ECMIS: computational approach for the identification of hotspots at protein-protein interfaces

Background

Various methods have been developed to computationally predict hotspot residues at novel protein-protein interfaces. However, there are various challenges in obtaining accurate prediction. We have developed a novel method which uses different aspects of protein structure and sequence space at residue level to highlight interface residues crucial for the protein-protein complex formation.

Results

ECMIS (Energetic Conservation Mass Index and Spatial Clustering) algorithm was able to outperform existing hotspot identification methods. It was able to achieve around 80% accuracy with incredible increase in sensitivity and outperforms other existing methods. This method is even sensitive towards the hotspot residues contributing only small-scale hydrophobic interactions.

Conclusion

Combination of diverse features of the protein viz. energy contribution, extent of conservation, location and surrounding environment, along with optimized weightage for each feature, was the key for the success of the algorithm. The academic version of the algorithm is available at http://caps.ncbs.res.in/download/ECMIS/ECMIS.zip.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-303) contains supplementary material, which is available to authorized users.     

Absence of cue-recruitment for extrinsic signals: sounds, spots, and swirling dots fail to influence perceived 3D rotation direction after training

The visual system can learn to use information in new ways to construct appearance. Thus, signals such as the location or translation direction of an ambiguously rotating wire frame cube, which are normally uninformative, can be learned as cues to determine the rotation direction. This perceptual learning occurs when the formerly uninformative signal is statistically associated with long-trusted visual cues (such as binocular disparity) that disambiguate appearance during training. In previous demonstrations, the newly learned cue was intrinsic to the perceived object, in that the signal was conveyed by the same image elements as the object itself. Here we used extrinsic new signals and observed no learning. We correlated three new signals with long-trusted cues in the rotating cube paradigm: one crossmodal (an auditory signal) and two within modality (visual). Cue recruitment did not occur in any of these conditions, either in single sessions or in ten sessions across as many days. These results suggest that the intrinsic/extrinsic distinction is important for the perceptual system in determining whether it can learn and use new information from the environment to construct appearance. Extrinsic cues do have perceptual effects (e.g. the "bounce-pass" illusion and McGurk effect), so we speculate that extrinsic signals must be recruited for perception, but only if certain conditions are met. These conditions might specify the age of the observer, the strength of the long-trusted cues, or the amount of exposure to the correlation.     

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n?=?52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n?=?11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.     

Enhanced intracellular delivery of the reactive oxygen species (ROS)-generating copper chelator D-penicillamine via a novel gelatin--D-penicillamine conjugate

D-Penicillamine (D-pen) is an established copper chelator. We have recently shown that the copper-catalyzed D-pen oxidation generates concentration-dependent hydrogen peroxide (H 2O 2). Additionally, D-pen coincubated with cupric sulfate resulted in cytotoxicity in human leukemia and breast cancer cells due to the extracellular generation of reactive oxygen species (ROS). The inherent physicochemical properties of D-pen such as its short in vivo half-life, low partition coefficient, and rapid metal catalyzed oxidation limit its intracellular uptake and the potential utility as an anticancer agent in vivo. Therefore, to enhance the intracellular delivery and to protect the thiol moiety of D-pen, we designed, synthesized, and evaluated a novel gelatin-D-pen conjugate. D-pen was covalently coupled to gelatin with a biologically reversible disulfide bond with the aid of a heterobifunctional cross-linker ( N-succinimidyl-3-(2-pyridyldithio)-propionate) (SPDP). Additionally, fluorescein-labeled gelatin-D-pen conjugate was synthesized for cell uptake studies. D-pen alone was shown not to enter leukemia cells. In contrast, the qualitative intracellular uptake of the conjugate in human leukemia cells (HL-60) was shown with confocal microscopy. The conjugate exhibited slow cell uptake (over the period of 48 to 72 h). A novel HPLC assay was developed to simultaneously quantify both D-pen and glutathione in a single run. The conjugate was shown to completely release D-pen in the presence of glutathione (1 mM) in approximately 3 h in PBS buffer, pH 7.4. The gelatin-D-pen conjugate resulted in significantly greater cytotoxicity compared to free D-pen, gelatin alone, and a physical mixture of gelatin and D-pen in human leukemia cells. Further studies are warranted to assess the potential of D-pen conjugate in the delivery of D-pen as a ROS generating anticancer agent.     

Copper chelation by D-penicillamine generates reactive oxygen species that are cytotoxic to human leukemia and breast cancer cells

Serum and tumor copper levels are significantly elevated in a variety of malignancies including breast, ovarian, gastric, lung, and leukemia. D-Penicillamine (D-pen), a copper-chelating agent, at low concentrations in the presence of copper generates concentration-dependent cytotoxic hydrogen peroxide (H(2)O(2)). The purpose of these studies was to investigate the in vitro cytotoxicity, intracellular reactive oxygen species (ROS) generation, and the reduction in intracellular thiol levels due to H(2)O(2) and other ROS generated from copper-catalyzed D-pen oxidation in human breast cancer cells (BT474, MCF-7) and human leukemia cells (HL-60, HL-60/VCR, HL-60/ADR). D-pen (< or = 400 microM) in the presence of cupric sulfate (10 microM) resulted in concentration-dependent cytotoxicity. Catalase was able to completely protect the cells, substantiating the involvement of H(2)O(2) in cancer cell cytotoxicity. A linear correlation between the D-pen concentration and the intracellular ROS generated was shown in both breast cancer and leukemia cells. D-pen in the presence of copper also resulted in a reduction in intracellular reduced thiol levels. The H(2)O(2)-mediated cytotoxicity was greater in leukemia cells compared to breast cancer cells. These results support the hypothesis that D-pen can be employed as a cytotoxic copper-chelating agent based on its ROS-generating ability.     

Synthesis of desformylflustrabromine and its evaluation as an alpha4beta2 and alpha7 nACh receptor modulator

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.