Clinical data survey and association with Li-Fraumeni Syndrome
Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder often resulting from germline mutations impairing the antitumor functions of p53. Li-Fraumeni syndrome was characterized by a marked increase in familial disposition to cancers, including rhabdomyosarcoma and Choroid plexus carcinomas (CPCs). The Intergroup Rhabdomyosarcoma Study Group has defined RMS as a malignant neoplasm showing exclusive evidence of skeletal muscle differentiation by histologic morphology or ancillary techniques such as immunohistochemical stains or molecular genetics. The intergroup rhabdomyosarcoma as a malignant neoplasm was showing exclusive evidence of skeletal muscle differentiation by histologic morphology ancillary techniques such as immunohistochemical stains or molecular genetics. The past three decades have been through the Intergroup Rhabdomyosarcoma Studies (IRS) formulated in the 1970s. Treatment of RMS with multiple modalities has transformed the dismal 25% 3-year life expectancy of the 1960s to overall survival (O.S.) rates of higher than 90% today.
Here our reference of a healthy five and three-year-old boy presented with swelling of the left and right eyelid that had been gradually enlarging over the previous two days (Fig. 1a-f). After raising the upper eyelid, visual acuity was 0.8, and ocular motility was unremarkable. On the following day, after oral antibiotic treatment initiation, visual acuity decreased to 0.63. There was a restriction in the left eye's upward gaze, indicating the presence of inflammatory orbital cellulitis. It can occur in 3 to 4% of children with acute rhinosinusitis and was a potentially fatal emergency (sinus vein thrombosis, meningitis). Initially, it was essential to differentiate between pre and post sepal disease using imaging, motility testing, and pupillary reaction. Eyelid swelling without elevated temperature can also be a sign of an orbital complication, and in cases of functional mono-vision, diplopia may not be noticed. Orbital cellulitis can be caused by dacryocystitis, sinusitis, or trauma. The diagnosis has to be confirmed by checking for inflammatory parameters, performing magnetic resonance imaging/computed tomography, and elevated temperature and blood culture for a specific intravenous antibiotic therapy. Interdisciplinary collaboration between pediatrics, otorhinolaryngology, and ophthalmology is of utmost importance in these patients.
Choroid plexus carcinomas (CPCs) are rare, aggressive pediatric brain tumors with no established curative therapy for relapsed disease and low survival rates. MRI revealed new lesions in the right and left ventricles; the spine and cerebral spinal fluid (CSF) were negative for the disease. She began treatment with relapse chemotherapy, which included bevacizumab, irinotecan, and temozolomide, during which time the tumor progressed. I have given one clinical example data of performed a retrospective analysis of the prospective data based on brain tumors from the pediatric neurosurgery department, Necker-Enfants Malades Hospital, Paris, from January 2011 to September 2014 choroid plexus lesions (Lin et al., 2019). Inclusion criteria were the following available M.R. imaging with Perfusion Weighted Imaging (PWI) arterial spin labeling (ASL) or dynamic susceptibility contrast (DSC) in the radiology department also no prior surgical resection (Fig. 1g-k), biopsy, or treatment of the tumor and a histologic diagnosis of choroid plexus papilloma or carcinoma.
But the genetic basis of CPC was poorly understood. CPC is known to have a strong association with Li-Fraumeni Syndrome and TP53 mutations, but understanding the underlying biology and molecular alterations in these cancers are incomplete. It is essential to report upon the computational methods for studying both how TP53 mutations functionally and structurally impact for which helps to further targeted therapy has been used. A based on recently reported one-missense rare mutation Arg267Trp identified in a Saudi family. Based on simulations of the crystal structure of p53, substitutions Arg26Trp cause significant changes in its tertiary structure (Fig. 2). It is likely that these changes will impair p53-DNA binding across the board, regardless of the sequence specificity. We observed a loss of transactivation for this mutation, which is consistent with this interpretation. Arg267Trp and Arg283His mutations of p53 are likely to adopt a grossly misfolded conformation, resulting in reduced or absent DNA binding. A hot spot mutant p53 273His was functionally similar to p53 258Asp because it prevented p53 transactivation on both promoters. A mutation in p53-DNA interaction models indicates that some mutants bind to p53REs differently. This particular mutation Arg26Trp further confirmed the study of structural and functional analysis by computational molecular dynamics and simulation.
Structural and functional effect of TP53 rare mutation
Each amino acid has a specific size, charge, and hydrophobicity-value. The original wild type residue Arg267 and newly introduced mutant residue Trp267 often differ in these properties. The mutant residue was more significant than the wild-type residue, and the wild-type residue charge was positive, the mutant residue charge was neutral. The mutant residue was more hydrophobic than the wild type residue. The wild type residue forms a hydrogen bond with Lysine at position 101. A size difference between wild type and mutant residue makes the new residue not in the correct position to make the same hydrogen bond as the original wild-type residue. Based on this difference in hydrophobicity will affect hydrogen bond formation. The wild type residue forms a salt bridge with Glutamic Acid at position 258. The difference in charge will disturb the ionic interaction made by the original, wild type residue. This mutation was located within a stretch of residues annotated in UniProt as a unique region interaction with CCAR2 (Fig. 3a-j). The differences in amino acid properties can disturb this region and disturb its function. The mutation; dbSNP: rs587780075. Li-Fraumeni syndrome (LFS) [MIM: 151623] was located in a region with known splice variants.
Further conservation of the wild type residue was much conserved, but a few other residue types have been observed at this position too. But in the mutant residue was not among other residue types observed in other homologous proteins. However, this residue that has some properties in common with mutated residue was observed. It means that in some rare cases, this mutation might occur without damaging the protein also located near a highly conserved position.
The mutated residue was located in a domain with classification (Table 1) that was important for the protein activity and in contact with another domain that was also important for the activity. The interaction between these domains could be disturbed by this mutation, which might affect the protein's function. The mutated residue was located in a domain that was important for the activity of the TP53 protein and in contact with residues in another domain. This interaction might have been necessary for the correct function of the protein. The mutation can affect this interaction and, as such, affect protein function. The mutated residue was nearly located in a domain that was an essential activity of TP53 protein and in contact with another domain known to be involved in binding. The interaction between these domains could be disturbed by the mutation, which might affect the signal transduction between the domains. The mutated residue was located in a domain that was important for the protein's activity and in contact with residues in a regulatory domain. It might be possible that the mutation disturbs this interaction and thereby affects the regulation of catalytic activity.
Table 1
Classify TP53 protein families and domains
Interpro Domain | Gene Ontology Term | Broad Gene Ontology Term |
P53, DNA-Binding Domain IPR011615 | Transcription Regulatory Region DNA Binding GO:0044212 | Binding GO:0005488 Nucleic Acid Binding GO:0003676 Molecular Function GO:0003674 |
P53 Tumor Suppressor Family IPR002117 | DNA Binding Transcription Factor Activity GO:0003700 DNA Binding GO:0003677 | Molecular Function GO:0003674 Binding GO:0005488 Nucleic Acid Binding GO:0003676 Molecular Function GO:0003674 |
P53/Runt-Type Transcription Factor, DNA-Binding Domain Superfamily IPR012346 | DNA Binding GO:0003677 DNA Binding Transcription Factor Activity GO:0003700 | Binding GO:0005488 Nucleic Acid Binding GO:0003676 Molecular Function GO:0003674 Molecular Function GO:0003674 |
P53-Like Transcription Factor, DNA Binding IPR008967 | DNA Binding Transcription Factor Activity GO:0003700 | Molecular Function GO:0003674 |
Mutations affecting TP53 interaction network and impact
We built a protein interaction network to investigate p53 mutations at interface residues encoded by 11 exons that target specific protein interactions. Because this network explicitly represents the residues on each partner that facilitate PPIs, the network has distinct classes of nodes, with colored circles representing proteins of amino acid residues (Fig. 4). We first centralized on the sub network of frequently altered cancer genes and their interaction partners were encoded 22 exons BRCA1 and BRCA2 encoded 27 exons (Table 2). This sub network included more than 100 proteins node, with at least cancer associated 60 genes and 90 interaction partners (Table 3). These network modules exhibit distinct patterns of mutation localization at interfaces, indicating that different selective pressures are acting to target mutation Arg267Trp in each case. In the following sections, we go over these two modules in full detail. Mutations in this gene have been linked to varying effects on p53 activity by simulation resulted was increased 80%. Some of the mutations result in p53 function gain, while others proteins BRCA1 was decreased 10% and BRCA2 was 30% suppress it. Even different amino acid substitutions at the same residue can result in distinct phenotypes. We used the protein-residue bipartite interaction network of p53 to investigate the biological consequences of various mutations. Mutations at these residues could specifically inhibit binding, thereby freeing the binding site to interact, by destabilizing the p53-BRCA1 and BRCA2 interactions, which is unfavorable for tumor progression.
Conformational changes and thermodynamics Stability
The difference in relative solvent accessibility between wild type and the mutant residue of all TP53 mutations was calculated using DynaMut: SDM. While analyzing the maximum destabilizing mutations among all the possible mutations at each residue position, it was noted that maximum destabilizing mutants at 260–310 amino acid residues positions (80.65%) show increases residue relative solvent accessibility (RSA). The maximum destabilizing mutants at the rest of amino acid 210–259 residue positions indicated a decrease in RSA. The maximum destabilizing mutants at 267 substitutions residue positions showed an increase in RSA, from polar/charged (wild type) to hydrophobic residues (mutants). As mutant hydrophobic residues with increased solvent accessibility often destabilize the protein, the destabilizing effects of mutation Arg267Trp were − 0.77 kcal/mol (Fig. 5a).
Additionally, these substitutions resulted in increased residue-depth (ranging from 85Å), concomitant with the decrease in solvent accessibility (18.78%), and torsion angle range from − 154.9–142.1 ω. Also, ENCoM provided that rapid and simplified access to insightful analysis of protein motion also enables rapid analysis of the impact of mutation R267W on a protein's dynamics and stability. Further, the resulting of this mutation vibrational entropy changes were. ΔΔSVib ENCoM: -0.057 kcal.mol-1.K-1 (Decrease of molecule flexibility). The mutational Residue Arg267 with distinct p53 protein structural properties was necessary for the function. Quantifying different structural with surface area and contact properties on a per-residue basis and analyzing their correlations provides a way to identify outlier residues that might be important for structure and function because buried residue with many contacts can be critical for protein stability. Scatter plots display values based on the thermostability of mutation of interest with values of the variables determining its x- and y-coordinates. A scatterplot matrix represents more than two variables using multiple scatter plots arranged in a grid, with one row and column per variable. The calculated properties such as the solvent-accessible area of the complex, network centrality measures, and the degree for each residue are plotted against each other in the scatter plot matrix for highlighting multiple positions that are disease mutations in rhodopsin onto the scatter plot (Fig. 5b).
Further, the residues that contribute to destabilizing the protein p53 native state. The protein electrostatic energy is taking into account each residue's contribution with a polar charged side chain (Fig. 5c). The charge-to-charge energy indicates the contribution of each ionizable residue to the p53 protein native state stability. The amino acid residues mutated to increase the p53 protein thermostability. It was indicated the residue, which present unfavorable energy values ΔGqq ≥ 0 and are, exposed to solvent with SASA ≥ 50%. In p53 protein, the highlighted mutated residue was Arg267Trp with its respective based on ΔGqq and SASA values. The total energy contribution to protein native state stability was given as a sum of the ΔGqq for each ionizable group n in the protein, named as ΔGelec. The ΔGelec for the p53 protein in pH 5.5 is -61.99kJ/mol. The total energy contribution ΔGelec was a pH-dependent measure. The ionization degree of each polar residue side chain and its ΔGqq vary with the pH input value. The ΔGelec as a function of pH for p53 mutates type protein (Fig. 5d).
Molecular Dynamics simulation of solvated Tp53 protein
This section will account for how mutation relates to the changes observed in translated regions of the TP53 gene at exon8. When the mutation shows a change in the translated protein (in exon), and regardless of whether it impacted, there was a missense mutation in the protein. A test on the missense mutation within the gene on chromosome 17 was also accomplished. It was also identified that the test output was linked with TP53 coding regions of the protein structure recovered from the Protein Data Bank (PDB). Hence we settled the structure from UniProtKB - P04637 (P53_HUMAN) (PDB ID: 1GZH. However, the resulting structure was regularly four chains A-D, yet our focused arrangement adjusted to just the "Chain A" arrangements for the merging parameters and the structure combination simulations. The TP53 related protein space "Chain A" structure mutation was within β-strand at Arg267Trp.
On the other hand, mutations for relating positions were accomplished using the Collaborative Computational Project, Number 4 (CCP4); QtMG, which was identified completely to inspect the changed model structures. We did a check on the residues as well as a β-strand mutation analysis for TP53 protein. Another method called the consensus was utilized on an extra essential residue with a hot residue to expand the thermodynamic strength of TP53. Notably, this system was straightforward in contrast with the already depicted methodologies. Arg267 in TP53 and another thermostable site (267Trp) were focused as basic residues and were substituted by Schrodinger (BioLuminate).
Later, the minimization of TP53 receding energy was performed at 300 800.706kJ/mol and later lowered to -410 036.014 kJ/mol after completing the mutation as calculated with the help of the BioLuminate. Various polar and non-polar residues were used to screen and base the energy minimization procedures that enabled fixing a refinement cut of 0.00 Å with a reasonable solvent reduction. The consequence of diminishing the limited energy estimation of the β-strand (Arg267Trp) demonstrated that the substitution of Arg to Trp267 might add to enhanced conservativeness and inflate protein folding. Additionally, β-strand locale mutation depended on the appropriate solvent, as indicated by MD simulations. Amid these reenactments, proteins ceaselessly fold and unfold and give a clear understanding of this procedure. When contrasted with the outcomes at a temperature of 300 K, the deviation of TP53 protein stretched from 2.0 Å to 10 ns. Interestingly, the deviation of mutation Arg267Trp was kept up at 1.2 Å until the simulation was completed (t = 10 ns). It was confirmed that Arg267Trp had achieved its folded state while the little peak at 1.2 ns showed that Arg267Trp settled the protein structure.
The resulting evidence demonstrated that the Arg267Trp mutant structure was steady and could keep up its adaptation at 300 K, at a pressure bar of 1.00047. Besides, with a surface tension of 5,000.0 Å, in an aggregate recreation time (ns) of 1.2/slipped by 0.0, and recording intervals (ps) energy of 1.2, in any case, there were inconspicuous changes that were seen between wild sort and mutant TP53 protein (β-strand; Arg267Trp). It can be seen when the superimposed structures with an RMSD estimation of 1.023 Å were resolved using the Molecular Operating Environment (MOE). Additionally, the mutation Arg 267 of the TP53 protein demonstrated a higher dissolvable and available surface zone than 267Trp of the mutant in the protein structure. The structural discharge of Arg267 brought about less hydrophobic properties within the protein structure. Simultaneously, the addition of 267Trp residue could expand the minimization and clearing of the unwanted deposits in the protein structure.
Other investigations of annotated expected solvent availability and pre-ascertained packing density revealed that Arg267Trp shape the density and reduce in cavities contrasted with the wild type frame. Accordingly, the substitution of a residue (Arg267) with Trp267 upgraded the mutant structure's packing and compactness and diminished the interior residue in the protein structure. The results of this mutation were contemplated utilizing a traditional molecular dynamics approach, whereby TP53 wild and mutated structures were surveyed through long simulations in an express solvent. It also involves investigating the varieties in the dynamics and protein stability by protein chain "A" structure and calculations of free energy. However, the solvate was used to amass the right watery solvent environment close to the TP53 protein area. The solvate distinguished the framework's measurement with parameter designs of water box changing to dissolve the molecule with an edge split-up of 10.0 Å.