The c-Jun N-terminal kinases (JNK) are important regulators of cell growth, proliferation, and apoptosis. JNKs are typically activated by a sequence of events that include phosphorylation of its T-P-Y motif by an upstream kinase, followed by homodimerization and translocation to the nucleus. Constitutive activation of JNK has been found in a variety of cancers including non-small cell lung carcinomas, gliomas, and mantle cell lymphoma. In vitro studies show that constitutive activation of JNK induces a transformed phenotype in fibroblasts and enhances tumorigenicity in a variety of cell lines. Interestingly, a subset of JNK isoforms was recently found to autoactivate rendering the proteins constitutively active. These constitutively active JNK proteins were found to play a pivotal role in activating transcription factors that increase cellular growth and tumor formation in mice. In this chapter, we describe techniques and methods that have been successfully used to study the three components of JNK activation. Use of these techniques may lead to a better understanding of the components of JNK pathways and how JNK is activated in cancer cells.
Ligand-dependent regulation of adenylyl cyclase by the large family of seven-transmembrane G protein-coupled receptors (GPCRs) represents a deeply conserved and widely deployed cellular signaling mechanism. Studies of adenylyl cyclase regulation by catecholamine receptors have led to a remarkably detailed understanding of the basic biochemistry of G protein-linked signal transduction and have elaborated numerous mechanisms of regulation. Endocytosis of GPCRs plays a significant role in controlling longer-term cellular responses, such as under conditions of prolonged or repeated receptor activation occurring over a course of hours or more. It has been more challenging to investigate regulatory effects occurring over shorter time intervals, within the minutes to tens of minutes spanning the time course of many acute cAMP-mediated signaling processes. A main reason for this is that biochemical methods used traditionally to assay changes in cytoplasmic cyclic AMP (cAMP) concentration are limited in spatiotemporal resolution, and typically require perturbing cellular structure and / or function for implementation. Recent developments in engineering genetically encoded cAMP biosensors linked to optical readouts, which can be expressed in cells or tissues and detected without cellular disruption or major functional perturbation, represent a significant step toward overcoming these limitations. Here we describe the application of two such cAMP biosensors, one based on enzyme complementation and luminescence detection and another using Förster resonance energy transfer and fluorescence detection. We focus on applying these approaches to investigate cAMP signaling by catecholamine receptors, and then on combining these analytical approaches with manipulations of receptor endocytic trafficking.
Endosome; cyclic AMP; clathrin; dynamin; fluorescence microscopy; luminescence imaging
Optical melting experiments provide measurements of thermodynamic parameters for nucleic acids. These thermodynamic parameters are widely used in RNA structure prediction programs and DNA primer design software. This review briefly summarizes the theory and underlying assumptions of the method and provides practical details for instrument calibration, experimental design, and data interpretation.
With the recent progress in identifying disease-causing genes in humans and in animal models, there are more and more opportunities for using retinal gene transfer to learn more about retinal physiology and also to develop therapies for blinding disorders. Success in preclinical studies for one form of inherited blindness have led to testing in human clinical trials. This paves the way to consider a number of other retinal diseases as ultimate gene therapy targets in human studies. The information presented here is designed to assist scientists and clinicians to use gene transfer to probe the biology of the retina and/or to move appropriate gene-based treatment studies from the bench to the clinic.
The switch-like regulation of protein activity by molecular signals is abundant in native proteins. The ability to engineer proteins with novel regulation has applications in bio-sensors, selective protein therapeutics, and basic research. One approach to building proteins with novel switch properties is creating combinatorial libraries of gene fusions between genes encoding proteins that have the prerequisite input and output functions of the desired switch. These libraries are then subjected to selections and/or screens to identify those rare gene fusions that encode functional switches. Combinatorial libraries in which an insert gene is inserted randomly into an acceptor gene have been useful for creating switches, particularly when combined with circular permutation of the insert gene. Methods for creating random domain insertion libraries are described. Three methods for creating a diverse set of insertion sites in the acceptor gene are presented and compared: DNase I digestion, S1 nuclease digestion, and multiplex inverse PCR. A PCR-based method for creating a library of circular permutations of the insert gene is also presented.
The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.
DNA damage repair is essential for the maintenance of genetic integrity in all organisms. Unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death or malignant transformation. DNA damage in the form of double-strand breaks (DSBs) can occur as a result of both exogenous insults, such as ionizing radiation and drug therapies, and normal metabolic processes including V(D)J recombination. Mammalian cells have multiple pathways for repairing DSBs, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). This chapter describes the use of reporter substrates for assaying the contributions of these pathways to DSB repair in mammalian cells, in particular murine embryonic stem cells. The individual contributions of NHEJ, HR, and SSA can be quantified using fluoreScence and PCR-based assays after the precise introduction of DSBs either by the I-SceI endonuclease or by the RAG recombinase. These reporters can be used to assess the effects of genetic background, dominant-negative constructs or physiological conditions on DSB repair in a wide variety of mammalian cells
Mitochondrial reactive oxygen species (ROS) are widely implicated in physiological and pathological pathways. We propose that it is critical to understand the specific sites of mitochondrial ROS production and their mechanisms of action. Mitochondria possess at least eight distinct sites of ROS production in the electron transport chain and matrix compartment. In this chapter, we describe the nature of the mitochondrial ROS-producing machinery and the relative capacities of each site. We provide detailed methods for the measurement of H2O2 release and the conditions under which maximal rates from each site can be achieved in intact skeletal muscle mitochondria.
Mesenchymal stem cells (MSCs) have become one of the most studied stem cells, especially toward the healing of diseased and damaged tissues and organs. MSCs can be readily isolated from a number of adult tissues by means of minimally invasive approaches. MSCs are capable of self-replication to many passages and, therefore, can potentially be expanded to sufficient numbers for tissue and organ regeneration. MSCs are able to differentiate into multiple cell lineages that resemble osteoblasts, chondrocytes, myoblasts, adipocytes, and fibroblasts and express some of the key markers typical of endothelial cells, neuron-like cells, and cardiomyocytes. MSCs have been used alone for cell delivery or seeded in biomaterial scaffolds toward the healing of tissue and organ defects. After an increasing number of the “proof of concept” studies, the remaining tasks are many, such as to determine MSC interactions with host cells and signaling molecules, to investigate the interplay between MSCs and biological scaffold materials, and to apply MSC-based therapies toward clinically relevant defect models. The ultimate goal of MSC-based therapies has valid biological rationale in that clusters of MSCs differentiate to form virtually all connective tissue during development. MSC-based therapies can only be realized our improved understanding of not only their fundamental properties such as population doubling and differentiation pathways but also translational studies that use MSCs in the de novo formation and/or regeneration of diseased or damaged tissues and organs.
Urinary exosome-like vesicles (ELVs), 20–200nm membrane bound particles shed by renal epithelium, are emerging as an important source of protein, mRNA, and miRNA biomarkers to monitor renal disease. However, purification of ELVs is compromised by the presence of large amounts of the urinary protein Tamm-Horsfall Protein (THP). THP molecules oligomerize into long, double-helical strands several microns long. These linear assemblies form a 3-dimensional gel which traps and sequesters ELVs in any centrifugation based protocol. Here we present a purification protocol that separates ELVs from THP and divides urinary ELVs into three distinct populations.
Plasma membrane expression of G protein-coupled receptors (GPCRs) is a dynamic process balancing anterograde and retrograde trafficking. Multiple interrelated cellular processes determine the final level of cell surface expression, including endoplasmic reticulum (ER) export/retention, receptor internalization, recycling, and degradation. These processes are highly regulated to achieve specific localization to subcellular domains (e.g., dendrites or basolateral membranes) and to affect receptor signaling. Analysis of potential ER trafficking motifs within GPCRs requires careful consideration of intracellular dynamics, such as protein folding, ER export and retention, and glycosylation. This chapter presents an approach and methods for qualitative and quantitative assessment of these processes to aid in accurate identification of GPCR trafficking motifs, utilizing the analysis of a hydrophobic extracellular trafficking motif in α2C adrenergic receptors as a model system.
Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and design of novel protein-synthesized materials. To date, limited atomic-resolution data have hindered experimental structure determination for proteins on mineral surfaces. Molecular simulation represents a complementary approach. In this chapter, we review RosettaSurface, a computational structure prediction-based algorithm designed to broadly sample conformational space to identify low-energy structures. We summarize the computational approaches, the published applications, and the new releases of the code in the Rosetta 3 framework. In addition, we provide a protocol capture to demonstrate the practical steps to employ RosettaSurface. As an example, we provide input files and output data analysis for a previously unstudied mineralization protein, osteocalcin. Finally, we summarize ongoing challenges in energy function optimization and conformational searching and suggest that the fusion between experiment and calculation is the best route forward.
Lipolysis is defined as the hydrolytic cleavage of ester bonds in triglycerides (TGs), resulting in the generation of fatty acids (FAs) and glycerol. The two major TG pools in the body of vertebrates comprise intracellular TGs and plasma/nutritional TGs. Accordingly, this leads to the discrimination between intracellular and intravascular/gastrointestinal lipolysis, respectively. This chapter focuses exclusively on intracellular lipolysis, referred to as lipolysis herein. The lipolytic cleavage of TGs occurs in essentially all cells and tissues of the body. In all of them, the resulting FAs are utilized endogenously for energy production or biosynthetic pathways with one exception, white adipose tissue (WAT). WAT releases FAs and glycerol to supply nonadipose tissues at times of nutrient deprivation. The fundamental role of lipolysis in lipid and energy homeostasis requires the accurate measurement of lipase activities and lipolytic rates. The recent discovery of new enzymes and regulators that mediate the hydrolysis of TG has made these measurements more complex. Here, we describe detailed methodology for how to measure lipolysis and specific enzymes’ activities in cells, organs, and their respective extracts.
Standard protein expression systems, such as E. coli, often fail to produce folded, mono-disperse, or functional eukaryotic proteins (see Small-scale Expression of Proteins in E. coli). The expression of these proteins is greatly benefited by using a eukaryotic system, such as mammalian cells, that contains the appropriate folding and posttranslational machinery. Here, we describe methods for both small- and large-scale transient expression in mammalian cells using polyethylenimine (PEI). We find this procedure to be more cost-effective and quicker than the more traditional route of generating stable cell lines. First, optimal transfection conditions are determined on a small-scale, using adherent cells. These conditions are then translated for use in large-scale suspension cultures. For further details on generating stable cell lines please (see Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® Recombination Cloning Technology and Flp-In T-REx® lines or Generating mammalian stable cell lines by electroporation).
Here, we provide a detailed account of how to denervate white and brown adipose tissue (WAT and BAT) and how to measure sympathetic nervous system (SNS) activity to these and other tissues neurochemically. The brain controls many of the functions of WAT and BAT via the SNS innervation of the tissues, especially lipolysis and thermogenesis, respectively. There is no clearly demonstrated parasympathetic innervation of WAT or the major interscapular BAT (IBAT) depot. WAT and BAT communicate with the brain neurally via sensory nerves. We detail the surgical denervation (eliminating both innervations) of several WAT pads and IBAT. We also detail more selective chemical denervation of the SNS innervation via intra-WAT/IBAT 6-hydroxy-dopamine (a catecholaminergic neurotoxin) injections and selective chemical sensory denervation via intra-WAT/IBAT capsaicin (a sensory nerve neurotoxin) injections. Verifications of the denervations are provided (HPLC-EC detection for SNS, ELIA for calcitonin gene-related peptide (proven sensory nerve marker)). Finally, assessment of the SNS drive to WAT/BAT or other tissues is described using the alpha-methyl-para-tyrosine method combined with HPLC-EC, a direct neurochemical measure of SNS activity. These methods have proven useful for us and for other investigators interested in innervation of adipose tissues. The chemical denervation approach has been extended to nonadipose tissues as well.
Actinomycetes, a group of filamentous, Gram-positive bacteria, have long been a remarkable source of useful therapeutics. Recent genome sequencing and transcriptomic studies have shown that these bacteria, responsible for half of the clinically used antibiotics, also harbor a large reservoir of gene clusters, which have the potential to produce novel secreted small molecules. Yet, many of these clusters are not expressed under common culture conditions. One reason why these clusters have not been linked to a secreted small molecule lies in the way that actinomycetes have typically been studied: as pure cultures in nutrient-rich media that do not mimic the complex environments in which these bacteria evolved. New methods based on multispecies culture conditions provide an alternative approach to investigating the products of these gene clusters. We have recently implemented binary interspecies interaction assays to mine for new secondary metabolites and to study the underlying biology in inter-actinomycete interactions. Here we describe the detailed biological and chemical methods comprising these studies.
secondary metabolites; interspecies interactions; amychelin; Streptomyces coelicolor; Amycolatopsis sp. AA4
The growth and survival of cancer cells is often driven by constitutive activity in the mitogen activated protein kinase (MAPK) and phospho-inositide 3-kinase (PI3K)/AKT signaling pathways. Activity in these signal transduction cascades is known to contribute to the uncontrolled growth and resistance to apoptosis that characterizes tumor progression. There is now a great deal of interest in therapeutically targeting these pathways in cancer using small molecule inhibitors. In this chapter we describe methods to measure constitutive MAPK and AKT activity in melanoma cell lines, with a focus upon Western blotting, phospho-flow cytometry and immunofluorescence staining techniques.
melanoma; MAPK; ERK; AKT; PLX4032; PLX4720; therapy
The primary purpose of this protocol is to prepare genomic DNA libraries that can then be analyzed by massively parallel next-generation sequencing on the Applied Bio-systems SOLiD platform. This protocol can be adapted to next-generation sequencing workflows to ultimately generate up to 1 billion 50 bp sequence tags from the ends of each of the DNA molecules in the library in a single next-generation sequencing run.
Recognition sequences for the site-specific DNA recombinases Cre and FLP are commonly incorporated into gene targeting vectors for the purposes of removing selection markers or generating conditional alleles. Gene targeting vectors typically contain a positive selection marker, such as the neomycin resistance gene, flanked by loxP sites. Thus, the selection marker can be removed by breeding to a mouse strain which expresses Cre recombinase in its germ line. Conditional knockout vectors typically have one or more exons flanked by loxP sites and the positive selection marker flanked by FRT sites. Thus, the selection marker is removed with FLP recombinase and the knockout allele is generated in tissues expressing Cre recombinase. Because the generation of mice by gene targeting in embryonic stem (ES) cells is an expensive and time-consuming process, it is important to confirm that the recombination sites in your targeting vector are functional prior to electroporation of ES cells. This chapter describes a simple method for testing the functionality of loxP and FRT sites in vivo using Cre- or FLP-expressing bacteria.
Technological breakthroughs in sequencing technologies have driven the advancement of molecular biology and molecular genetics research. The advent of high-throughput Sanger sequencing (for information on the method, see Sanger Dideoxy Sequencing of DNA) in the mid- to late-1990s made possible the accelerated completion of the human genome project, which has since revolutionized the pace of discovery in biomedical research. Similarly, the advent of next generation sequencing is poised to revolutionize biomedical research and usher a new era of individualized, rational medicine.
The term next generation sequencing refers to technologies that have enabled the massively parallel analysis of DNA sequence facilitated through the convergence of advancements in molecular biology, nucleic acid chemistry and biochemistry, computational biology, and electrical and mechanical engineering. The current next generation sequencing technologies are capable of sequencing tens to hundreds of millions of DNA templates simultaneously and generate >4 gigabases of sequence in a single day. These technologies have largely started to replace high-throughput Sanger sequencing for large-scale genomic projects, and have created significant enthusiasm for the advent of a new era of individualized medicine.
Theoretical analyses of targeting agent pharmacokinetics provides specific guidance with respect to desirable design objectives such as agent size, affinity, and target antigen. These analyses suggest that IgG-sized macromolecular constructs exhibit the most favorable balance between systemic clearance and vascular extravasation, resulting in maximal tumor uptake. Quantitative predictions of the effects of dose and binding affinity on tumor uptake and penetration are also provided. The single bolus dose required for saturation of xenografted tumors in mice can be predicted from knowledge of antigen expression level and metabolic half-life. The role of high binding affinity in tumor uptake can be summarized as: essential for small peptides, less important for antibodies, and negligible for nanoparticles.
Over the past decade, an increasing number of studies have shown that G-protein-coupled receptors including opioid and cannabinoid receptors associate to form heteromers. Moreover, G-protein-coupled receptor heteromerization leads to the modulation of the binding, signaling, and trafficking properties of individual receptors. Although very little information is available about the physiological role of receptor heteromers, some studies have shown that the levels of some heteromers are upregulated in disease states such as preeclamptic pregnancy, schizophrenia, Parkinson’s, ethanol-induced liver fibrosis, and development of tolerance to morphine. The recent generation of antibodies that selectively recognize distinct heteromers and, of peptides that selectively disrupt them, have started to elucidate the contribution of heteromers to the disease state. Here, we describe the methods for the generation of heteromer-selective antibodies and elucidation of their levels and localization under normal and pathological conditions.
Structural biology of GPCRs has made significant progress upon recently developed technologies for GPCRs expression/purification and elucidation of GPCRs crystal structures. The crystal structures provide a snapshot of the receptor structural disposition of GPCRs itself or with cocrystallized ligands, and the results are congruent with biophysical and computer modeling studies reported about GPCRs conformational and dynamics flexibility, regulated activation, and the various stabilizing interactions, such as “molecular switches.” The molecular switches generally constitute the most conserved domains within a particular GPCR superfamily. Often agonist-induced receptor activation proceeds by the disruption of majority of these interactions, while antagonist and inverse agonist act as blockers and structural stabilizers, respectively. Several elegant studies, particularly for the β2AR, have demonstrated the relationship between ligand structure, receptor conformational changes, and corresponding pharmacological outcomes. Thus, it is of great importance to understand GPCRs activation related to cell signaling pathways. Herein, we summarize the steps to produce functional GPCRs, generate suitably fluorescent labeled GPCRs and the procedure to use that to understand if ligand-induced activation can proceed by activation of the GPCRs via ionic lock switch and/or rotamer toggle switch mechanisms. Such understanding of ligand structure and mechanism of receptor activation will provide great insight toward uncovering newer pathways of GPCR activation and aid in structure-based drug design.