The role of Wnt palmitoleylated loop conserved disulfide bonds in Wnt-frizzled complex structural dynamics: Insights from molecular dynamics simulations.

Wnts are lipid-modified proteins rich in cysteine, regulating developmental processes, and are involved in various pathological conditions. Wnts structure resembles a hand, with a palmitoleylated thumb and an index finger-like domain interacting with frizzled (FZ) receptors. Previous research shows the palmitoleyl group and the disulfides importance in Wnt folding, secretion, and function, but the structural basis is not fully understood. Here, we utilized classical molecular dynamics simulation (800-ns in total) to investigate how the thumb palmitoleyl and its close conserved disulfides (183-190, 181-195) regulated Wnt-FZ interaction and structural dynamics. Using Steered molecular dynamics experiment followed by a relaxing procedure, we also explored if these disulfides are important in Wnt-FZ complex formation. According to our results, the palmitoleyl group contributes significantly to stabilize Wnt-FZ interaction, and the disulfides modulate this contribution. We also demonstrated that disulfide 183-190 regulates the Wnt thumb fluctuation, hydrogen bond network, and secondary structure. The DCCM analysis depicted disulfide 183-190 roles in regulating native-like collective movement in the palmitoleylated loop, which changed after this disulfide removal. The pulling-relaxing experiment showed that both the disulfides, and especially, the disulfide 183-190, are highly important for long-range salt-bridge interaction establishment between Wnt Lys182 and FZ Glu64, led palmitoleyl group appropriate positioning to FZ, suggested this disulfide essential role in Wnt-FZ complex formation. Together, our findings provide new insights to how thumb-positioned disulfides contribute to Wnt-FZ complex formation, structural dynamics, and stability, introducing disulfide 183-190 as a consequential element to target in drug design and development against Wnt signalling.

A simple 2-step purification process of α-amylase from Bacillus subtilis: Optimization by response surface methodology.

Purification of extracellular α-amylase from Bacillus subtilis was carried out via fractional precipitation by acetone and ion exchange chromatography. These steps provide fast precipitation as well as purification of α-amylase to improve enzyme purity, activity and stability. Compared with two-phase methods in which the yield was less than 1, this method resulted in a yield of more than 3. Moreover, 95% of acetone was recovered that enhanced the economy of the downstream process. Using the data provided by 2D electrophoresis, purification was done by a single step ion exchange chromatography. The enzyme exhibited a molecular mass (SDS-PAGE) of 50KD and the pI of 5. Maximum "yield" and "purification fold" were achieved through optimization of operation parameters such as volume and flowrate of loaded protein using response surface methodology (RSM). 0.5ml of loaded protein at a flow rate of 0.5 ml/min was purified as 48 folds and achieved a specific activity of 524 U/mg.

The next generation personalized models to screen hidden layers of breast cancer tumorigenicity.

BACKGROUND: Breast cancer (BC) is a challenging disease and major cause of death amongst women worldwide who die due to tumor relapse or sidelong diseases. BC main complexity comes from the heterogeneous nature of breast tumors that demands customized treatments in the form of personalized medicine. REVIEW OF THE LITERATURE AND DISCUSSION: Spatiotemporally dynamic and heterogeneous nature of BC tumors is shaped by their clonal evolution and sub-clonal selections and shapes resistance to collective or group therapies that drives cancer recurrence and tumor metastasis. Personalized intervention promises to administer medications that selectively target each individual patient tumor and even further each colonized secondary tumor. Such personalized regimens will require creation of in vitro and in vivo models genuinely recapitulating characteristics of each tumor type as initiating platforms for two main purposes: to closely monitor the tumorigenic processes that shape tumor heterogeneity and evolution as the main driving forces behind tumor chemo-resistance and relapse, and subsequently to establish patient-specific preventive and therapeutic measures. While application of tumor modeling for personalized drug screening and design requires a separate review, here we discuss the personalized utilities of xenograft modeling in investigating BC tumor formation and progression toward metastasis. We will further elaborate on the impact of innovative technologies on personalized modeling of BC tumorigenicity at improved resolution. CONCLUSION: Heterogeneous nature of each BC tumor requires personalized intervention implying that modeling breast tumors is inevitable for better disease understanding, detection and cure. Patient-derived xenografts are just the initiating piece of the puzzle for ideal management of breast cancer. Emerging technologies promise to model BC more personalized than before.

The investigation of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels: a computational simulation.

Kappa-Hefutoxin1 is a K(+) channel-blocking toxin from the scorpion Heterometrus fluvipes. It is a 22-residue protein that adapts a novel fold of two parallel helices linked by two disulfide bridges without beta-sheets. Recognition of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels, Kv1.1, Kv1.2, and Kv1.3, was studied by 3D-Dock software package. All structures of kappa-Hefutoxin1 were considered during the simulations, which indicated that even small changes in the structure of kappa-Hefutoxin1 considerably affected both the recognition and the binding between kappa-Hefutoxin1 and the Kv1 channels. kappa-Hefutoxin1 is located around the extracellular part of the Kv1 channels, making contacts with its helices. Lys 19, Tyr 5, Arg 6, Trp 9, or Arg 10 in the toxin and residues Asp 402, His 404, Thr 407,Gly 401, and Asp 386 in each subunit of the Kv potassium channel are the key residues for the toxin-channel recognition. Moreover, the simulation result demonstrates that the hydrophobic interactions are important in interaction of negatively charged toxins with potassium channels. The results of our docking/molecular dynamics simulations indicate that our 3D model structure of the kappa-Hefutoxin1-complex is both reasonable and acceptable and could be helpful for smarter drug design and the blocking agents of Kv1 channels.

Effective factors in thermostability of thermophilic proteins.

Thermostability of proteins in general and especially thermophilic proteins has been subject of a wide variety of studies based on theoretical and experimental investigation. Thermostability seems to be a property obtained through many minor structural modifications rather than certain amino acids substitution. In comparison with its mesophile homologue in a thermostable protein, usually a number of amino acids are exchanged. A wide variety of theoretical studies are based on comparative investigation of thermophilic proteins characteristics with their mesophilic counterparts in order to reveal their sequences, structural differences and consequently, to relate these observed differences to the thermostability properties. In this work we have compared a dataset of thermophilic proteins with their mesophilic homologues and furthermore, a mesophilic proteins dataset was also compared with its mesophilic homologue. This strategy enabled us first, to eliminate noise or background differences from signals and moreover, the important factors which were related to the thermostability were recognized too. Our results reveal that thermophilic and mesophilic proteins have both similar polar and nonpolar contribution to the surface area and compactness. On the other hand, salt bridges and main chain hydrogen bonds show an increase in the majority of thermophilic proteins in comparison to their mesophilic homologues. In addition, in thermophilic proteins hydrophobic residues are significantly more frequent, while polar residues are less. These findings indicate that thermostable proteins through evolution adopt several different strategies to withstand high temperature environments.

Chemical modification of bacterial alpha-amylases: changes in tertiary structures and the effect of additional calcium.

A comparative study was performed on the effect of calcium on native and chemically modified forms of mesophilic and thermophilic alpha-amylases. Circular dichroism (CD) and irreversible thermoinactivation studies were carried out in the absence and presence of 10 mM calcium. From the CD experiments, changes in the tertiary structure of these enzymes, brought about by modification, were concluded. Furthermore, these changes were found to be influenced by the presence of calcium. Sorbitol was very effective in affording protection against irreversible thermoinactivation of native and modified forms of the enzymes, both in the absence and presence of calcium. Results are discussed in terms of the usefulness of this new approach involving a combination of medium and chemical modification for protein stabilization and enhancement of catalytic potential.

Chemical modification of lysine residues in Bacillus alpha-amylases: effect on activity and stability.

Chemical modification of lysine residues in two bacterial alpha-amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13 +/- 1 residues in BAA and 10 +/- 1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement.

Prediction of protein surface accessibility with information theory.

A new, simple method based on information theory is introduced to predict the solvent accessibility of amino acid residues in various states defined by their different thresholds. Prediction is achieved by the application of information obtained from a single amino acid position or pair-information for a window of seventeen amino acids around the desired residue. Results obtained by pairwise information values are better than results from single amino acids. This reinforces the effect of the local environment on the accessibility of amino acid residues. The prediction accuracy of this method in a jackknife test system for two and three states is better than 70 and 60 %, respectively. A comparison of the results with those reported by others involving the same data set also testifies to a better prediction accuracy in our case.

Homology-based molecular modelling of PLP-dependent histidine decarboxylase from Mmorganella morganii.

The 3-D structural information is a prerequisite for a rational ligand design. In the absence of experimental data, model building on the basis of a known 3-D structure of a homologous protein is at present the only reliable method to obtain structural information. A homology model building study of the pyridoxal 5'-phosphate (PLP)-dependent histidine decarboxylase from Morganella morganii (HDC-MM) has been carried out based on the crystal structure of the aspartate aminotransferase from Escherichia coli (AAT-EC). The primary sequences of AAT-EC and HDC-MM were aligned by automated alignment procedure. A 3-D model of HDC-MM was constructed by copying the coordinates of the residues from the crystal structure of AAT-EC into the corresponding residues in HDC-MM. After energy-minimization of the resulting 3-D model of HDC-MM, possible active site residues were identified by fitting the substrate (l-histidine) into the proposed active-site. In our model, several residues, which have an important role in the AAT-EC active-site, are located in positions spatially identical to those in AAT-EC structure. The back-bone of the modelled active site pocket is constructed by residues; Gly-92, Gly-93, Thr-93, Ser-115, Asp-200, Ala-202, Ser-229 and Lys-232 together with residues Asn-8, His-119, Thr-171, His-198, Leu-203, His-231, Ser-236 and Ile-238. In the ligand binding site, it appears that the HDC-MM model will position l-histidine (substrate) in the area consisting of the residues; Glu-29, Ser-30, Leu-38, His-231 and Lys-232. The nitrogen atom of the imidazole ring (N2) of the substrate is predicted to interact with the carboxylate group of Ser-30. The alpha-carboxylate of histidine points toward the Lys-232 to have electrostatic interaction with its side chain nitrogen atom (N(Z)). In conclusion, this combination of sequence and 3-D structural homology between AAT-EC and HDC-MM model could provide insight in assigning the probable active site residues.

Quantum mechanical study of the intermediates formed following the reaction of the histidine decarboxylase's substrate and inhibitors with coenzyme.

Histidine decarboxylase catalyses the decarboxylation of l-histidine to histamine using pyridoxal-5'-phosphate (PLP) as coenzyme. The PM3 quantum mechanical conformation method of analysis and heat of formation calculation were carried out for intermediates which are probably formed during the interaction of histidine (substrate), (s)-alpha-methylhistidine, (s)-alpha-hydrazinohistidine, (s)-alpha-fluoromethylhistidine and (s)-alpha-difluoromethylhistidine (inhibitors) with PLP-dependent histidine decarboxylase from Morganella morganii. The results suggest that the structures of the intermediates before and after decarboxylation were found to exist in a conformation showing a planar arrangement of the double bonds with the pyridoxylidene ring and the bond to the carboxyl group being perpendicular to this plane. After decarboxylation, all the double bonds are in the plane of the pyridoxylidene ring which facilitates the electron displacement for the following protonation at C(alpha). The values of the enthalpy for intermediates would increase the probability of their formation in the enzyme's active site which are consistent with all available stereochemical and mechanistic data.