Page 12 - GPD-1-1

Gene & Protein in Disease Bioinformatics study of PCNP

protein structure was prepared using the GROMOS96 43a1 and enriched pathway analysis of PCNP, the Network
force field solvated by the single point charge water model and Analyst server (https://www.networkanalyst.ca/) was
was fixed in a periodic cubic solvated box. Other parameters employed [34,35] . It is a publicly available online tool widely
were neutralized by adding the salt (NaCl) concentration used for gene expression profiling, transcriptional factor
(0.15 M), energy minimization for 5000 steps of the prepared analysis, PPI networks, pathway analysis, toxicogenomics,
system, and equilibration type NVT/NPT at 300 K and 1 bar and pharmacogenomics studies [36,37] . In addition, the
of pressure. The final MDS step was processed for 50 ns of Enrichr web server (https://maayanlab.cloud/Enrichr/)
[35]
time. The MDS was analyzed by calculating the root mean was utilized to validate the Network Analyst results .
square deviation (RMSD) and root mean square fluctuation
(RMSF) values of the protein structure. 2.7. Phylogenetic analysis
To determine the evolutionary history of PCNP between
2.4. Physicochemical characterization and humans and other vertebrates, the phylogenetic tool,
secondary structure prediction molecular evolutionary genetics analysis (MEGA) X, was
[38]
The basic physicochemical properties, such as theoretical used . PCNP has no reported paralogs in the biological
isoelectric point (pI), molecular weight, total number database (ENSEMBL) or in the available literature.
of positive and negative residues, extinction coefficient Orthologs of PCNP were used for tree construction
instability index, aliphatic index, and grand average and sequences of eight orthologous candidate proteins
hydropathy, were computed using the ProtParam tool were retrieved from the NCBI database. Alignment of
of ExPASy (http://web.expasy.org/protparam/) [26-28] . For multiple sequences was carried out using the MUSCLE
the secondary structure prediction of PCNP, SOPMA program implemented within MEGA X. For pairwise
bioinformatics tool (https://npsa-prabi.ibcp.fr/cgi-bin/ sequence identity, Sequence Demarcation Tool Version 1.2
[39]
npsa_automat.pl?page=/NPSA/npsa_sopma.html) was used (SDTv1.2; http://web.cbio.uct.ac.za/~brejnev/) was used .
with its default query parameters . This tool uses the self- The best-fit substitution model was estimated in MEGA
[29]
optimized prediction method and a neural network method and selection was done based on the Neighbor-Joining
(PHD) to predict the secondary structure of proteins [29,30] . Method. The rate of variation among sites was modeled
with a gamma distribution (shape parameter = 1). All
2.5. Coexpression and protein-protein interaction positions that contained alignment gaps and missing data
(PPI) analysis were eliminated from the analysis. A phylogenetic tree was
The coexpression analysis of the PCNP was performed constructed using the unweighted pair group method with
by the GeneMANIA server (https://genemania.org/) by arithmetic mean, and the validity of the tree generated was
[40,41]
setting Homo sapiens as the species organism and the tested by bootstrap analysis of 1000 pseudoreplicates .
maximum number of resultant genes and attributes as 10. 3. Results and discussion
It is a web interface that has a high-accuracy prediction
algorithm and a large database for gene functional analysis, 3.1. Structure prediction
including coexpression, genetic interaction, colocalization, Four tools were utilized to generate the 3D structure of
physical interaction, pathway interaction, and shared the PCNP protein. From the results of all these tools, we
protein domains of the submitted query . Furthermore, selected the best score-generating model and subjected it
[31]
the PPI of PCNP was evaluated by accessing the search to further validation through the ERRAT and PROCHECK
tool for the retrieval of interacting genes (STRING) servers. In addition, the quality model QMEAN score
protein database (https://string-db.org/). The PPI network was evaluated for the best scoring models. The graphical
was constructed based on the data from tandem affinity illustration of these four best models is presented in
purification assay, affinity chromatography technology Figure 1. Following the 3D model analysis, it was discovered
assay, and coimmunoprecipitation assay. The STRING that the I-TASSER generated model had the highest quality
database query terms were cutoff at 0.4 and the maximum factor score among the four best-scoring models, as shown
additional interaction was 10 . Cytoscape software in Figure 1D and Table 2. Moreover, the I-TASSER model
[32]
version 3.8.2 (https://cytoscape.org/) was employed to structure was subjected to MDS for profound validation
construct the coexpression and PPI networks . analysis.
[33]
2.6. Gene ontology (GO) and pathway enrichment 3.2. Molecular dynamics simulation
analysis An MDS was conducted for the analysis of the stability
To assimilate the biological data of GO, including biological and flexibility of the I-TASSER-generated PCNP protein
processes, molecular functions, and cellular components, model. The RMSD and RMSF of the protein structure

Volume 1 Issue 1 (2022) 4 https://doi.org/10.36922/gpd.v1i1.65