For most of the pathogenic variants, a strongly destabilizing mutation corresponds to the loss of function, whereas a modest change in stability may generate changes in protein conformation affecting the binding affinity with interacting molecules (protein, RNA and DNA). The influence of amino acid modifications on protein stability, on the other hand, is critical information for precision medicine .
TCGA based expression analysis and alterations in the FBXW7 gene
To have an overview of FBXw7 expression in different solid tumours, we analysed its expression in samples from the TCGA, which revealed that FBXw7 is coupled with differential expression as well as cervical, endometrial, and ovarian cancer in women (Fig. 1).
We used the NCBI dbSNP database to evaluate reported FBXW7 variants, which include SNPs in the intronic region, 49,125 SNPs, and 701 SNPs in the non-coding area. Within the coding sequence, there were nearly 394 missense (nsSNPs) and 257 synonymous SNPs. The current study included clinically significant nsSNPs and mutants to investigate their impact on the FBXW7 structure. In addition to that, uncharacterized nsSNPs were assessed for their structural and functional level impact (Table.1). In addition to that, 57 nSSNP’s located in the F-box and WD40 domain were also included in the current study.
Detection of harmful substances SNPs
SIFT, PANTHER, PolyPhen-2, SNPs & GO, PROVEAN, and PredictSNP were used to screen the mutant's impact at the sequencing and structural levels. As per prediction, ten proposed variants with clinical significance are considered as deleterious variants in all computational prediction methods (Table.2). The F-box and WD40 domain variants' impacts were also predicted and tabulated.
Screening of mutant residues prevalence within FBXW7 domains
InterPro, a domain identification tool, uses the protein family’s functional analysis concept to predict a protein's domains as well as its active sites. To assess the position of variants within the conserved domains of FBXW7, we used the InterPro tool, which can identify motifs and domains of a protein. It is projected that two functional domains of FBXW7, which are 1PR001810 indicate F-BOX domain (278-325), and IPR17986 indicates WD40-repeat-containing domain (376-659) and FBXW7 variants of current study were positioned on second domain (Fig.2). Within the propeller phosphorylation-binding region, three arginine Missense point mutant residues (R465, R479, and R505) prohibit Fbxw7 from binding substrate . The F-box domain contains nsSNPs and a few premature truncation cause variants. So far, nearly twenty-one premature truncation variants have been reported in the WD40 domain. Sixty-eight nsSNPs were reported in the WD40 domain, five nsSNP’s in the F-Box domain, thus their deleterious effect was assessed using consensus prediction results. (Supplementary table.1)
Exploring the impact of the nsSNPs on protein stability
I-Mutant is a tool that assesses the influence of mutations on protein stability using a neural network technique. I-Mutant calculates the degree of protein instability and displays the projected free energy change value (G) as well as the prediction's sign: decrease or increase. The ΔΔG value predicted by I-Mutant revealed that all Clinvar proposed variants decreased stability (Table.3). In addition, we examined all the nsSNPs found in both domains of FBXW7.The support vector machine-based algorithm used here effectively predicted the transformation in protein stability free energy of the proposed FBXW7 mutants. The elevated negative DDG score suggests it is highly deleterious. (Supplementary table.2)
Evolutionary conservation analysis
The ConSurf web server revealed the evolutionary conservation of amino acid residues in native FBXW7. We looked at the evolutionary conservation and solvent accessibility of the FBXW7 mutant's structural and functional residues. R465 and R505 were found to be exposed and functional, whereas both mutants’ residues were buried and structural. All these mutant residues have a high level of conservation (Table.4). Furthermore, both residues are expected to be relatively conserved and exposed, whereas both mutants are anticipated to be conserved and buried (Fig.3).
Structural changes are reflected through the energy state of the FBXW7 mutant models in the SwissPDB viewer assessment. Mutant models were visualized using Pymol and RMSD values were tabulated (Table.5). The potential energy of the wild type FBXW7 structure was observed at -11346.87 kJ/mol following energy minimization. Similarly, other mutants showed that R465H (−17.713 kJ/mol), which considerably diminished total energy, but R465P (−17.161 kJ/ mol) (Fig. 5), R465L (−16,953kJ/mol), and R505H (−16.667 kJ/mol) were the top three mutants that impacted the FBXW7 structure by increasing the total energy after energy minimization (Table.6).
Structural effect of point mutation on human FBXW7 protein
The Project HOPE server revealed that substituting Glycine in R505G, R465G can cause FBXW7 protein stiffness to be disrupted. Furthermore, R505L, R465H, R465L and R465P can be mutations located within a specification that is frequent in the protein repeat and is known as WD3. The mutation into a new residue might interrupt this repeat and, consequently, their substrate binding capability (Fig.4). Interference with both salt bridge and hydrogen bonding was seen in R505C mutants. The ability of a protein structure to offer specific activities depends on its flexibility and rigidity nature. Besides this, the R505H and R505S mutants expose distinct properties and so they might drastically alter the functional FBXW7 associating domain (Table.7). Missense 3D tool stated that R465L, R465C, R465G, R465H, R465P, R505L, R505C variant created a buried Proline that delivers constrained backbone conformation. Moreover, R465L, R465C, R465G, R465H, R465P, R505L, R505C interrupted the sidechain and main-chain H-bond (s) established by wild type buried Arginine residue and R505L, R505G and R505S substitution triggered the expansion of cavity size (Table.8). The R505C substitution caused a shift from buried to exposed states, with the ARG exposed (RSA 23.7%) and the introduced mutant CYS buried (RSA 5.1%). Similarly, in R505L, ARG is exposed (RSA 23.7%) and LEU is buried (RSA 6.7%). The R465P substitution converts the 'E' (extended strand in parallel and/or anti-parallel -sheet conformation) to the '' (no secondary structure). nsSNPs found in both domains of FBXW7 were also evaluated for structural level changes and are included in the supplementary file.1
FBXW7 mutants' structural level impact on its molecular interactors
Cytoscape was employed to construct the protein interaction network of FBXW7 protein, and it projected that the F-box family of proteins is functionally linked with 311 proteins, among which the top ten hub proteins NOTCH1, c-Myc, CCNE1, STYX, KLF5, SREB1, NFKB2, SKP1, CUL1 were included for the current study to explore the FBXW7 structural variants impact protein protein interaction (Table.9). The Cytoscape based protein-protein interaction network was based on the graph theory parameters, namely degree, average shortest path length, betweenness centrality, closeness centrality, and neighbourhood connectivity (Fig.5).
F-box protein Fbw7 (gene FBXW7), which forms the E3 ubiquitin ligase complex SCFFbw7 with Skp1 and Cullin1 . Uterine carcinosarcoma (UCS) is a biphasic, high-grade endometrial cancer with de-differentiated sarcoma and carcinoma features. UCS tumours are serous-like, according to next-generation sequencing, and common somatic mutations are found in them. According to next-generation sequencing, UCS tumours are considered as serious-like and observed with somatic mutations in certain genes, namely TP53, PIK3CA, FBXW7, PTEN, and ARID1A .
Metaplastic breast carcinoma (MBC) and Uterine carcinosarcoma (UCS) anchorage have repeated somatic genetic changes influencing TP53 and a few other genes associated with the PI3K, Wnt, and Notch pathways. The histologically dissimilar components identified in MBCs and UCSs were discovered to be clonally related, and, at least in some cases, their mesenchymal component presumably arose from the epithelial component. Despite this, a few differences, specifically genetic alterations, distinguish MBCs and UCSs, as well as the role of the corresponding pathway in tumour induction The frequency of FBXW7 and PPP2R1A mutations, HER2 augmentation, and the absence of HRD distinguish UCSs from MBCs . Endometrial carcinoma (EC) is a clinically diverse disease with a wide range of histological subtypes, and this heterogeneity may play a role in the accumulation of genetic alterations in the mutations were linked to late-stage cancer, vascular invasion, and lymph node metastatic disease . Reported studies have revealed that about 15%-29% of serous ECs, 11%-39% of uterine carcinosarcomas, 13%- 25% of clear cell endometrial cancers, and 0%-15% of endometrioid endometrial cancers show somatic mutations in FBXW7 . Besides the most common serous ECs, somatic mutational hotspots are listed as 423, 465, 479, and 505 residues . genes FBXW7, PTEN, PIK3CA, TP53, KRAS, CTNNB1, FBFR2, and RB1.FBWX7
The coding region of the FBXW7 gene contains isoform-specific 5-exons, a nuclear localization signal, an F-box motif, and a WD40 domain. The Phosphorylated substrates such as cyclin E, Notch, cJun, MYC, PS1, and SREBF bind to this functional region. GSK3/FBXW7-dependent degradation of sterol regulatory element-binding protein 1 (SREBP1) suppresses lipogenesis in cancer cells when mTOR complex 2 is inhibited . WD40 repeats, generating a propeller shape that may be observed on the surface or lumen of the h-propeller structure and is embedded with R465, R479, and R505 residues. As a result, such point mutations could impair substrate binding and interfere with wild type FBXW7 activity, implying a central negative effect. The WDR protein FBXW7 has a part in human cancer since it is the prime aberrated protein in the ubiquitin / proteasome system (UPS) seen among cancer patients . The tumour suppressor protein FBXW7 interacts to substrate protein via phosphorylation and initiate breakdown process. FBXW7 gene loss-of-function mutations cause an abnormal build-up of cyclin E, which is found in 18% of colorectal malignancies, 15% of uterine endometrial carcinoma, and 40% of uterine carcinosarcoma [41,42].Surprisingly, these changed residues lead to the outside of the WD40-sheet domain, signalling that they are close to FBXW7-interacting substrate proteins.R505H, R505L, R505S, R505G, R505C, R465L, R465P, R465H, R465G, R465C mutations in FBXW7 resulted in changes in hydrophobic behaviour and electrostatic surface interactions, as well as a change in substrate binding.
FBXW7 mutants increases cancer-originating cell action in association with Notch1 oncogenes. Through skin carcinogenesis, FBXW7 regulates keratinocyte proliferation and differentiation, utilising both repressive and stimulatory signals, primarily through maintaining the proliferation-enhancing drive of c-Myc and the tumor-suppressive action of NOTCH . Inactivation of FBXW7 has been shown to increase tumour resistance to anti-tubulin chemotherapeutic drugs. Furthermore, in cancer types that enhance resistance to gamma-secretase inhibitors, deletion of FBXW7 has been revealed to be a crucial prognostic marker (GSIs). These promising findings show that targeting NOTCH and/or FBXW7 to overcome MDR is a viable option. The biological importance of NOTCH and FBXW7 dysregulation in inducing MDR in tumours is discussed in the following sections [44,45]. Still, deregulation of FBXW7 and NOTCH activity can occur because of circumstances such as a mutation that disrupts the homeostatic state, leading to neoplastic transformation.
A recent study emphasises the specific variants, like FBXW7 R505L, localized in WD repeat 4 of the Fbxw7 protein. The Exon 9 based variant R465C of FBXW7 was reported numerous times for missense mutation occurrence (18.6%). Likewise, R465H and R505C were the next most familiar FBXW7 missense mutations (16.3% each) among the gynaecological conditions. Fbxw7 is rendered inactive by R505L, as evidenced by the activation of the NOTCH pathway in cultured cells  and the inability to bind substrates . Another damaging mutation, R505C, is discovered in the WD repeat 4 of the Fbxw7 protein. R505C disrupts FBXW7-substrate interaction and impairs FBXW7 substrate degradation, leading to increased Notch intracellular domain and Myc expression , aberrant subnuclear localization , and decreased KLF5 degradation . Mutant R465C is found in the Fbxw7 protein's WD repeat 3. In R465C mice, Fbxw7 protein function is lost, as indicated by a lack of Fbxw7-substrate interaction and poor substrate degradation by Fbxw7, resulting in prolonged Notch1 intracellular domain and Myc expression , as well as decreased degradation of Klf5 . In culture, aberrant subnuclear localization resulted in lower inhibition of migration, invasion, and colony formation compared to wild-type Fbxw7 [48,50]. The WD repeat 3 of the Fbxw7 protein contains R465H (which corresponds to R385H in isoform 2. R465H prevents FBXW7 substrate degradation by preventing FBXW7-substrate contact, resulting in extended NICD and MYC expression , as well as KLF5 degradation . H468R is in the WD repeat 3 of the Fbxw7 protein. H468R causes the Fbxw7 protein to lose function, as evidenced by the inability to induce the degradation of cyclin E, c-Myc, Mcl-1, and Braf in cultured cells , confers resistance to some BET inhibitors in cultured cells , and causes impaired NICD degradation in cultured cells, potentially leading to increased Notch1 signalling . R479H is found in the Fbxw7 protein's WD repeat domain. This variation results in FBXW7 substrate degradation and a lack of FBXW7-substrate interaction, resulting in Notch-driven reporter activation . R479Q is present in the WD repeat 3 of the Fbxw7 protein. R479Q prevents FBXW7 from degrading substrates, resulting in prolonged Notch1 intracellular domain and Myc expression [46,53], as well as aberrant subcellular nuclear localization and loss of Notch1 intracellular domain binding in culture . The FBXW7 R505H mutation is found in Fbxw7's WD repeat 4. R505H has been found in sequencing studies [54, 55, 56], but it has not been biochemically described. Therefore, its impact on the function of the Fbxw7 protein is unknown. R505G is found in the Fbxw7 protein's WD repeat 4. In cell culture studies, variant R505G causes enhanced proliferation, migration, invasion, and colony formation , implying that the Fbxw7 protein function will be lost. R465L is found in the Fbxw7 protein's WD repeat domain 3. This variant results in FBXW7 substrate degradation and a lack of FBXW7-substrate interaction, resulting in Notch-driven reporter activation . The WD repeat domain of the Fbxw7 protein contains R465P. Other R465 hotspots inactivate Fbxw7, but R465P has yet to be explored. As a result, R465P is predicted to result in function loss [46, 57, 58]. R465Y has not been detected in the WD repeat domain of the Fbxw7 protein, however other R465 hotspots inactivate Fbxw7. As a result, R465Y is likely to cause function loss [46, 57, 58]. The WD repeat domain of the Fbxw7 protein contains R479L. This mutation causes the Fbxw7 protein to lose function, as seen by increased ubiquitination and lower protein stability . R479P is found in the Fbxw7 protein's WD repeat domain. In culture, R479P causes enhanced proliferation, migration, invasion, and colony formation , implying that Fbxw7 protein function will be lost.
NFκB2 precursor protein is recognised as one of the main Fbw7 substrates. We identified NFκB2 as a downstream ubiquitin substrate of Fbw7 since it is a physiological interactor of SCFFbw7 . Furthermore, the transcription factor nuclear factor kappa B (NF-kB2) is well-known for controlling cell survival, tumour invasion, and treatment resistance via the regulation of many oncogenic gene products . Tumour suppressor FBXW7 is a component of the SCF (Skp1, Cullin 1, F-box protein) ubiquitin ligase complex, which regulates the degradation of a variety of substrates that, if not correctly regulated, might contribute to carcinogenesis. We show that FBXW7 mutations increase phosphorylated SRC-3, Cyclin E1, and c-MYC levels. Increased amounts of phosphorylated proteins have been linked to recurrent FBXW7 mutations, the majority of which occur in druggable pathways . STYX has been associated to colorectal cancer cell proliferation, migration, invasion, and apoptosis, and several studies have suggested that STYX serves as a latent oncogene that inhibits apoptosis in colorectal and breast cancer, particularly via interacting with the FBXW7 protein. Collectively, these explanations reveal that the STYX/FBXW7 axis is engaged in the promotion of EC cells and might participate in another tumour development as well by controlling the NOTCH-mTOR signalling pathway. And hence, STYX being involved in numerous cancers, its part in modifying the NOTCH-mTOR interaction via FBXW7 permits further than consideration .
FBXW7 is a substrate differentiation factor of an E3 ubiquitin ligase of the SKP1-cullin-F-box (SCF) type that is responsible for the ubiquitin-dependent degradation of cyclin E. (encoded by CCNE1). When all uterine serous carcinomas were evaluated together, molecular genetic anomalies in the cyclin E pathway caused by FBXW7 point mutations, FBXW7 deletions, or CCNE1 amplification were found in more than half of them. Stimulation of cyclin E, whether by preventing its ubiquitin-dependent protein degradation owing to FBXW7 mutations or by elevating its expression due to gene amplification, may play a key role in uterine serous carcinoma carcinogenesis. SNP array analysis and immunohistochemistry based CCNE1 expression variation analysis also ensure the same functionality . Zhao et al., discovered that FBW7 phosphorylates GSK3 and recruits KLF5 for ubiquitin-mediated proteasomal destruction . Aberrant expression of FBW7 diminishes the KLF5 protein level and its half-life, while deactivation of FBW7 surges the KLF5 protein and half-life . These two groups’ findings support the idea that FBW7 is a critical negative regulator regulating KLF5-mediated cell proliferation. Mutations in FBXW7 increase KLF5 expression.
FBXW7 variants impact on chemoresistance:
FBXW7's antitumor effects are mostly achieved through controlling the network of proteins degradation, many of the members of proteins such as cyclin E, c-Myc, and Notch have oncogenic functions, Mutated FBXW7 is also known to induce oncoprotein stabilisation in tumours, resulting in chemoresistance induction. As a result, FBXW7 protein downregulation may contribute to tumour development and chemoresistance. As a result, FBXW7 has been recommended as a possible therapeutic target for improving chemotherapeutic drug sensitivity and efficacy . The FBXW7 mutation, which primes the dysfunctional FBXW7 by producing missense mutations in three arginine residues (R465, R479, and R505), could be the basis for the accumulation of a wide range of substrates, which could be essential in chemoresistance . Richter et al identified FBXW7 as a new FBXO45 substrate and investigated its function in cancerous cells . In another study, researchers found that blocking FBXO45-arbitrated FBXW7 depletion could help with drug resistance in chemotherapeutic treatment by increasing mitotic cell death. In other research, some mutations may affect the degradation of FBXW7 targets differently than the hot area arginine mutations (R465, R479, and R505). Design of small inhibitors that target specific downstream signalling pathways and/or affect FBXW7 substrate preference could be aided by a better understanding of the conformational changes that generate these symptoms . Earlier research has investigated the role of Fbw7 in cancer chemoresistance in a variety of cancers [71,72]. c-Myc, nuclear factor erythroid 2-related factor 2, myeloid leukaemia cell differentiation protein Mcl-1 (Mcl-1), and transcription factor SOX-9 are all participating in chemoresistance in cancers such pancreatic cancer, gastric cancer, and colorectal cancer [73,74]. According to Tong J et al, FBXW7 mutations in colorectal cancer cells impede Mcl-1 degradation, boosting the development of resistance to regorafenib-based targeted therapy . FBXW7 ablation in ovarian cancer cells inhibited c-Myc degradation, which was like the findings in colon cancer cell lines. This renders the cells more resistant to vincristine-induced cell death. On the one hand, the above-mentioned research studies underlined the crucial role of FBXW7 in chemotherapeutic drug therapeutic effects, while on the other hand, they provided much-needed information for developing viable techniques to increase cancer cell sensitivity to vincristine . Standard chemotherapy was shown to be more resistant in FBXW7-deficient leukaemia-initiating cells (LICs), but imatinib was found to be more sensitive . Combining FBXW7 genetic ablation and imatinib is more effective than any of these approaches alone in cancer animal models, according to studies. When all these aspects are considered, it becomes evident that FBXW7 plays an important role.
Out of fifty-eight nsSNP’s reported within the WD40 domain of FBXW7, ten were related to gynaecological cancer and had clinical significance; the remaining sixty deleterious nsSNPs were assessed for their mutational impact on FBXW7 and tabulated in the supplementary file 1. Our comprehensive study assessed all identified deleterious nsSNPs localized in the WD40 domain decreased stability of the FBXW7 structure. Only three of the five nsSNPs within the F-box domain have been shown to have a negative impact on the FBXW7 structure. The significance of arginine substitution among malignancies was revealed by proteins with the highest ratio of arginine substitution events in the CCLE database. Arginine has highly unusual properties studies showed that arginine is most frequently lost in mutations in cancer. Out of forty-three arginine residues twenty-six substitutions reported in FBXW7.TP53 and PTEN are the other proteins with arginine substitutions. In our studies we found twelve nsSNP’s as deleterious out of eighteen arginine substitutions within the domain region of FBXW7.Increasing evidence about the FBXW7 variant's role in cancer induction and chemoresistance emphasizes the need for this kind of comprehensive study, which would be helpful in refining the currently available therapeutic regimen for gynaecological cancer.