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Past Issue in 2017
Volume: 7, Issue: 3
Biochemistry
PRODIGY: A Contact-based Predictor of Binding Affinity in Protein-protein Complexes
Biomolecular interactions between proteins regulate and control almost every biological process in the cell. Understanding these interactions is therefore a crucial step in the investigation of biological systems and in drug design. Many efforts have been devoted to unravel principles of protein-protein interactions. Recently, we introduced a simple but robust descriptor of binding affinity based only on structural properties of a protein-protein complex. In Vangone and Bonvin (2015), we demonstrated that the number of interfacial contacts at the interface of a protein-protein complex correlates with the experimental binding affinity. Our findings have led one of the best performing predictor so far reported (Pearson’s Correlation r = 0.73; RMSE = 1.89 kcal mol-1). Despite the importance of the topic, there is surprisingly only a limited number of online tools for fast and easy prediction of binding affinity. For this reason, we implemented our predictor into the user-friendly PRODIGY web-server. In this protocol, we explain the use of the PRODIGY web-server to predict the affinity of a protein-protein complex from its three-dimensional structure. The PRODIGY server is freely available at: http://milou.science.uu.nl/services/PRODIGY.
Cancer Biology
Polysome Fractionation to Analyze mRNA Distribution Profiles
Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.
In vitro Assessment of RNA Polymerase I Activity
In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the smallest species of ribosomal RNA (5S rRNA). This protocol describes an in vitro assay to evaluate the rRNA transcriptional activity of RNA polymerase I. The method measures the quantity of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I, in optimal conditions for the enzyme activity and in the presence of a toxin, α-amanitin, which inhibits RNA polymerase II and III without affecting RNA polymerase I (Novello and Stirpe, 1970).
Microbiology
Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract
This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of both pattern recognition receptor systems (Cirl et al., 2008; Waldhuber et al., 2016). We infected wild type and knock-out mice with wildtype CFT073 and a mutant CFT073 strain lacking tcpC. This protocol describes how the mice were infected, how CFT073 was prepared and how the infection was monitored. The protocol was derived from our previously published work and allowed us to demonstrate that TcpC is a powerful virulence factor by increasing the bacterial burden of CFT073 in the urine and kidneys. Moreover, TcpC was responsible for the development of kidney abscesses since infection of mice with wildtype but not tcpC-deficient CFT073 mutants caused this complication.
Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies
The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure (Geertsma et al., 2015). The procedure may be adapted to purify and crystallize other membrane protein complexes.
Enriching Acidophilic Fe(II)-oxidizing Bacteria in No-flow, Fed-batch Systems
Low-pH microbial Fe(II) oxidation occurs naturally in some Fe(II)-rich acid mine drainage (AMD) ecosystems across so-called terraced iron formations. Indigenous acidophilic Fe(II)-oxidizing bacterial communities can be incorporated into both passive and active treatments to remove Fe from the AMD solution. Here, we present a protocol of enriching acidophilic Fe(II)-oxidizing bacteria in no-flow, fed-batch systems. Mixed cultures of naturally occurring microbes are enriched from the fresh surface sediments at AMD sites using a chemo-static bioreactor (Eppendorf BioFlo®/Celligen® 115 Fermentor) with respect to constant stirring speed, temperature, pH and unlimited dissolved oxygen. Ferrous sulfate is discontinuously added to the reactor as the primary substrate to enrich for acidophilic Fe(II)-oxidizing bacteria. Successfully and efficiently enriching acidophilic Fe(II)-oxidizing bacteria helps to exploit this biogeochemical process into AMD treatment systems.
Determination of the in vitro Sporulation Frequency of Clostridium difficile
The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This protocol describes a method to accurately enumerate both viable vegetative cells and spores separately and subsequently calculate a sporulation frequency of a mixed C. difficile population from various in vitro growth conditions (Edwards et al., 2016b).
Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers
Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical information to understand this unique mechanism involved in their infectious process. In this protocol, we describe a method to measure the force generated by Mycoplasma mobile, the fastest gliding species in Mycoplasma. This protocol should be useful for the studies of many kinds of gliding microorganisms.
Molecular Biology
Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells
A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).
Neuroscience
Cued Rat Gambling Task
The ability of salient cues to serve as powerful motivators has long been recognized in models of drug addiction, but little has been done to investigate their effects on complex decision making. The Cued rat Gambling Task (CrGT) is an operant behavioural task which pairs salient, audiovisual cues with the delivery of sucrose pellet rewards on complex schedules of reinforcement that involve both sugar pellet ‘wins’ and timeout penalty ‘losses’. The task was designed with the intention of providing insight into the influence of such cues on decision making in a manner that models human gambling.
Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.
Plant Science
Miniature External Sapflow Gauges and the Heat Ratio Method for Quantifying Plant Water Loss
External sapflow sensors are a useful tool in plant ecology and physiology for monitoring water movement within small stems or other small plant organs. These gauges make use of heat as a tracer of water movement through the stem and can be applied in both a laboratory and a field setting to generate data of relatively high temporal resolution. Typical outputs of these data include monitoring plant water use on a diurnal time scale or over a season (e.g., in response to increasing water deficit during drought) to gain insight into plant physiological strategies. This protocol describes how to construct the gauges, how best to install them and some expected data outputs.
Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies
GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about 350-450 resides and can be subdivided into five distinct motifs with their name derived from the most prominent amino acids: LRI (leucine-rich region I), VHIID, LRII (leucine-rich region II), PFYRE and SAW and mainly function in the interaction between GRAS proteins and their partners (Sun et al., 2012).By phylogenetic analysis, the GRAS family can be divided into more than ten subfamilies, of which SCL4/7 is one important subgroup and functions in response to environmental stresses. Here we describe a detailed protocol for the expression and purification of the GRAS domain of Os-SCL7, a SCL4/7 member in rice, which enables us to crystallize it and determine its structure.
Stem Cell
A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue
Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.
Systems Biology
Bioinformatic Analysis for Profiling Drug-induced Chromatin Modification Landscapes in Mouse Brain Using ChlP-seq Data
Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful technology to profile genome-wide chromatin modification patterns and is increasingly being used to study the molecular mechanisms of brain diseases such as drug addiction. This protocol discusses the typical procedures involved in ChIP-seq data generation, bioinformatic analysis, and interpretation of results, using a chronic cocaine treatment study as a template. We describe an experimental design that induces significant chromatin modifications in mouse brain, and the use of ChIP-seq to derive novel information about the chromatin regulatory mechanisms involved. We describe the bioinformatic methods used to preprocess the sequencing data, generate global enrichment profiles for specific histone modifications, identify enriched genomic loci, find differential modification sites, and perform functional analyses. These ChIP-seq analyses provide many details into the chromatin changes that are induced in brain by chronic exposure to cocaine, and generates an invaluable source of information to understand the molecular mechanisms underlying drug addiction. Our protocol provides a standardized procedure for data analysis and can serve as a starting point for any other ChIP-seq projects.