Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short ß-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains.

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.

Polysaccharides from the Marine Environment with Pharmacological, Cosmeceutical and Nutraceutical Potential.

Carbohydrates, also called saccharides, are molecules composed of carbon, hydrogen, and oxygen. They are the most abundant biomolecules and essential components of many natural products and have attracted the attention of researchers because of their numerous human health benefits. Among carbohydrates the polysaccharides represent some of the most abundant bioactive substances in marine organisms. In fact, many marine macro- and microorganisms are good resources of carbohydrates with diverse applications due to their biofunctional properties. By acting on cell proliferation and cycle, and by modulating different metabolic pathways, marine polysaccharides (including mainly chitin, chitosan, fucoidan, carrageenan and alginate) also have numerous pharmaceutical activities, such as antioxidative, antibacterial, antiviral, immuno-stimulatory, anticoagulant and anticancer effects. Moreover, these polysaccharides have many general beneficial effects for human health, and have therefore been developed into potential cosmeceuticals and nutraceuticals. In this review we describe current advances in the development of marine polysaccharides for nutraceutical, cosmeceutical and pharmacological applications. Research in this field is opening new doors for harnessing the potential of marine natural products.

Structure-function relationship and evolutionary history of the human selenoprotein M (SelM) found over-expressed in hepatocellular carcinoma.

In humans we know 25 selenoproteins that play important roles in redox regulation, detoxification, immune-system protection and viral suppression. In particular, selenoprotein M (SelM) may function as thiol disulfide oxidoreductase that participates in the formation of disulfide bonds, and can be implicated in calcium responses. However, it presents a redox motif (CXXU), where U is a selenocysteine, and may also function as redox regulator because its decreased or increased expression regulated by dietary selenium alters redox homeostasis. No data are reported in literature about its involvement in cancer but only in neurodegenerative diseases. In this paper we evaluated the SelM expression in two hepatoma cell lines, HepG2 and Huh7, compared to normal hepatocytes. The results suggested its involvement in hepatocellular carcinoma (HCC) as well as its possible use to follow the progression of this cancer as putative marker. The aim of this study has been to analyze the structure-function relationships of SelM. Hence, firstly we studied the evolutionary history of this protein by phylogenetic analysis and GC content of genes from various species. So, we modeled the three-dimensional structure of the human SelM evaluating its energetic stability by molecular dynamics simulations. Moreover, we modeled some of its mutants to obtain structural information helpful for structure-based drug design.

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.

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.

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.