Agonist Regulation of Gene Expression of Adrenergic Receptors and G Proteins

Abstract: Study of transmembrane signaling via G proteins has focused to a large extent upon investigations of individual G protein-linked receptor-effector systems. Agonist-induced desensitization and down-regulation of β-adrenergic receptors, for example, have been studied extensively and adopted as a general model for G protein-linked receptor regulation. This review focuses not only on agonist regulation of adrenergic receptor gene expression, but also on how agonists regulate opposing adrenergic receptor-mediated pathways. This important feature of G protein-mediated pathways, i.e., cross-regulation and integration of information among several pathways, will be discussed in the context of what has been learned in the adrenergic receptor-coupled pathways.     

Trypsin In Lecithin Based w/o Microemulsions. Fluorescence and Enzyme Activity Studies

Lecithin based microemulsions were used as model systems for enzymic studies. The phase behavior of the system: purified soya bean lecithin/propan-1-ol/isooctane/water was examined. It was found that the ability of the system to solubilize water was strongly affected by the lecithin and alcohol concentrations. Trypsin was entrapped in lecithin microemulsion systems of different composition and tested for proteolytic activity on the hydrolysis of lysine-p-nitroanilide (LNA). The kinetic constants were determined and in most cases the ratio k_cat/Km was higher than that observed in aqueous solution. The optimum enzyme activity was found at pH 9 for the system formulated with 5% w/w lecithin in isooctane, while increasing w_o, where w_o = [H₂o]/[Lecithin], the enzyme activity followed a bell-shaped pattern with a maximum at w_o= 20. The stability of trypsin in microemulsions was higher in the low water containing systems. Using the fluorescence quenching technique it was found that the system compartmentalization depended on the water content and the presence of the enzyme. Time-resolved luminescence decay studies were carried out to clarify the effect of the water content and the presence of the enzyme molecules on the micro-emulsion structure. The analysis of the luminescence data was done with a “percolation” model of stretched exponential. A dramatic variation of the water/oil interface occurred above the percolation threshold, while the addition of the enzyme induced a more restricted microenvironment.     

Conformational analysis of bioactive peptides in reverse micelles as mimics of cell membrane environments

Summary Conformational preferences of secretin as a model peptide have been analyzed by CD and IR spectroscopy in reverse micelles of AOT/isooctane/water and compared to those in aqueous TFE, in SDS micelles and in DMPG vesicles. Among the systems examined, reverse micelles and phospholipid vesicles displayed almost identical conformational equilibria. Very high lipid-to-peptide ratios can be obtained in reverse micelles with full retention of optical transparency, even at millimolar peptide concentrations, thus indicating this system to be an interesting mimic of cell membrane environments for spectroscopic analysis of bioactive peptide conformations.Abbreviations TFE trifluoroethanol - SDS sodium dodecyl sulfate - DMPG dimyristoylphosphatidylglycerol - AOT bis(2-ethylhexyl)sulfosuccinate - CMC critical micellar concentration - VIP vasoactive intestinal peptide     

Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1 through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically precluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-modified compound, revealing a promising opportunity to target novel regions of the ligand binding pocket to alter LRH-1 target gene expression.     

Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth

EphA2 receptor kinase could become a novel target for anti-glioblastoma treatment. Doxazosin previously identified acts like the endogenous ligand of EphA2 and induces cell apoptosis. Through lead structure modification a derivative of Doxazosin possessing unique dimeric structure showed an improvement in the activity. In the current study, we expanded the dimeric scaffold by lead optimization to explore the chemical space of the conjoining moieties and a slight variation to the core structure. 27 new derivatives were synthesized and examined with EphA2 overexpressed and wild type glioblastoma cell lines for cell proliferation and EphA2 activation. Three new compounds 3d, 3e, and 7bg showed potent and selective activities against the growth of EphA2 overexpressed glioblastoma cells. Dimer 3d modification replaces the long alkyl chain with a short polyethylene glycol chain. Dimer 7bg has a relatively longer polyethylene glycol chain in comparison to compound 3d and the length is more similar to the lead compound. Whereas dimer 3e has a rigid aromatic linker exploring the chemical space. The diversity of the linkers in the active suggest additional hydrogen binding sites has a positive correlation to the activity. All three dimers showed selective activity in EphA2 overexpressed cells, indicating the activity is correlated to the EphA2 targeting effect.     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus, microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

Understanding cytokine and growth factor receptor activation mechanisms

Our understanding of the detailed mechanism of action of cytokine and growth factor receptors – and particularly our quantitative understanding of the link between structure, mechanism and function – lags significantly behind our knowledge of comparable functional protein classes such as enzymes, G protein-coupled receptors, and ion channels. In particular, it remains controversial whether such receptors are activated by a mechanism of ligand-induced oligomerization, versus a mechanism in which the ligand binds to a pre-associated receptor dimer or oligomer that becomes activated through subsequent conformational rearrangement. A major limitation to progress has been the relative paucity of methods for performing quantitative mechanistic experiments on unmodified receptors expressed at endogenous levels on live cells. In this article, we review the current state of knowledge on the activation mechanisms of cytokine and growth factor receptors, critically evaluate the evidence for and against the different proposed mechanisms, and highlight other key questions that remain unanswered. New approaches and techniques have led to rapid recent progress in this area, and the field is poised for major advances in the coming years which promise to revolutionize our understanding of this large and biologically and medically important class of receptors.