: to create a pathway
install.packages("BiocManager") library("BiocManager") BiocManager::install("KEGGREST") BiocManager::install("KEGGgraph") BiocManager::install("Rgraphviz") install.packages("igraph")
#keggFind function: Finds Entries With Matching Query Keywords.
Output: path:map00010: 'Glycolysis / Gluconeogenesis’
#Viewing the KEGG PATHWAY, in R:
file1 <- system.file("extdata/hsa04010.xml",package="KEGGgraph") graph1 <- parseKGML2Graph(file1,expandGenes=TRUE) graph1 plotKEGGgraph(graph1, y = "neato", shortLabel = TRUE, useDisplayName=TRUE)
#system.file function: Finds the full file names of files in packages. This checks the existence of the specified files with file.exists. So file paths are only returned if there are sufficient permissions to establish their existence. #parseKGML2Graph function: This function is a wrapper for parseKGML and KEGGpathway2Graph. It takes two actions: first it reads in the KGML file and parses it into an object of KEGGPathway-class, the second step it calls KEGGpathway2Graph function to return the graph model.
#The following pathway is the Human Glycolysis Pathway, plotted in R:
A graphNEL graph with directed edges
Number of Nodes = 265 Number of Edges = 876
TASK II: QUERYING THE NODES AND EDGES, TO FIND THE NODES PLAYING KEY ROLE IN REGULATION:
# To find the number of incoming edges for a node. These data have significance in understanding the role of the node in the pathway
inward <- sapply(inEdges(graph1), length) maxinward <- sort(inward, decreasing=T) maxinward[1:5]
#inEdges function: Returns a list of all incoming edges for the specified nodes. #sapply function:The purpose is to apply function to every element and obtain the output. #sort function: Sort (or order) a vector into ascending or descending order.
#Obtaining information about a particular node / nodes. The node 'has:5923' has maximum number of incoming edges.
getnodedata <- keggGet("hsa:5923") getnodedata
[]$NAME  "RASGRF1, CDC25, CDC25L, GNRP, GRF1, GRF55, H-GRF55, PP13187, ras-GRF1"
[]$DEFINITION  "(RefSeq) Ras protein specific guanine nucleotide releasing factor 1"
"Ras-specific guanine nucleotide-releasing factor 1"
"Homo sapiens (human)"
[]$PATHWAY hsa04010 hsa04014 hsa04510
"MAPK signaling pathway" "Ras signaling pathway"
[]$BRITE  "KEGG Orthology (KO) [BR:hsa00001]"  " 09130 Environmental Information Processing"  " 09132 Signal transduction"  " 04014 Ras signaling pathway"  " 5923 (RASGRF1)"  " 04010 MAPK signaling pathway"
 " 5923 (RASGRF1)"  " 09140 Cellular Processes"  " 09144 Cellular community - eukaryotes"
 " 04510 Focal adhesion"  " 5923 (RASGRF1)"
[]$POSITION  "15q25.1"
[]$MOTIF  "Pfam: RasGEF RhoGEF PH RasGEF_N GOLGA2L5 PH_11 IQ PH_13 Sec34"
[]$DBLINKS  "NCBI-GeneID: 5923" "NCBI-ProteinID: NP_002882"  "OMIM: 606600" "HGNC: 9875"  "Ensembl: ENSG00000058335" "Vega: OTTHUMG00000144172"  "Pharos: Q13972(Tbio)" "UniProt: Q13972 A8K270"
[]$AASEQ A AAStringSet instance of length 1
width seq  1273 MQKGIRLNDGHVASLGLLARKDGTRKGYLSKRSS...QAKVTQYLLDQSFV MDEESLYESSLRIEPKLPT
[]$NTSEQ A DNAStringSet instance of length 1
 3822 ATGCAGAAGGGGATCCGGCTGAATGATGGCCACG...TCTCTCCGAATAGA ACCAAAACTCCCCACCTGA
#getKEGGedgeData function: The 'get' methods extracts KEGG edge attributes from a graph produced by calling parseKGML2Graph or KEGGpathway2Graph.getedgedata <- getKEGGedgeData(graph1)
TASK III: REPRESENTING A NODE WITH THE HIGHEST NUMBER OF REGULATIONS IN A GRAPHICAL FORMAT:
#Creating a compilation of the nodes and edges, exporting data as .txt file to the current working directory class(getedgedata) n <- as.matrix(getedgedata)
class(n) write.table(n, “edgedata.txt")
Output: 'list' ‘matrix’ And exported file, as follows:
The exported text is with the details of connectivity between nodes. Since, we are studying here about a node being regulated, we have to consider data, where this node is entry2ID. Please refer to the image above.
#Creating a graph which represents graphically the regulation a given node by other nodes. We have chosen a single node (edges directed towards from this node).
g1<-graph(edges=c("hsa:10368","hsa:5923", "hsa:10369","hsa:5923", "hsa: 27091","hsa:5923", "hsa:27092","hsa:5923", "hsa:55799","hsa:5923","hsa: 59283","hsa:5923","hsa:59284","hsa:5923","hsa:59285","hsa:5923","hsa:773","hsa: 5923","hsa:774","hsa:5923","hsa:775","hsa:5923","hsa:776","hsa:5923","hsa: 777","hsa:5923","hsa:778","hsa:5923","hsa:779","hsa:5923","hsa:781","hsa: 5923","hsa:782","hsa:5923", "hsa:783","hsa:5923", "hsa:784","hsa:5923","hsa: 785","hsa:5923","hsa:786","hsa:5923","hsa:8911","hsa:5923","hsa:8912","hsa: 5923","hsa:8913","hsa:5923", "hsa:9254","hsa:5923","hsa:93589","hsa:5923"), directed=F)
Below are the list of 5 nodes, each regulated by 26 other nodes: Ras protein specific guanine nucleotide releasing factor 1 Ras protein specific guanine nucleotide releasing factor 2 RAS guanyl releasing protein 1 RAS guanyl releasing protein 2 RAS guanyl releasing protein 4
From this study, we understand that the most regulated nodes are associated with the RAS protein subfamily.
All Ras protein family members belong to a class of protein called small GTPase. They are involved in cellular signal transduction. When Ras is activated by incoming signals, it subsequently switches on other proteins, which ultimately turn on genes involved in cell growth. Regulation of Ras protein is very important. Mutations in ras genes can lead to the production of permanently activated Ras proteins, a cause of cancer. Hence, Ras proteins are normally tightly regulated by factors that switch off the GTPase activity.