Ca 2+ /Calmodulin Directly Interacts with the Pleckstrin Homology Domain of AKT1

AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca(2+).calmodulin (Ca(2+).CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903-38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca(2+).CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca(2+)-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969-5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca(2+).CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca(2+).CaM. In addition, Ca(2+).CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca(2+).CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane

In vitro selection of proteins with desired characteristics using mRNA-display

mRNA-display is an amplification-based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two-hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA-display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with very high diversity. The purpose of this article is to provide a summary of the field and practical framework of mRNA-display-based selections. We summarize the advantages and limitations of selections using mRNA-display as well as the recent applications, namely, the identification of novel affinity reagents, target-binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. Practically, we provide a detailed procedure for performing mRNA-display-based selections with the aim of identifying protease substrates and binding partners of a target protein. Furthermore, we describe how to confirm the function of the selected protein sequences by biochemical assays and bioinformatic tools

Método de elementos finitos en dos dimensiones para estudio de propagación en potenciales electrostáticos

Este artículo presenta la motivación y algunas consideraciones del método de elementos finitos con su aplicación para el caso bidimensional. Una vez se plantea, se describe cómo se desarrolla el aplicativo de manera computacional. Los resultados obtenidos son comparados con un caso típico de propagación de potencial electrostático mostrado en el artículo método iterativo para el cálculo de potenciales electrostáticos [1]

Highly-Efficient Purification of Native Polyhistidine-Tagged Proteins by Multivalent NTA-Modified Magnetic Nanoparticles

A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His×6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His×6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His×6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His×6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His×6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His×6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads

Morphing Low-Affinity Ligands into High-Avidity Nanoparticles by Thermally Triggered Self-Assembly of a Genetically Encoded Polymer

Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in non-target tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy - Dynamic Affinity Modulation (DAM) - using Elastin-like polypeptide diblock copolymers (ELPBCs) that self-assemble from a low-affinity to high-avidity state by a tunable thermal “switch”, thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELPBCs on their receptor-mediated binding and cellular uptake. The data presented herein show that: (1) ligand presentation does not disrupt ELPBC self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micron-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM, and provide rational design parameters for future applications of this technology for targeted drug delivery

Tribody: Robust Self-Assembled Trimeric Targeting Ligands with High Stability and Significantly Improved Target-Binding Strength

The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribody), against epidermal growth factor receptor (EGFR) and prostate-membrane specific antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target binding strength compared with the corresponding monomers. Cellular binding studies confirmed that the trimeric targeting ligands have superior binding strength towards their respective receptors. Significantly, EGFR-binding tribody was considerably accumulated in tumor in xenograft mice bearing EGFR positive tumors, indicating its effective cancer targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribody can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications

The S-Star Cluster at the Center of the Milky Way: On the nature of diffuse NIR emission in the inner tenth of a parsec

Sagittarius A*, the super-massive black hole at the center of the Milky Way, is surrounded by a small cluster of high velocity stars, known as the S-stars. We aim to constrain the amount and nature of stellar and dark mass associated with the cluster in the immediate vicinity of Sagittarius A*. We use near-infrared imaging to determine the $K_\mathrm{s}$-band luminosity function of the S-star cluster members, and the distribution of the diffuse background emission and the stellar number density counts around the central black hole. This allows us to determine the stellar light and mass contribution expected from the faint members of the cluster. We then use post-Newtonian N-body techniques to investigate the effect of stellar perturbations on the motion of S2, as a means of detecting the number and masses of the perturbers. We find that the stellar mass derived from the $K_\mathrm{s}$-band luminosity extrapolation is much smaller than the amount of mass that might be present considering the uncertainties in the orbital motion of the star S2. Also the amount of light from the fainter S-cluster members is below the amount of residual light at the position of the S-star cluster after removing the bright cluster members. If the distribution of stars and stellar remnants is strongly enough peaked near Sagittarius A*, observed changes in the orbital elements of S2 can be used to constrain both their masses and numbers. Based on simulations of the cluster of high velocity stars we find that at a wavelength of 2.2 $\mu$m close to the confusion level for 8 m class telescopes blend stars will occur (preferentially near the position of Sagittarius A*) that last for typically 3 years before they dissolve due to proper motions.Comment: 14 pages, 11 figures, minor changes to match the published version in Astronomy & Astrophysic

LA HIDROGENACION SELECTIVA DE ACEITES NATURALES A TRAVES DE CATALIZADORES HETEROGENEOS

En este artículo se presenta una revisión bibliográfica sobre los procesos y los catalizadores heterogéneos que permiten hidrogenar selectivamente el grupo carbonilo de sustratos insaturados presentes en aceites vegetales tales como ácidos y ésteres grasos con el fin de sintetizar productos de la química fina. Se discuten además algunos resultados importantes relacionados con los mecanismos que se han propuesto recientemente para la reacción de hidrogenación selectiva, así como algunas pautas para trabajos futuros

Activation of Ventral Tegmental Area 5-HT2C Receptors Reduces Incentive Motivation

FUNDING AND DISCLOSURE The research was funded by Wellcome Trust (WT098012) to LKH; and National Institute of Health (DK056731) and the Marilyn H. Vincent Foundation to MGM. The University of Michigan Transgenic Core facility is partially supported by the NIH-funded University of Michigan Center for Gastrointestinal Research (DK034933). The remaining authors declare no conflict of interest. ACKNOWLEDGMENTS We thank Dr Celine Cansell, Ms Raffaella Chianese and the staff of the Medical Research Facility for technical assistance. We thank Dr Vladimir Orduña for the scientific advice and technical assistance.Peer reviewedPublisher PD