Helical Packing Regulates Structural Transitions In Bax

Apoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to transition from a cytosolic form to a membrane-bound form remain poorly understood. Elucidating this process is a necessary step in the development of BAX as a novel therapeutic target for the treatment of cancer, as well as autoimmune and neurodegenerative disorders. A three-part study, utilizing computational modeling and biological assays, was used to examine how BAX, and similar proteins, transition to membranes. The first part tested the hypothesis that the C-terminal α9 helix regulates the distribution and activity of BAX by functioning as a molecular switch to trigger conformational changes that enable the protein to redistribute from the cytosol to mitochondrial membrane. Computational analysis, tested in biological assays, revealed a new finding: that the α9 helix can dock into a hydrophobic groove of BAX in two opposite directions – in a self-associated, forward orientation and a previously, unknown reverse orientation that enables dimerization and apoptosis. Peptides, made to mimic the α9-helix, were able to induce the mitochondrial translocation of BAX, but not when key residues in the hydrophobic groove were mutated. Such findings indicate that the α9 helix of BAX can function as a molecular switch to mediate occupancy of the hydrophobic groove and regulate the membrane-binding activity of BAX. This new discovery contributes to the understanding of how BAX functions during apoptosis and can lead to the design of new therapeutic approaches based on manipulating the occupancy of the hydrophobic groove. The second and third parts of the study used computational modeling to examine how the helical stability of proteins relates to their ability to functionally transition. Analysis of BAX, as a prototypical transitioning protein, revealed that it has a broad variation in the distribution of its helical interaction energy. This observation led to the hypothesis tested, that proteins which undergo 3D structural transitions during execution of their function have broad variations in the distribution of their helical interaction energies. The result of this study, after examination of a large group of all-alpha proteins, was the development of a novel, predictive computational method, based on measuring helical interactions energies, which can be used to identify new proteins that undergo structural transitioning in the execution of their function. When this method was used to examine transitioning in other members the BCL-2 family, a strong agreement with the published experimental findings resulted. Further, it was revealed that the binding of a ligand, such as a small peptide, to a protein can have significant stabilizing or destabilizing influences that impact upon the activation and function of the protein. This computational analysis thus contributes to a better understanding of the function and regulation of the BCL-2 family members and also offers the means by which peptide mimics that modulate protein activity can be designed for testing in therapeutic endeavors

Activation energies define kinetic (in)stabilities of therapeutic antibodies

Antibody degradation pathways are many-fold and can result in loss of function, efficacy and even to adverse effects in patients. Among others, aggregation and fragmentation is still the major challenge. The identification of the primary degradation pathway can be very complex. Monoclonal antibodies (mAbs) are large, multi-domain macromolecules. Due to their complex nature and inherent properties, temperature induced unfolding leads to rather complex unfolding kinetics. In this study, we determined activation energies (Ea) of thermal intrinsic fluorescence (IF) unfolding profiles unique for each individual antibody. The analyzed activation energies give insights both into kinetic (in)stabilities of single domains and the overall structure of the antibody. To realize this, we used a novel developed experimental setup to perform temperature dependent fluorescence unfolding profiles of various mAbs. In conclusion, the activation energies can be used as descriptors for kinetic (in)stabilities of therapeutic antibodies. Moreover, we show that lower activation energies correlate to monomer loss in long-term storage stabilities and can thus likely be used for shelf-life prediction

Specific Delivery Of Therapeutic Rnas To Cancer Cells Via The Dimerization Mechanism Of Phi29 Motor Prna

The application of small RNA in therapy has been hindered by the lack of an efficient and safe delivery system to target specific cells. Packaging RNA (pRNA), part of the DNA-packaging motor of bacteriophage phi29(φ29), was manipulated by RNA nanotechnology to make chimeric RNAs that form dimers via interlocking right- and left-hand loops. Fusing pRNA with receptor-binding RNA aptamer, folate, small interfering RNA (siRNA), ribozyme, or another chemical group did not disturb dimer formation or interfere with the function of the inserted moieties. Incubation of cancer cells with the pRNA dimer, one subunit of which harbored the receptor-binding moiety and the other harboring the gene-silencing molecule, resulted in their binding and entry into the cells, and subsequent silencing of anti/proapoptotic genes. The chimeric pRNA complex was found to be processed into functional double-stranded siRNA by Dicer (RNA-specific endonuclease). Animal trials confirmed the suppression of tumorigenicity of cancer cells by ex vivo delivery. It has been reported [Shu, D., Moll, W.-D., Deng, Z., Mao, C., and Guo, P. (2004). Nano Lett. 4:1717-1724] that RNA can be used as a building block for bottom-up assembly in nanotechnology. The assembly of protein-free 25-nm RNA nanoparticles reported here will allow for repeated long-term administration and avoid the problems of short retention time of small molecules and the difficulties in the delivery of particles larger than 100 nm. © Mary Ann Liebert, Inc

The Interaction Of Lck And The Cd4 Co-Receptor Alters The Dose Response Of T-Cells To Interleukin-7

CD8 and CD4 T-cells grow optimally under different concentrations of the cytokine, interleukin-7 (IL-7). While CD8 T-cells expand at high doses of IL-7, CD4 T-cells favor low doses. To examine the reason for the preference of CD4 T-cells for lower doses of the cytokine, we used IL-7 dependent T-cells to study signal transduction upon a range of IL-7 concentrations. We found that the high dose responsiveness of CD8 T-cells to IL-7 could be altered if these cells also expressed CD4. Using the phosphorylation of STAT5 as an indicator of growth, we found that the co-receptor associated kinase, LCK, contributed to phospho-STAT5 levels. Phospho-STAT5 was elevated at high dose IL-7 for CD8 T-cells and at low dose IL-7 for CD4 T-cells, which was reversed upon LCK inhibition. Examining the direct association of LCK with CD4 using a T- cell line that over-expresses CD4, we determined that CD4 could directly sequester LCK. Non-CD4 T-cells were not restricted in this manner and levels of phospho-STAT5 increased proportionally to the IL-7 dose. Our studies, therefore, show that the response of a T-cell to IL-7 can be modulated by the availability of LCK. © 2010 Elsevier B.V

Ligand biased and probe-dependent modulation of the chemokine receptor CXCR3 signaling by negative allosteric modulators

Over the last decade functional selectivity (or bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands targeting G protein-coupled receptors (GPCRs). The chemokine receptor CXCR3 is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate the receptor signaling. At the same time CXCR3 is an attractive therapeutic target in autoimmune diseases and cancer. Here we report the discovery of a small molecule (1b) that can selectively inhibit CXCL11-dependent G protein activation over β-arrestin recruitment (with a 190-fold selectivity). The compound also demonstrates probe-dependent activity, i.e. it inhibits CXCL11- over CXCL10-mediated G protein activation with a 12-fold selectivity. Together with previously reported biased negative allosteric modulator from our group, the present study provides additional support to our hypothesis of multiple binding orientations for synthetic ligands of CXCR3

Active-State Model of a Dopamine D2 Receptor - Gai Complex Stabilized by Aripiprazole-Type Partial Agonists

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gai protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gai-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy

Development Of Flavonoid-Based Inverse Agonists Of The Key Signaling Receptor Us28 Of Human Cytomegalovirus

A series of 31 chalcone- and flavonoid-based derivatives were synthesized in good overall yields and screened for their inverse agonist activity on the US28 receptor of human cytomegalovirus (HCMV). With one exception (e.g., 2-(5-bromo-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-one), halogen-substituted flavonoids were typically more potent inverse agonists than their related hydro derivatives. While toxicity could be used to partially explain the inverse agonist activity of some members of the series, 5-(benzyloxy)-2-(5-bromo-2- methoxyphenyl)-4H-chromen-4-one (11b) acted on the US28 receptor as a nontoxic, inverse agonist. The full inverse agonism (efficacy, -89%) and potency (EC 50 = 3.5 μM) observed with flavonoid 11b is especially important as it provides both a new tool to study US28 signaling and a potential platform for the future development of HCMV-targeting drugs. © 2013 American Chemical Society