Contribution of Natural Inhibitors to the Understanding of the PI3K/PDK1/PKB Pathway in the Insulin-mediated Intracellular Signaling Cascade

The critical initial steps in insulin action include phosphorylation of adapter proteins and activation of phosphatidylinositol 3-kinase (PI3K). One of important components in this process is a protein called Akt/protein kinase B (PKB). The work of numerous different researchers indicates a role of PKB in regulating insulin-stimulated glucose uptake. The crucial role of lipid second messengers in PKB activation has been dissected through the use of the PI3K-specific inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Receptor-activated PI3K synthesizes the lipid second messenger PtdIns[3,4,5]-trisphosphate, leading to the recruitment of PKB to the membrane. Membrane attachment of PKB is mediated by its pleckstrin homology domain binding to PtdIns[3,4,5]-trisphosphate or PtdIns[3,4]-bisphosphate with high affinity. Activation of PKB alpha is then achieved at the plasma membrane by phosphorylation of Thr308 in the activation-loop of the kinase domain and Ser473 in the carboxy-terminal regulatory region, respectively. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) is responsible for T308 phosphorylation. The usage of specific inhibitors and natural compound has significantly contributed to investigate the molecular mechanism of PI3K/PDK1/PKB signaling pathway, leading to the putative therapeutics benefits of patients. This review focuses on the contribution of natural inhibitor or compound in our understanding of the mechanism by which insulin induces, especially in PI3K/PDK1/PKB signaling.     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

Selective Antifolates for Chemically Labeling Proteins in Mammalian Cells

Antifolate labels : Molecules that bind specifically and with high affinity to proteins can be developed into powerful tools for chemical biology. The interaction between substituted 5‐benzyl pyrimidines and dihydrofolate reductase can be exploited for chemically labeling fusion proteins in mammalian cells.

     

Analogies Between Digital Radio and Chemical Orthogonality as a Method for Enhanced Analysis of Molecular Recognition Events

Acoustic wave biosensors are a real-time, label-free biosensor technology, which have been exploited for the detection of proteins and cells. One of the conventional biosensor approaches involves the immobilization of a monolayer of antibodies onto the surface of the acoustic wave device for the detection of a specific analyte. The method described within includes at least two immobilizations of two different antibodies onto the surfaces of two separate acoustic wave devices for the detection of several analogous analytes. The chemical specificity of the molecular recognition event is achieved by virtue of the extremely high (nM to pM) binding affinity between the antibody and its antigen. In a standard ELISA (Enzyme-Linked ImmunoSorbent Assay) test, there are multiple steps and the end result is a measure of what is bound so tightly that it does not wash away easily. The fact that this “gold standard” is very much not real time, masks the dance that is the molecular recognition event. X-Ray Crystallographer, Ian Wilson, demonstrated more than a decade ago that antibodies undergo conformational change during a binding event[1, 2]. Further, it is known in the arena of immunochemistry that some antibodies exhibit significant cross-reactivity and this is widely termed antibody promiscuity. A third piece of the puzzle that we will exploit in our system of acoustic wave biosensors is the notion of chemical orthogonality. These three biochemical constructs, the dance, antibody promiscuity and chemical orthogonality will be combined in this paper with the notions of in-phase (I) and quadrature (Q) signals from digital radio to manifest an approach to molecular recognition that allows a level of discrimination and analysis unobtainable without the aggregate. As an example we present experimental data on the detection of TNT, RDX, C4, ammonium nitrate and musk oil from a system of antibody-coated acoustic wave sensors.     

Synthesis and Characterization of Mesoporous Silica Functionalized with Calix[]arene Derivatives

This work reports a new method to covalently attach calix[4]arene derivatives onto MCM-41, using a diisocyanate as a linker. The modified mesoporous silicates were characterized by fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA) and elemental analysis. The FTIR spectra and TGA analysis verified that the calix[4]arene derivates are covalently attached to the mesoporous silica. The preservation of the MCM-41 channel system was checked by X-ray diffraction and nitrogen adsorption analysis.     

In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAF^V600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.     

Characterization of a Low Shrinkage Dental Composite Containing Bismethylene Spiroorthocarbonate Expanding Monomer

In this study, a novel dental composite based on the unsaturated bismethylene spiroorthocarbonate expanding monomer 3,9-dimethylene-1,3,5,7-tetraoxa-spiro[5,5]undecane (BMSOC) and bisphenol-S-bis(3-meth acrylate-2-hydroxypropyl)ether (BisS-GMA) was prepared. CQ (camphorquinone) of 1 wt % and DMAEMA (2-(dimethylamino)ethyl methacrylate) of 2 wt % were used in a photoinitiation system to initiate the copolymerization of the matrix resins. Distilled water contact angle measurements were performed for the wettability measurement. Degree of conversion, volumetric shrinkage, contraction stress and compressive strength were measured using Fourier Transformation Infrared-FTIR spectroscopy, the AccuVol and a universal testing machine, respectively. Within the limitations of this study, it can be concluded that the resin composites modified by bismethylene spiroorthocarbonate and BisS-GMA showed a low volumetric shrinkage at 1.25% and a higher contact angle. The lower contraction stress, higher degree of conversion and compressive strength of the novel dental composites were also observed.     

In Vivo Modification of Native Carrier Protein Domains

Insider information : Selective labeling of endogenous proteins within cells has been an elusive goal. Here carrier protein labeling has been optimized for visualization, isolation, and protein sequencing.

     

A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1 , based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe— P2 , based on a SNAP-tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.     

Preparation and characterization of bottle‐brush polymer via host−guest self‐assembly between β‐cyclodextrin and adamantane

Supramolecular assemblies with a bottle‐brush structure are obtained by inclusion complexation between β‐cyclodextrin and adamantane. β‐cyclodextrin‐modified chitosan is synthesized via the aldimine condensation reaction between β‐cyclodextrin monoaldehyde and chitosan as the host. The guest is prepared through the esterification reaction between methoxypoly(ethylene glycol) and 1‐adamantanecarboxylic acid chloride. The supramolecular assemblies are formed through the inclusion of adamantane‐modified methoxypoly(ethylene glycol) into the β‐cyclodextrin cavity on the chitosan chain. Fourier transform infrared and ¹H NMR spectra were used to prove that the host, guest and assemblies were successfully obtained. UV?visible spectra were employed to confirm the formation of assemblies. Furthermore, the size of the particles in the assembled solution, the change before and after self‐assembly, and the effect of the addition of competitive molecules were studied by dynamic light scattering measurements. The results indicate that supramolecular assemblies have formed successfully which might be used to realize the biomimetic structure of the articular cartilage proteoglycan. © 2014 Society of Chemical Industry     

Synthesis of Analogues of Gingerol and Shogaol,the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.     

Fabrication of Calix[]arene Derivative Monolayers to Control Orientation of Antibody Immobilization

Three calix[4]arene (Cal-4) derivatives which separately contain ethylester (1), carboxylic acid (2), and crownether (3) at the lower rim with a common reactive thiol at the upper rim were synthesized and constructed to self-assembled monolayers (SAMs) on Au films. After spectroscopic characterization of the monolayers, surface coverage and orientation of antibody immobilized on the Cal-4 derivative SAMs were studied by surface plasmon resonance (SPR) technique. Experimental results revealed that the antibody could be immobilized on the Cal-4 derivatives spontaneously. The orientation of absorbed antibody on the Cal-4 derivative SAMs is related to the SAM’s dipole moment. The possible orientations of the antibody immobilized on the Cal-4 derivative 1 SAM are lying-on or side-on, while on the Cal-4 derivative 2 and Cal-4 derivative 3 head-on and end-on respectively. These experimental results demonstrate the surface dipole moment of Cal-4 derivative appears to be an important factor to antibody orientation. Cal-4 derivatives are useful in developing site direct protein chips.     

An Artificial Supramolecular Nanozyme Based on β-Cyclodextrin-Modified Gold Nanoparticles

An artificial nanozyme model was developed by the supramolecular complexation of a β-cyclodextrin-modified gold nanoparticle and metal catalytic centers. The cyclodextrin-based monolayer was first constructed on the surface of gold nanoparticle by using the thiol modified cyclodextrin, subsequently the cyclodextrin-modified gold nanoparticle was utilized as a backbone to install metal catalytic centers by supramolecular assembly of the copper complex of triethylnetetramine-adamantane and β-cyclodextrin receptors immobilized on the surface of gold nanospheres via hydrophobic interaction. The catalytic behaviors of β-cyclodextrin-modified gold nanoparticles with adjacent multi-metal catalytic centers were investigated as an esterase mimic. Strong hydrolase activities for catalyzing the cleavage of an active ester 4,4′-dinitrodiphenyl carbonate (DNDPC) were observed. A detailed kinetic study on nanozyme-catalyzed hydrolysis of ester DNDPC has been described.     

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.     

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

A new enzymatic protein ligation tool , sortase, has recently emerged from Gram‐positive bacteria. This article outlines the technique, sortase‐mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self‐cleavable tag for protein expression, protein–PNA hybrids, neoglycoconjugates, and cell‐surface protein labeling, etc.

     

Visualizing biochemical activities in living cells through chemistry

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.     

Endo‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement

β‐Glucoside‐configured cyclophellitols are activity‐based probes (ABPs) that allow sensitive detection of β‐glucosidases. Their applicability to detect proteins fused with β‐glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M‐777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4‐methylumbelliferyl β‐d ‐lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre‐blocking with conduritol β‐epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β‐glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high‐resolution detection moieties) should assist further research in living cells and organisms.     

Studies of arginine-arene interactions through synthesis and evaluation of a series of galectin-binding aromatic lactose esters

Aromatic lactose 2-O-esters were synthesized and used to probe arene-arginine interactions with the galectin family of proteins. They were found to be low microM inhibitors of galectin-1, -3, and -9N-terminal domain and moderate inhibitors of galectin-7, but not inhibitors of galectin-8N-terminal, which lacks an arginine residue close to the critical, esterified lactose 2-O-position. Molecular modeling of galectins in complex with aromatic lactose 2-O-esters, as well as binding studies with a galectin-3 R186S mutant, confirmed that the inhibitory efficiency of the lactose 2-O-esters was due to the formation of strong interactions between the aromatic ester moieties and the arginine guanidinium groups of galectin-1 and -3. An important common feature shared by galectin-1 and -3 was that the arginines formed in-plane ion pairs with two side-chain carboxylates, which resulted in extended planar pi-electron surfaces that did not require solvation by water; these surfaces were ideal for stacking with aromatic moieties of the ligands. The results provide a basis for the design of lectin inhibitors and drugs that exploit interactions with arginine side-chains via aromatic moieties, which are involved in intramolecular protein salt bridges.