The structural basis of Akt PH domain interaction with calmodulin

Akt plays a key role in the Ras/PI3K/Akt/mTOR signaling pathway. In breast cancer, Akt translocation to the plasma membrane is enabled by the interaction of its pleckstrin homology domain (PHD) with calmodulin (CaM). At the membrane, the conformational change promoted by PIP₃ releases CaM and facilitates Thr308 and Ser473 phosphorylation and activation. Here, using modeling and molecular dynamics simulations, we aim to figure out how CaM interacts with Akt’s PHD at the atomic level. Our simulations show that CaM-PHD interaction is thermodynamically stable and involves a β-strand rather than an α-helix, in agreement with NMR data, and that electrostatic and hydrophobic interactions are critical. The PHD interacts with CaM lobes; however, multiple modes are possible. IP₄, the polar head of PIP₃, weakens the CaM-PHD interaction, implicating the release mechanism at the plasma membrane. Recently, we unraveled the mechanism of PI3Kα activation at the atomistic level and the structural basis for Ras role in the activation. Here, our atomistic structural data clarify the mechanism of how CaM interacts, delivers, and releases Akt—the next node in the Ras/PI3K pathway—at the plasma membrane.     

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.     

Constructing structural networks of signaling pathways on the proteome scale

Proteins function through their interactions, and the availability of protein interaction networks could help in understanding cellular processes. However, the known structural data are limited and the classical network node-and-edge representation, where proteins are nodes and interactions are edges, shows only which proteins interact; not how they interact. Structural networks provide this information. Protein-protein interface structures can also indicate which binding partners can interact simultaneously and which are competitive, and can help forecasting potentially harmful drug side effects. Here, we use a powerful protein-protein interactions prediction tool which is able to carry out accurate predictions on the proteome scale to construct the structural network of the extracellular signal-regulated kinases (ERK) in the mitogen-activated protein kinase (MAPK) signaling pathway. This knowledge-based method, PRISM, is motif-based, and is combined with flexible refinement and energy scoring. PRISM predicts protein interactions based on structural and evolutionary similarity to known protein interfaces.     

Enriching the human apoptosis pathway by predicting the structures of protein-protein complexes

Apoptosis is a matter of life and death for cells and both inhibited and enhanced apoptosis may be involved in the pathogenesis of human diseases. The structures of protein-protein complexes in the apoptosis signaling pathway are important as the structural pathway helps in understanding the mechanism of the regulation and information transfer, and in identifying targets for drug design. Here, we aim to predict the structures toward a more informative pathway than currently available. Based on the 3D structures of complexes in the target pathway and a protein-protein interaction modeling tool which allows accurate and proteome-scale applications, we modeled the structures of 29 interactions, 21 of which were previously unknown. Next, 27 interactions which were not listed in the KEGG apoptosis pathway were predicted and subsequently validated by the experimental data in the literature. Additional interactions are also predicted. The multi-partner hub proteins are analyzed and interactions that can and cannot co-exist are identified. Overall, our results enrich the understanding of the pathway with interactions and provide structural details for the human apoptosis pathway. They also illustrate that computational modeling of protein-protein interactions on a large scale can help validate experimental data and provide accurate, structural atom-level detail of signaling pathways in the human cell.     

Principles of K-Ras effector organization and the role of oncogenic K-Ras in cancer initiation through G1 cell cycle deregulation

Illustrated here is the critical role of oncogenic KRAS in the initiation of cancer through deregulation of the G1 cell cycle, and elements and scenarios taking place under physiological conditions and in KRAS-driven cancer. Raf, PI3K and RalGDS are major K-Ras effectors. They bind at the same Ras site. What decides the cell selection among them? This temporal and spatial decision is critical since in some cellular context the outcome of their signaling pathways may oppose each other. Key among them is the concentration of calcium/calmodulin, negative feedback loops, where a downstream member of the pathway inhibits its upstream activator and cross-inhibition, where inhibition entails blocking another pathway. These three elements, in addition to spatial restrictions by K-Ras-membrane interactions, are not independent; they integrate to provide blueprints for cell decisions. Importantly, elucidation of signaling requires not only K-Ras binary interactions; but the structures and dynamics of its multiprotein complexes.     

MMP?REDOX/NO Interplay in Periodontitis and Its Inhibition with Satureja hortensis L. Essential Oil

Satureja hortensis L. is an aromatic plant with antibacterial and antibiofilm activities against periodontopathogens. Here, we attempted to find out whether the antioxidant properties of S. hortensis L. essential oil (EO) could be used to inhibit matrix metalloproteinase (MMP) activities and prevent the induction of cell death by a pro‐oxidant insult. First, a landscape analysis of MMP and REDOX/nitric oxide (NO)‐related genes was performed (MRN model), and array data from periodontitis patients were plotted over the newly developed model. Thereafter, the antigelatinolytic activity of S. hortensis L. EO and its preventive effect against hydrogen peroxide (H₂O₂)‐induced cell death were tested in vitro (HaCaT cells). Up‐regulation of MMP genes in the MRN network (except for MMP‐10, ‐15, ‐16, ‐20, ‐25, and ‐26) and differential expression of genes coding for antioxidant enzymes were found among others in periodontitis samples. MMP2 and MMP9 were central genes in the MRN network model. Moreover, treatments with 1 and 5 μl/ml of S. hortensis L. EO inhibited both MMP‐2 and MMP‐9 activities, and H₂O₂‐induced cell death in vitro. We concluded that S. hortensis L. EO could be a promising host‐modulating agent, since oxidative stress and excessive MMP expression/activity are typical hallmarks of periodontal pathogenesis.     

The Structural Pathway of Interleukin 1 (IL-1) Initiated Signaling Reveals Mechanisms of Oncogenic Mutations and SNPs in Inflammation and Cancer

Interleukin-1 (IL-1) is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance) the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor). We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. In silico mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP) mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.     

Anti-biofilm properties of Satureja hortensis L. essential oil against periodontal pathogens

Essential oils of several plants are widely used in ethnomedicine for their antimicrobial and anti-inflammatory properties. However, very limited data exist on their use in connection to periodontal diseases. The aim of the present study was to investigate the bacterial growth inhibiting and anti-biofilm effects of Satureja hortensis L. (summer savory), Salvia fruticosa M. (sage), Lavandula stoechas L. (lavender), Myrtus communis L., and Juniperus communis L. (juniper) essential oils. Chemical compositions of the essential oils were analyzed by gas chromatography–mass spectrometry, minimum inhibitor concentrations (MICs) with the agar dilution method, and anti-biofilm effects by the microplate biofilm assay. The toxicity of each essential oil was tested on cultured keratinocytes. Of the 5 essential oils, S. hortensis L. essential oil had the strongest growth inhibition effect. Subinhibitory dose of S. hortensis L. essential oil had anti-biofilm effects only against Prevotella nigrescens. Essential oils did not inhibit keratinocyte viability at the concentrations of 1 and 5 μl/ml, however at the concentration of 5 μl/ml epithelial cells detached from the culture well bottom. The present findings suggest that S. hortensis L. essential oil inhibits the growth of periodontal bacteria in the concentration that is safe on keratinocytes, however, in the subinhibitory concentration its anti-biofilm effect is limited.     

Electrical remodeling of cardiac myocytes from mice with heart failure due to the overexpression of tumor necrosis factor-alpha

Mice that overexpress the inflammatory cytokine tumor necrosis factor-alpha in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca2+ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K+ and/or inward Ca2+ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K+ currents from left ventricular myocytes of TNF mice revealed an approximately 50% decrease in the rapidly activating, rapidly inactivating transient outward K+ current Ito and in the rapidly activating, slowly inactivating delayed rectifier current IK,slow1, an approximately 25% decrease in the rapidly activating, slowly inactivating delayed rectifier current IK,slow2, and no significant change in the steady-state current Iss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca2+ current ICa,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K+ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca2+ handling, contribute to arrhythmias.