Structure and dynamics of γ-secretase with presenilin 2 compared to presenilin 1

Severe early-onset familial Alzheimer's disease (FAD) is caused by more than 200 different mutations in the genes coding for presenilin, the catalytic subunit of the 4-subunit protease complex γ-secretase, which cleaves the C99 fragment of the amyloid precursor protein (APP) to produce Aβ peptides. γ-Secretase exists with either of two homologues, PS1 and PS2. All cryo-electron microscopic structures and computational work has so far focused on γ-secretase with PS1, yet PS2 mutations also cause FAD. A central question is thus whether there are structural and dynamic differences between PS1 and PS2. To address this question, we use the cryo-electron microscopic data for PS1 to develop the first structural and dynamic model of PS2-γ-secretase in the catalytically relevant mature membrane-bound state at ambient temperature, equilibrated by three independent 500 ns molecular dynamics simulations. We find that the characteristic nicastrin extra-cellular domain breathing mode and major movements in the cytosolic loop between TM6 and TM7 occur in both PS2- and PS1-γ-secretase. The overall structures and conformational states are similar, suggesting similar catalytic activities. However, at the sequence level, charge-controlled membrane-anchoring is extracellular for PS1 and intracellular for PS2, which suggests different subcellular locations. The tilt angles of the TM2, TM6, TM7 and TM9 helices differ in the two forms of γ-secretase, suggesting that the two proteins have somewhat different substrate processing and channel sizes. Our MD simulations consistently indicated that PS2 retains several water molecules near the catalytic site at the bilayer, as required for catalysis. The possible reasons for the differences of PS1 and PS2 are discussed in relation to their location and function

Analysis of polyphenolic metabolites from in vitro gastrointestinal digested soft fruit extracts identify malvidin-3-glucoside as an inhibitor of PTP1B

Acknowledgements We are grateful to the Scottish Government Rural and Environment Science and Analytical Services (RESAS), the University of Aberdeen and Nutricia Research Foundation for funding. We thank Graham Horgan from Biomathematics and Statistics Scotland for their assistance with the statistical analysis. We thank Gordon J. McDougall and Rex M. Brennan, from The James Hutton Institute for supplying the soft fruits.Peer reviewedPostprin

ESMP: A high-throughput computational pipeline for mining SSR markers from ESTs

With the advent of high-throughput sequencing technology, sequences from many genomes are being deposited to public databases at a brisk rate. Open access to large amount of expressed sequence tag (EST) data in the public databases has provided a powerful platform for simple sequence repeat (SSR) development in species where sequence information is not available. SSRs are markers of choice for their high reproducibility, abundant polymorphism and high inter-specific transferability. The mining of SSRs from ESTs requires different high-throughput computational tools that need to be executed individually which are computationally intensive and time consuming. To reduce the time lag and to streamline the cumbersome process of SSR mining from ESTs, we have developed a user-friendly, web-based EST-SSR pipeline “EST-SSR-MARKER PIPELINE (ESMP)”. This pipeline integrates EST pre-processing, clustering, assembly and subsequently mining of SSRs from assembled EST sequences. The mining of SSRs from ESTs provides valuable information on the abundance of SSRs in ESTs and will facilitate the development of markers for genetic analysis and related applications such as marker-assisted breeding

Mining for SSRs and FDMs from expressed sequence tags of Camellia sinensis

Simple Sequence Repeats (SSRs) developed from Expressed Sequence Tags (ESTs), known as EST-SSRs are most widely used and potentially valuable source of gene based markers for their high levels of crosstaxon portability, rapid and less expensive development. The EST sequence information in the publicly available databases is increasing in a faster rate. The emerging computational approach provides a better alternative process of development of SSR markers from the ESTs than the conventional methods. In the present study, 12,851 EST sequences of Camellia sinensis, downloaded from National Center for Biotechnology Information (NCBI) were mined for the development of Microsatellites. 6148 (4779 singletons and 1369 contigs) non redundant EST sequences were found after preprocessing and assembly of these sequences using various computational tools. Out of total 3822.68 kb sequence examined, 1636 (26.61%) EST sequences containing 2371 SSRs were detected with a density of 1 SSR/1.61 kb leading to development of 245 primer pairs. These mined EST-SSR markers will help further in the study of variability, mapping, evolutionary relationship in Camellia sinensis. In addition, these developed SSRs can also be applied for various studies across species

Kanamycin-Mediated Conformational Dynamics of Escherichia coli Outer Membrane Protein TolC.

TolC is a member of the outer membrane efflux proteins (OEPs) family and acts as an exit duct to export proteins, antibiotics, and substrate molecules across the Escherichia coli cell membrane. Export of these molecules is evidenced to be brought about through the reversible interactions and binding of substrate-specific drug molecules or antibiotics with TolC and by being open for transport, which afterward leads to cross-resistance. Hence, the binding of kanamycin with TolC was monitored through molecular docking (MD), the structural fluctuations and conformational changes to the atomic level. The results were further supported from the steady-state fluorescence binding and isothermal titration calorimetry (ITC) studies. Binding of kanamycin with TolC resulted in a concentration dependent fluorescence intensity quenching with 7 nm blue shift. ITC binding data maintains a single binding site endothermic energetic curve with binding parameters indicating an entropy driven binding process. The confirmational changes resulting from this binding were monitored by a circular dichroism (CD) study, and the results showed insignificant changes in the α-helix and β-sheets secondary structure contents, but the tertiary structure shows inclusive changes in the presence of kanamycin. The experimental data substaintially correlates the RMSD, R g, and RMSF results. The resulting conformational changes of the TolC-kanamycin complexation was stabilized through H-bonding and other interactions

Screening of novel inhibitors targeting Human Papillomavirus 16 E6/AP/P53 ternary complex towards development of therapeutic strategies against HPV-mediated oncogenesis

Cervical cancer is the fourth most common cancer in women worldwide. It is well known that high-risk HPV is the main etiological agent for this infectious viral carcinoma. Human papillomaviruses are small (50 nm) double-stranded DNA viruses composed of a genome of 8 kilobase pair, enclosed inside a non-enveloped capsid protein. The genome includes three portions: (a) early genes (E1, E2, E4, E5, E6, E7) those regulate the vegetative and productive phase of viral life cycle; (b) late genes (L1, L2) which encode the capsid protein and (c) a noncoding regulatory region called long control region (LCR) involved in the regulation of viral replication and transcription. The HPV oncoproteins E6 and E7 recognize numerous host proteins, in large part by hijacking cellular domain-motif interaction networks. E6 and E7 oncoproteins disrupt cell cycle checkpoint control by inhibiting CDKs inhibitors (P21, P27) and degrading P53. In the process of E6 mediated degradation, E6 binds to a short leucine (L)-rich LxxLL consensus sequence within the cellular ubiquitin ligase E6AP3. Subsequently, the E6/E6AP heterodimer recruits and degrades p53. The LxxLL peptide of E6AP is sufficient to render E6 liable to interact with p53 ‘core’ (DNA binding) domain of p53 required for the interaction with E6/E6AP9–11. In the present study, we explored specific novel inhibitors targeting three different druggable pocket i.e., E6-binding cleft, LxxLL pocket of AP and the p53-binding cleft of E6/E6AP/p53 ternary complex using AutoDock tool. A total of five novel compounds with higher binding energy were identified as potential competitive inhibitors against HPV16 E6/AP/P53 ternary complex. The combinatorial strategies targeting these druggable pockets are expected to open up better avenues for the development of therapeutic strategies against HPV-mediated oncogenesis in near future

Insights into the mode of recognition of DIII of dengue E protein with GRP78: A molecular dynamics approach

Dengue virus (DENV), a single stranded RNA positive-strand virus belongs to Flaviviridae causes Dengue in several parts of South-East Asia. During infection, dengue virus proteins interact with host cellular constituents, thus promotes the remodeling of the cell to facilitate virus production. Recent studies have shown that DENV E protein interact the cellular chaperone GRP78. GRP78 that plays a dual role in virus life cycle i.e., virus entry and virus replication, is a novel host factor that could be a potential therapeutic target. Currently, the three-dimensional interaction between GRP78 and DENV E protein remains largely unknown. It is assumed that DENV E protein interacts with the C-terminus of GRP78, and the C-terminus of GRP78 is believed to be the predominant protein interacting domain, while the N-terminus is believed to contain regulatory domains that mediate C-terminal binding. Although the exact E protein domain mediating binding to GRP78 is not known, it has been proposed that GRP78 and DENV E protein interact through the immunoglobulin like structure in the DENV E protein that resides in domain III (DIII).  So, the present study was undertaken to unravel molecular basis of GRP78 and DENV E protein interaction through molecular modeling, protein-protein docking and Molecular dynamics simulations. The three-dimensional structures of DIII of E protein from DENVI was modelled and docked against crystal structure of GRP78 (PDB ID: 3LDL) using ClusPro. The top ranked pose from ClusPro was again refined using HADDOCK. Molecular dynamics simulation was performed to understand mode of recognition and dynamics stability of the refined DIII-GRP78 complex in aqueous solution for 10 ns. The critical residues i.e., Thr303/Lys46, Lys295/ Lys152 and Lys399/Asn239 identified in this study are indispensable for DIII mediated interaction of dengue virus with host protein GRP78. The results from this study is expected shed deep insights into the crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective anti-viral therapy for DENV in near future

Computing the Pathogenicity of Alzheimer’s Disease Presenilin 1 Mutations

Alzheimer’s disease (AD) is one of the major global health challenges of the 21st century. More than 200 distinct mutations in presenilin 1 (PSEN1) cause severe early-onset familial AD (FAD) and are thus of central interest to the etiology of AD. PSEN1 is the catalytic subunit of γ-secretase that produces β-amyloid peptide (Aβ), and the mutations tend to increase the produced Aβ42/Aβ40 ratio. The molecular reasons for the pathogenesis of these mutations are unknown. We studied a close-to-complete data set of PSEN1 mutations using 21 different computational methods hypothesized to reproduce pathogenesis, using both sequence- and structure-based methods with the full γ-secretase complex as input. First, we tested whether pathogenicity can be estimated accurately using all possible mutations in PSEN1 as a direct control. Several methods predict the pathogenicity of the mutations (pathogenic vs all other possible mutations) well, with accuracies approaching 90%. We then designed a stricter test for predicting the severity of the mutations estimated by the average clinical age of symptom onset for mutation carriers. Surprisingly, we can predict the clinical age of symptom onset at 95% confidence or higher with several methods. Accordingly, our results show that simple biochemical properties of the amino acid changes rationalize an important part of the pathogenicity of FAD-causing PSEN1 mutations. Although pathogenic mutations generally destabilize γ-secretase, all of the tested protein stability methods failed to predict pathogenicity. Thus, either the static cryogenic-electron-microscopy-derived molecular-dynamics-equilibrated structures used as input fail to capture the stability effect of mutated side chains or protein stability is simply not a key factor in the pathogenicity. Our findings suggest that the chemical causes of FAD may be modeled and lend promise to the development of a semiquantitative model predicting the age of onset of mutation carriers that could eventually become of care-strategic value