BAPJ69-4A: A yeast two-hybrid strain for both positive and negative genetic selection

Genetic selection systems, such as the yeast two-hybrid system, are efficient methods to detect protein-protein and protein-ligand interactions. These systems have been further developed to assess negative interactions, such as inhibition, using the URA3 genetic selection marker. Previously, chemical complementation was used to assess positive selection in Saccharomyces cerevisiae. In this work, a new S. cerevisiae strain, called BAPJ69-4A, containing three selective markers ADE2, HIS3, and URA3 as well as the lacZ gene controlled by Gal4 response elements, was developed and characterized using the retinoid X receptor (RXR) and its ligand 9-cis retinoic acid (9cRA). Further characterization was performed using RXR variants and the synthetic ligand LG335. To assess the functionality of the strain, RXR was compared to the parent strain PJ69-4A in adenine, histidine, and uracil selective media. In positive selection, associating partners that lead to cell growth were observed in all media in the presence of ligand, whereas partners that did not associate due to the absence of ligand displayed no growth. Conversely, in negative selection, partners that did not associate in 5-FOA medium did not display cell death due to the lack of expression of the URA3 gene. The creation of the BAPJ69-4A yeast strain provides a high-throughput selection system, called negative chemical complementation, which can be used for both positive and negative selection, providing a fast, powerful tool for discovering novel ligand receptor pairs for applications in drug discovery and protein engineering.     

Nerve growth factor receptor TrkA exists as a preformed, yet inactive, dimer in living cells

The tropomyosin-related kinase A (TrkA) receptor and its ligand, nerve growth factor (NGF), play crucial roles in the development and function of the nervous system. NGF is believed to activate TrkA by bridging two TrkA monomers, leading to TrkA transphosphorylation. However, here we show that the majority of TrkA receptors exist as preformed, yet inactive, homodimers prior to NGF binding by using three different approaches such as chemical crosslinking and enzyme fragment complementation assay. Furthermore, TrkA homodimers are formed in endoplasmic reticulum before newly synthesized receptors reach the cell surface. These findings shed light on molecular mechanisms underlying transmembrane signaling by TrkA.

Structured summary

TrkAphysically interacts with TrkA by protein complementation assay (View interaction)TrkAphysically interacts with TrkA by bimolecular fluorescence complementation (View interaction)TrkAphysically interacts with TrkA by cross-linking study (View interaction)     

Essential roles of bile acids and their nuclear receptors,FXR and PXR,in the cholestatic liver disease

Bile acids (BAs) are steroid acids found predominantly in the bile of mammals and other vertebrates. Though BAs have been known as digestive juice, recent studies have revealed that BAs act as signaling molecules to control metabolism and inflammation. Today, BAs are considered as potential therapeutic molecules for treatment of complex metabolic liver disease. However, the detergent properties of BAs lead to hepatic injury and intrahepatic cholestasis when BAs are accumulated in the liver with impaired bile flow into gall bladder. Cholestasis is a pathological condition of hepatic retention of cytotoxic bile acids. To date, hydrophilic ursodeoxycholic acid has been currently used to treat cholestasis, but the efficacy of UDCA for cholestasis is still limited. Given that BAs are endogenous ligands of several nuclear receptors, including Farnesoid X receptor and Pregnane X receptor, novel synthetic ligands for those nuclear receptors are promising for the treatment of cholestatic liver diseases.     

CAR and PXR: the xenobiotic-sensing receptors

The xenobiotic receptors CAR and PXR constitute two important members of the NR1I nuclear receptor family. They function as sensors of toxic byproducts derived from endogenous metabolism and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. In contrast, the steroid receptors, exemplified by the estrogen receptor (ER) and glucocorticoid receptor (GR), are the sensors that tightly monitor and respond to changes in circulating steroid hormone levels to maintain body homeostasis. This divergence of the chemical- and steroid-sensing functions has evolved to ensure the fidelity of the steroid hormone endocrine regulation while allowing development of metabolic elimination pathways for xenobiotics. The development of the xenobiotic receptors CAR and PXR also reflect the increasing complexity of metabolism in higher organisms, which necessitate novel mechanisms for handling and eliminating metabolic by-products and foreign compounds from the body. The purpose of this review is to discuss similarities and differences between the xenobiotic receptors CAR and PXR with the prototypical steroid hormone receptors ER and GR. Interesting differences in structure explain in part the divergence in function and activation mechanisms of CAR/PXR from ER/GR. In addition, the physiological roles of CAR and PXR will be reviewed, with discussion of interactions of CAR and PXR with endocrine signaling pathways.     

Channels as taste receptors in vertebrates

Taste reception is fundamental for proper selection of food and beverages. Chemicals detected as taste stimuli by vertebrates include a large variety of substances, ranging from inorganic ions (e.g., Na⁺, H⁺) to more complex molecules (e.g., sucrose, amino acids, alkaloids). Specialized epithelial cells, called taste receptor cells (TRCs), express specific membrane proteins that function as receptors for taste stimuli. Classical view of the early events in chemical detection was based on the assumption that taste substances bind to membrane receptors in TRCs without permeating the tissue. Although this model is still valid for some chemicals, such as sucrose, it does not hold for small ions, such as Na⁺, that actually diffuse inside the taste tissue through ion channels. Electrophysiological, pharmacological, biochemical, and molecular biological studies have provided evidence that indeed TRCs use ion channels to reveal the presence of certain substances in foodstuff. In this review, we focus on the functional and molecular properties of ion channels that serve as receptors in taste transduction.     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.     

Powered by gas--a ligand for a fruit fly nuclear receptor

The difficulty in identifying ligands for nuclear hormone receptors remains an obstacle to understanding their function. For example, in the fruit fly Drosophila melanogaster, only one of its nuclear receptors has a known ligand. In this issue of Cell, report that the fruit fly E75 nuclear receptor contains heme in its ligand binding pocket and that the oxidation state of this molecule controls E75 activity. They also show that E75-heme responds to the small diatomic gases, nitric oxide and carbon monoxide. This study sheds light on how heme, gas signaling, and nuclear receptors interact to control metabolic and developmental pathways.     

Gamma Aminobutyric Acid (GABA) and Plant Responses to Stress

4-aminobutyrate (GABA) is a non-protein amino acid that is widely distributed throughout the biological world. In animals, GABA functions as the predominant inhibitory neurotransmitter in the central nervous system by acting through the GABA receptors. The neuromuscular system enables animals to escape from environmental stresses. Being nonmotile, plants have evolved chemical responses to mitigate stress. Mechanisms by which GABA may facilitate these responses are discussed in this review. Environmental stresses increase GABA accumulation through two different mechanisms. Stresses causing metabolic and/or mechanical disruptions, resulting in cytosolic acidification, induce an acidic pH-dependent activation of glutamate decarboxylase and GABA synthesis. Extremely marked declines in cytosolic pH occur under oxygen deprivation, which is the primary stress factor in flooded soils, and this stress induces the greatest accumulation of GABA. Other stresses, including cold, heat, salt, and mild or transient environmental factors, such as touch, wind, rain, etc. rapidly increase cellular levels of Ca²⁺. Increased cytosolic Ca²⁺ stimulates calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. A review of the kinetics of GABA accumulation in plants reveals a stress-specific pattern of accumulation that is consistent with a physiological role for GABA in stress mitigation. Recent physiological and genetic evidence indicates that plants may possess GAB A-like receptors that have features in common with the animal receptors. The mechanism of action of animal GABA receptors suggests a model for rapid amplification of ion-mediated signals and GABA accumulation in response to stress. Metabolic pathways that link GABA to stress-related metabolism and plant hormones are identified. The survival value of stress-related metabolism is dependent on metabolic changes occurring before stress causes irreversible damage to plant tissue. Rapid accumulation of GABA in stressed tissue may provide a critical link in the chain of events leading from perception of environmental stresses to timely physiological responses.     

Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria

Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields – evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in energy, pharmaceuticals, chemical commodities, biomining, and bioremediation.     

Differential regulation and properties of angiopoietin-like proteins 3 and 4

Angiopoietin-like protein 3 and 4 (Angptl3 and Angptl4) are two members of the angiopoietin-like family of proteins. These two closely related proteins have been reported to similarly affect lipid metabolism through their capacity to inhibit lipoprotein lipase. We undertook a series of studies to compare the structure, function, and regulation of Angptl3 and Angptl4. Previously, we reported that Angptl4 exists as variable-sized oligomers that contain intermolecular disulfide bonds. We now have evidence that although there are no intermolecular disulfide bonds evident in Angptl3, higher molecular weight forms do exist. In addition, Angptl4 exhibits a widespread distribution of tissue expression, while Angptl3 is exclusively expressed in the liver. Treatments with various ligands of nuclear receptors reveal that Angptl3 is a target gene of liver X receptor, while Angptl4 expression is activated by ligands of all peroxisome proliferator-activated receptors. Expression of Angptl4 in adipose tissue and liver is induced by fasting, while Angptl3 expression is not appreciably affected by nutritional status. We suggest that the differential regulation of Angptl3 and Angptl4 by sites of expression, nutritional status, and ligands of nuclear receptors may confer unique roles of each in lipoprotein metabolism.