A Tiny Viral Protein, SARS-CoV-2-ORF7b: Functional Molecular Mechanisms.

This study presents the interaction with the human host metabolism of SARS-CoV-2 ORF7b protein (43 aa), using a protein-protein interaction network analysis. After pruning, we selected from BioGRID the 51 most significant proteins among 2753 proven interactions and 1708 interactors specific to ORF7b. We used these proteins as functional seeds, and we obtained a significant network of 551 nodes via STRING. We performed topological analysis and calculated topological distributions by Cytoscape. By following a hub-and-spoke network architectural model, we were able to identify seven proteins that ranked high as hubs and an additional seven as bottlenecks. Through this interaction model, we identified significant GO-processes (5057 terms in 15 categories) induced in human metabolism by ORF7b. We discovered high statistical significance processes of dysregulated molecular cell mechanisms caused by acting ORF7b. We detected disease-related human proteins and their involvement in metabolic roles, how they relate in a distorted way to signaling and/or functional systems, in particular intra- and inter-cellular signaling systems, and the molecular mechanisms that supervise programmed cell death, with mechanisms similar to that of cancer metastasis diffusion. A cluster analysis showed 10 compact and significant functional clusters, where two of them overlap in a Giant Connected Component core of 206 total nodes. These two clusters contain most of the high-rank nodes. ORF7b acts through these two clusters, inducing most of the metabolic dysregulation. We conducted a co-regulation and transcriptional analysis by hub and bottleneck proteins. This analysis allowed us to define the transcription factors and miRNAs that control the high-ranking proteins and the dysregulated processes within the limits of the poor knowledge that these sectors still impose.

The INCH Trial - Induction Chemotherapy in Patients With Bulky Anal Canal Cancer: Evaluation of the Pilot Phase.

AIM: To assess feasibility, complications and efficacy of induction chemotherapy followed by standard chemoradiotherapy in patients with bulky anal canal cancer. PATIENTS AND METHODS: Patients with squamous cell carcinoma of the anal canal, staged bulky tumor with or without nodal involvement were prospectively enrolled. Before standard chemoradiotherapy, patients received induction chemotherapy with 3 cycles of 75 mg/m2 cisplatin and 750 mg/m2 5-fluorouracil. Patients were followed-up routinely until recurrence or death. RESULTS: Seven patients with bulky anal canal cancer were evaluable for this pilot phase of the study. All patients had human papillomavirus-negative disease. Five completed the scheduled induction chemotherapy and all patients completed the programmed concomitant chemoradiotherapy. None had severe hematological toxicity. The majority of patients (6/7) had tumor downsizing after induction treatment. Six months after chemoradiotherapy, complete response was documented in three patients and salvage surgery was performed in two cases. With a median follow-up of 38 months (range=28-48 months), two patients are disease-free survivors. CONCLUSION: Induction chemotherapy has the potential to become a standard approach in patients with bulky human papillomavirus-negative anal canal cancer.

System-Wide Pollution of Biomedical Data: Consequence of the Search for Hub Genes of Hepatocellular Carcinoma Without Spatiotemporal Consideration.

Biomedical institutions rely on data evaluation and are turning into data factories. Big-data storage centers, supercomputing systems, and increased algorithmic efficiency allow us to analyze the ever-increasing amount of data generated every day in biomedical research centers. In network science, the principal intrinsic problem is how to integrate the data and information from different experiments on genes or proteins. Data curation is an essential process in annotating new functional data to known genes or proteins, undertaken by a biobank curator, which is then reflected in the calculated networks. We provide an example of how protein-protein networks today have space-time limits. The next step is the integration of data and information from different biobanks. Omics data and networks are essential parts of this step but also have flawed protocols and errors. Consider data from patients with cancer: from biopsy procedures to experimental tests, to archiving methods and computational algorithms, these are continuously handled so require critical and continuous "updates" to obtain reproducible, reliable, and correct results. We show, as a second example, how all this distorts studies in cellular hepatocellular carcinoma. It is not unlikely that these flawed data have been polluting biobanks for some time before stringent conditions for the veracity of data were implemented in Big data. Therefore, all this could contribute to errors in future medical decisions.

Evaluating the associations between human circadian rhythms and dysregulated genes in liver cancer cells.

Network analysis is a useful approach in cancer biology as it provides information regarding the genes and proteins. In our previous study, a network analysis was performed on dysregulated genes in HepG2 cells, a hepatoblastoma cell line that lacks the viral infection, compared with normal hepatocytes, identifying the presence of 26 HUB genes. The present study aimed to identify whether these previously identified HUB genes participate in the network that controls the human circadian rhythms. The results of the present study demonstrated that 20/26 HUB genes were associated with the metabolic processes that control human circadian rhythms, which supports the hypothesis that a number of cancer types are dependent from circadian cycles. In addition, it was revealed that the CLOCK circadian regulator gene was associated, via cytoskeleton associated protein 5 (CKAP5), with the HUB genes of the HepG2 network, and that CKAP5 was associated with three other circadian genes (casein kinase 1ε, casein kinase 1δ and histone deacetylase 4) and 10 HepG2 genes (SH2 domain containing, ZW10 interacting kinetochore protein, aurora kinase B, cell division cycle 20, centromere protein A, inner centromere protein, mitotic arrest deficient 2 like 1, baculoviral IAP repeat containing 5, SPC24 NDC80 kinetochore complex component and kinesin family member 2C). Furthermore, the genes that associate the circadian system with liver cancer were demonstrated to encode intrinsically disordered proteins. Finally, the results of the present study identified the microRNAs involved in the network formed by the overlapping of HepG2 and circadian genes.

Conformational analysis of the human chemokine receptor CXCR3.

In the last years, some studies showed the patho-genetic role of CXCR3 bound to its ligands in many human inflammatory diseases and cancers. Thus, the blockage of the CXCR3 interaction site to its ligands is seen as a possible therapeutic target for the treatment of cancer. The presence of flexible regions in the chemokine receptors determines their capability to develop specific mechanisms of action. We have recently focused on the features of the N-terminal region of human CXCR3 free in solution, where we demonstrate the presence of numerous conformational ensembles, dynamically stabilized by H-bonds. Since up to now no structure was experimentally determined for CXCR3, we decided to approach the study of its conformational behavior by molecular dynamics simulations, in a lipid bilayer, surrounded of water, at neutral pH and 300K. Furthermore, we modeled the CXCR3/CXCL11 complex, where CXCL11 is one of its natural ligands. The aim of this work is to have a vision as realistic as possible in dynamic terms of the biological mechanism that drives the search for the ligand, its interaction and the formation of a stable complex between CXCR3 and CXCL11. Overall, our approach has been able to describe the structural events which dynamically characterize the molecular mechanisms involved in the binding of CXCR3 to CXCL11 and the critical role exerted by its N-terminal region in "hunting" and capturing the ligand.

Comparison of the seleno-transcriptome expression between human non-cancerous mammary epithelial cells and two human breast cancer cell lines.

Breast cancer is the second most common cause of mortality in women; therefore, the identification of novel putative markers is required to improve its diagnosis and prognosis. Selenium is known to protect mammary epithelial cells from oxidative DNA damage, and to inhibit the initiation phase of carcinogenesis by stimulating DNA repair and apoptosis regulation. Consequently, the present study has focused attention on the selenoprotein family and their involvement in breast cancer. The present study performed a global analysis of the seleno-transcriptome expression in human breast cancer MCF-7 and MDA-MB231 cell lines compared with healthy breast MCF-10A cells using reverse transcription-quantitative polymerase chain reaction. The present data revealed the presence of differently expressed genes in MCF-7 and MDA-MB231 cells compared with MCF-10A cells: Four downregulated [glutathione peroxidase (GPX)1, GPX4, GPX5 and GPX7] and three upregulated (deiodinase iodothyronine, type II, GPX2 and GPX3) genes. Additionally, interactomic investigation were performed by the present study to evaluate the association between the downregulated and upregulated genes, and to identify putative HUB nodes, which represent the centers of association between the genes that are capable of direct control over the gene networks. Network analysis revealed that all differentially regulated genes, with the exception of selenoprotein T, are implicated in the same network that presents three HUB nodes interconnected to the selenoprotein mRNAs, including TP53, estrogen receptor 1 and catenin-ß1 (CTNNB1). Overall, these data demonstrated for the first time, a profile of seleno-mRNAs specific for human breast cells, indicating that these genes alter their expression on the basis of the ER-positivity or negativity of breast cancer cells.

Combining doxorubicin with a phenolic extract from flaxseed oil: Evaluation of the effect on two breast cancer cell lines.

Breast cancer is one of the most frequently diagnosed forms of cancer and different treatments are used to block its progression. However, it still represents a very common cause of death in women. Doxorubicin (Dox) is reported as an effective agent in breast cancer treatment nonetheless it induces many side­effects. For this reason, many laboratories are engaged in understanding how it is possible to decrease the drug concentration, considering that one of the possible solutions is to use drug synergy, combining it with natural substances. Recently we showed that a phenolic extract from flaxseed (FS) oil, named PEFSO, induced on MCF­7 cell line an increase of apoptosis with related modification of G0/G1 phase cell cycle, and the activation of signaling and pro­oxidant pathways. In this study we present data on the combined effect of Dox and PEFSO on two different breast cancer cell lines to define the conditions to use lower doses of this chemotherapeutic agent. We report the data relating to the ability of this mixture to induce cytotoxicity and apoptosis, cell cycle modification, mitochondrial membrane depolarization and activation of extrinsic and/or intrinsic apoptotic pathway.

A study on the structural features of SELK, an over-expressed protein in hepatocellular carcinoma, by molecular dynamics simulations in a lipid-water system.

Human SELK is a small trans-membrane selenoprotein characterized by a single trans-membrane helix, while the N-terminal region protrudes into the lumen and the long C-terminal domain into the cytoplasm. SELK is over-expressed in some cancers, like hepatocellular carcinoma; however its precise role in cancer development is presently unknown. SELK is involved in promoting the calcium flux, catalyzing palmitoylation reactions and protein degradation in the endoplasmic reticulum (ER). Therefore, this protein should bind many different proteins like p97/VCP in the supramolecular complex involved in the ER degradation pathway. To study the structural features of SELK in the membrane, we have modeled the protein and then subjected it to molecular dynamics simulations in a lipid-water system. The model shows a N-terminal domain with three ß-strands and a short helix, a well-defined trans-membrane helix and a C-terminal domain that lacks a persistent secondary structure and contains long disordered regions. The trajectory analysis during the simulation evidences that: (i) the N-terminal region explores a limited conformational space and is stabilized by intra-peptide H-bonds as well with membrane lipids and water, (ii) the trans-membrane helix was found to be quite stable and (iii) the disordered C-terminal region is stabilized by H-bonds with clustered water molecules as well as by rapidly interchanging intra-peptidic H-bonds, with a structural tendency to compact around the four HUB residues found for this domain. Moreover, N-terminal and C-terminal clusters are distributed differently in the conformational space suggesting that their dynamics are coupled complicatedly through the membrane. Further analyses have shown that the N-terminal has a tendency to pivot around the insertion with the TM-helix through the fluctuations of the three ß-strands, which, in turn, show features similar to WW-domains. These results will be useful to study the SELK, SELS and VCP complex representing an interesting druggable target for cancer.

Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines.

Hepatocellular carcinoma (HCC) is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK), whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3'UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3'UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium.

Differential Response of Two Human Breast Cancer Cell Lines to the Phenolic Extract from Flaxseed Oil.

Many studies have evidenced that the phenolic components from flaxseed (FS) oil have potential health benefits. The effect of the phenolic extract from FS oil has been evaluated on two human breast cancer cell lines, MCF7 and MDA-MB231, and on the human non-cancerous breast cell line, MCF10A, by SRB assay, cellular death, cell cycle, cell signaling, lipid peroxidation and expression of some key genes. We have evidenced that the extract shows anti-proliferative activity on MCF7 cells by inducing cellular apoptosis, increase of the percentage of cells in G0/G1 phase and of lipid peroxidation, activation of the H2AX signaling pathway, and upregulation of a six gene signature. On the other hand, on the MDA-MB2131 cells we verified only an anti-proliferative activity, a weak lipid peroxidation, the activation of the PI3K signaling pathway and an up-regulation of four genes. Overall these data suggest that the extract has both cytotoxic and pro-oxidant effects only on MCF7 cells, and can act as a metabolic probe, inducing differences in the gene expression. For this purpose, we have performed an interactomic analysis, highlighting the existing associations. From this approach, we show that the phenotypic difference between the two cell lines can be explained through their differential response to the phenolic extract.

Deducing the functional characteristics of the human selenoprotein SELK from the structural properties of its intrinsically disordered C-terminal domain.

The intrinsically disordered proteins (IDPs) cannot be described by a single structural representation but, due to their high structural fluctuation, through conformational ensembles. Certainly, molecular dynamics (MD) simulations represent a useful tool to study their different conformations capturing the conformational distribution. Our group is focusing on the structural characterization of proteins belonging to the seleno-proteome due to their involvement in cancer. They present disordered domains central for their biological function, and, in particular, SELK is a single-pass transmembrane protein that resides in the endoplasmic reticulum membrane (ER) with a C-terminal domain exposed to the cytoplasm that is known to interact with different components of the endoplasmic reticulum associated to the protein degradation (ERAD) pathway. This protein is found to be up-expressed in hepatocellular carcinoma and in other cancers. In this work we performed a detailed analysis of the C-terminal domain sequence of SELK and discovered that it is characterized by many prolines, and four negatively and eleven positively charged residues, which are crucial for its biological activity. This region can be considered as a weak polyelectrolyte and, specifically, a polycation, with high disordered propensity and different phosphorylation sites dislocated along the sequence. Then, we modeled its three-dimensional structure by performing MD simulations in water at neutral pH to analyze the structural stability as well as to identify the presence of HUB residues that play a key structural role as evidenced by the residue-residue interaction network analysis. Through this approach, we demonstrate that the C-terminal domain of SELK (i) presents a poor content of regular secondary structure elements, (ii) is dynamically stabilized by a network of intra-molecular H-bonds and H-bonds with water molecules, (iii) is highly fluctuating and, therefore, can be described only through a conformational ensemble, where we evidenced a distribution of equilibrium conformers which continuously inter-change their conformations. Finally to verify the specific role played by the negative charges, we also performed MD simulations at acidic pH. Overall, all the obtained results evidenced that SELK has the dynamic structural features to be defined as a HUB protein able to interact with multiple members. Therefore, considering the possible role that this protein can have in cancer development and progression, it can represent a target for drug design studies.

Serum Cytokinome Profile Evaluation: A Tool to Define New Diagnostic and Prognostic Markers of Cancer Using Multiplexed Bead-Based Immunoassays.

In recent years, many researchers are focusing their attention on the link between inflammation and cancer. The inflammation is involved in the tumor development and suppression, by stimulating the immune response. In particular, the transition from chronic inflammation to cancer produces angiogenic and growth factors able to repair the tissue and to promote cancer cell survival, implantation, and growth. In this contest, the cytokines contribute to the development of these processes becoming active before and during the inflammatory process and playing an important function at the various disease levels. Thus, these proteins can represent specific markers of tumor development and progression. Therefore the "cytokinome" term is used to indicate the evaluation of cytokine pattern by using an "omics" approach. Newly, specific protein chips of considerable and improved sensitivity are being developed to determine simultaneously several and different cytokines. This can be achieved by a multiplex technology that, through the use of small amounts of serum or other fluids, is used to determine the presence and the levels of underrepresented cytokines. Since this method is an accurate, sensitive, and reproducible cytokine assay, it is already used in many different studies. Thus, this review focuses on the more latest aspects related to cytokinome profile evaluation in different cancers.

Anti-Inflammatory Effects of a Methanol Extract from the Marine Sponge Geodia cydonium on the Human Breast Cancer MCF-7 Cell Line.

Many research groups are working to find new possible anti-inflammatory molecules, and marine sponges represent a rich source of biologically active compounds with pharmacological applications. In the present study, we tested different concentrations of the methanol extract from the marine sponge, Geodia cydonium, on normal human breast epithelial cells (MCF-10A) and human breast cancer cells (MCF-7). Our results show that this extract has no cytotoxic effects on both cell lines whereas it induces a decrease in levels of VEGF and five proinflammatory cytokines (CCL2, CXCL8, CXCL10, IFN-γ, and TNF-α) only in MCF-7 cells in a dose-dependent manner, thereby indicating an anti-inflammatory effect. Moreover, interactomic analysis suggests that all six cytokines are involved in a network and are connected with some HUB nodes such as NF-kB subunits and ESR1 (estrogen receptor 1). We also report a decrease in the expression of two NFKB1 and c-Rel subunits by RT-qPCR experiments only in MCF-7 cells after extract treatment, confirming NF-kB inactivation. These data highlight the potential of G. cydonium for future drug discovery against major diseases, such as breast cancer.

Intrinsically disordered amphiphilic peptides as potential targets in drug delivery vehicles.

Intrinsically disordered proteins/peptides play a crucial role in many physiological and pathological events and may assume a precise conformation upon binding to a specific target. Recently, we have described the conformational and functional properties of two linear ester peptides provided with the following sequences: Y-G-E-C-P-C-K-OAllyl (PepK) and Y-G-E-C-P-C-E-OAllyl (PepE). Both peptides are characterized by the presence of the "CPC" motif together with a few amino acids able to promote disorder. The CPC sequence is a binding motif for the CXCR4 receptor that represents a well-known target for cancer therapies. In this paper, we report on synthetic amphiphilic peptides that consist of lipophilic derivatives of PepE and PepK bearing two stearic alkyl chains and/or an ethoxylic spacer. These peptide amphiphiles form stable supramolecular aggregates; they present conformational features that are typical of intrinsically disordered molecules as shown by CD spectroscopy. Solution fluorescence and DLS studies have been performed to evaluate Critical Micellar Concentrations and the dimension of supramolecular aggregates. Moreover, preliminary in vitro cell-based assays have been conducted to investigate the molecular recognition processes involving the CXCR4 receptor. In the end, the results obtained have been compared with the previous data generated by the corresponding non-amphiphilic peptides (PepE and PepK).

The gene expression profiling of hepatocellular carcinoma by a network analysis approach shows a dominance of intrinsically disordered proteins (IDPs) between hub nodes.

We have analyzed the transcriptomic data from patients with hepatocellular carcinoma (HCC) after viral HCV infection at the various stages of the disease by means of a networking analysis using the publicly available E-MTAB-950 dataset. The data was compared with those obtained in our group from HepG2 cells, a cancer cell line that lacks the viral infection. By sequential pruning of data, and also taking into account the data from cells of healthy patients as blanks, we were able to obtain a distribution of hub genes for the various stages that characterize the disease and finally, we isolated a metabolic sub-net specific to HCC alone. The general picture is that the basic organization to energetically and metabolically sustain the cells in both the normal and diseased conditions is the same, but a complex cluster of sub-networks controlled by hub genes drives the HCC progression with high metabolic flexibility and plasticity. In particular, we have extracted a sub-net of genes strictly correlated to other hub genes of the network from HepG2 cells, but specific for the HCC and mainly devoted to: (i) control at chromatin levels of cell division; (ii) control of ergastoplasmatic stress through protein degradation and misfolding; (iii) control of the immune response also through an increase of mature T-cells in the thymus. This sub-net is characterized by 26 hub genes coding for intrinsically disordered proteins with a high ability to interact with numerous molecular partners. Moreover, we have also noted that periphery molecules, that is, with one or very few interactions (e.g., cytokines or post-translational enzymes), which do not have a central role in the clusters that make up the global metabolic network, essentially have roles as information transporters. The results evidence a strong presence of intrinsically disordered proteins with key roles as hubs in the sub-networks that characterize the various stages of the disease, conferring a structural plasticity to the net nodes but an inherent functional versatility to the whole metabolic net. Thus, our present article provides a novel way of targeting the intrinsic disorder in HCC networks to dampen the cancer effects and provides new insight into the potential mechanisms of HCC. Taken together, the present findings suggest novel targets to design strategies for drug design and may support a rational intervention in the pharmacotherapy of HCC and other associated diseases.

Evaluation of the selenotranscriptome expression in two hepatocellular carcinoma cell lines.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. Since the selenium is able to fight the oxidative damage which is one of the major origins of cell damage as well as cancer, we have recently focused our attention on selenoprotein family and their involvement in HCC. In the present paper we have carried out a global analysis of the selenotranscriptome expression in HepG2 and Huh7 cells compared to the normal human hepatocytes by reverse transcription-qPCR (RT-qPCR). Our data showed that in both cells there are three downregulated (DIO1, DIO2, and SELO) and ten upregulated (GPX4, GPX7, SELK, SELM, SELN, SELT, SELV, SEP15, SEPW1, and TrxR1) genes. Additionally, interactomic studies were carried out to evaluate the ability of these down- and upregulated genes to interact between them as well as to identify putative HUB nodes representing the centers of correlation able to exercise a direct control over the coordinated genes.

The Cytokinome Profile in Patients with Hepatocellular Carcinoma and Type 2 Diabetes.

Understanding the dynamics of the complex interaction network of cytokines, defined as ''cytokinome'', can be useful to follow progression and evolution of hepatocellular carcinoma (HCC) from its early stages as well as to define therapeutic strategies. Recently we have evaluated the cytokinome profile in patients with type 2 diabetes (T2D) and/or chronic hepatitis C (CHC) infection and/or cirrhosis suggesting specific markers for the different stages of the diseases. Since T2D has been identified as one of the contributory cause of HCC, in this paper we examined the serum levels of cytokines, growth factors, chemokines, as well as of other cancer and diabetes biomarkers in a discovery cohort of patients with T2D, chronic hepatitis C (CHC) and/or CHC-related HCC comparing them with a healthy control group to define a profile of proteins able to characterize these patients, and to recognize the association between diabetes and HCC. The results have evidenced that the serum levels of some proteins are significantly and differently up-regulated in all the patients but they increased still more when HCC develops on the background of T2D. Our results were verified also using a separate validation cohort. Furthermore, significant correlations between clinical and laboratory data characterizing the various stages of this complex disease, have been found. In overall, our results highlighted that a large and simple omics approach, such as that of the cytokinome analysis, supplemented by common biochemical and clinical data, can give a complete picture able to improve the prognosis of the various stages of the disease progression. We have also demonstrated by means of interactomic analysis that our experimental results correlate positively with the general metabolic picture that is emerging in the literature for this complex multifactorial disease.

Conformational ensembles explored dynamically from disordered peptides targeting chemokine receptor CXCR4.

This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target.

Structure-fluctuation-function relationships of seven pro-angiogenic isoforms of VEGFA, important mediators of tumorigenesis.

Vascular endothelial growth factor A (VEGFA) has different biological activities and plays a central role in tumor proliferation, angiogenesis and metastasis. Different VEGFA isoforms are generated by alternative splice site selection of exons 6, 7 and 8. In this paper, we analyzed the physical and chemical properties of the VEGFA exon 6 sequence, and modeled the three-dimensional structures of the regions corresponding to exons 6, 7 and 8 of six different pro-angiogenic isoforms of VEGFA in comparison to the experimental structure of VEGFA_165 by a combined approach of fold recognition and comparative modeling strategies and molecular dynamics simulations. Our results showed that i) exon 6 is a very flexible polycation with high disordered propensity, features well conserved in all mammals, ii) the structures of all the isoforms are stabilized by H-bond sub-networks organized around HUB residues and, iii) the charge content of exon 6 modulates the intrinsic structural preference of its flexible backbone, which can be described as an ensemble of conformations. Moreover, complexes between NRP-1 and VEGFA isoforms were modeled by molecular docking to study what isoforms are able to bind NRP-1. The analysis of complexes evidenced that VEGFA_121, VEGFA_145, VEGFA_183, VEGFA_189 and VEGFA_206, containing exons 7 and 8a, are able to interact with NRP-1 because they have the key regions of exons 7b and/or 8a. An overview of the isoforms shows how the fluctuations are the main guidance of their biological function. MD simulations also provide insights into factors that stabilize the binding regions of isoforms.