InSiDDe: A server for designing artificial disordered proteins

InSiDDe (In Silico Disorder Design) is a program for the in silico design of intrinsically disordered proteins of desired length and disorder probability. The latter is assessed using IUPred and spans values ranging from 0.55 to 0.95 with 0.05 increments. One to ten artificial sequences per query, each made of 50 to 200 residues, can be generated by InSiDDe. We describe the rationale used to set up InSiDDe and show that an artificial sequence of 100 residues with an IUPred score of 0.6 designed by InSiDDe could be recombinantly expressed in E. coli at high levels without degradation when fused to a natural molecular recognition element (MoRE). In addition, the artificial fusion protein exhibited the expected behavior in terms of binding modulation of the specific partner recognized by the MoRE. To the best of our knowledge, InSiDDe is the first publicly available software for the design of intrinsically disordered protein (IDP) sequences. InSiDDE is publicly available online

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results

One-step generation of error-prone PCR libraries using Gateway® technology

<p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway^®technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated <it>E. coli </it>cell transformations and plasmid purifications.</p> <p>Results</p> <p>We describe a method for making epPCR libraries in Gateway^®plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.</p> <p>Conclusions</p> <p>The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.</p

GFP-Fragment Reassembly Screens for the Functional Characterization of Variants of Uncertain Significance in Protein Interaction Domains of the BRCA1 and BRCA2 Genes

Genetic testing for BRCA1 and BRCA2 genes has led to the identification of many unique variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is conditioned by the amount of necessary data, which are difficult to obtain if a variant is rare. As an alternative, variants mapping to the coding regions can be examined using in vitro functional assays. BRCA1 and BRCA2 proteins promote genome protection by interacting with different proteins. In this study, we assessed the functional effect of two sets of variants in BRCA genes by exploiting the green fluorescent protein (GFP)-reassembly in vitro assay, which was set-up to test the BRCA1/BARD1, BRCA1/UbcH5a, and BRCA2/DSS1 interactions. Based on the findings observed for the validation panels of previously classified variants, BRCA1/UbcH5a and BRCA2/DSS1 binding assays showed 100% sensitivity and specificity in identifying pathogenic and non-pathogenic variants. While the actual efficiency of these assays in assessing the clinical significance of BRCA VUS has to be verified using larger validation panels, our results suggest that the GFP-reassembly assay is a robust method to identify variants affecting normal protein functioning and contributes to the classification of VUS

An Antibiotic Discovery Campaign Targeting VirF, the Main Transcriptional Regulator of Virulence in Shigella flexneri

Shigella flexneri is a gram-negative enteropathogen that infects the human colonic epithelium. It is estimated that Shigella spp. infect 165 million people a year worldwide. Symptoms of shigellosis include bloody dysentery, dehydration, and ultimately death if the infection is not treated properly. The current recommended first-line treatment for shigellosis is ciprofloxacin; however, many new multi-drug resistant strains of Shigella have begun to emerge. The emergence of these strains, along with the lack of novel antibiotics in the drug pipeline, makes the need for new effective treatments for shigellosis a priority. Genetic knockout studies have shown that VirF, the main transcriptional activator of the Shigella pathogenesis cascade, is necessary for virulence, but not bacterial viability. We hypothesized that a novel anti-virulence therapy for shigellosis could be developed through the inhibition of VirF. To identify inhibitors of VirF, we performed a high-throughput screening (HTS) campaign testing over 140,000 small molecules and 20,000 natural product extracts using a Shigella-based, VirF-driven, β-galactosidase reporter assay. Following a series of confirmation screens, we identified five compounds from the HTS campaign that had VirF inhibitory properties. Using tissue culture-based models of the S. flexneri infection process, we were able to show that three of compounds were able to attenuate the virulence of S. flexneri, thereby, validating VirF as a target for the treatment of shigellosis. To further characterize the hits from the HTS campaign a series of established in vitro assays were adapted and optimized for VirF. An electrophoretic mobility shift assay and a fluorescence polarization assay were used to monitor VirF binding to the virB promoter, and a fluorescence intercalator displacement assay was used to determine if the hits could directly bind DNA. Using these assays, we were able to determine the mechanism of inhibition (blockade of VirF binding to the virB promoter) and preliminary structure-activity relationship trends for one of the hits, and report the first dissociation constant for VirF binding to the virB promoter (2.8 ± 1.0 μM).PhDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120884/1/aae_1.pd

Characterization of an Italian founder mutation in the RING-finger domain of BRCA1

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area

Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained

Applying Functional Assay Evidence to Interpret Sequence Variants Identified in Hereditary Cancer Genes

The demand for the interpretation of sequence variants identified by next-generation sequencing is gradually increasing in clinical laboratories. The American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) 2015 guidelines provide a basis for using functional assays as strong evidence for variant classification. However, it is challenging to use the evidence because the protein’s function and the functional assays used to prove it are too diverse. Therefore, this study reviewed various functional assays that can aid in classifying sequence variants in clinical laboratories. This review focuses on the 1) general functional assays associated with basic protein functions and processing and 2) functional assays related to the specific pathogenic mechanisms of four genes (TP53, BRCA1, CDH1, and PTEN) associated with hereditary cancer.ope

Programmable DNA looping using engineered bivalent dCas9 complexes

DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops. Cleavage-defective Cas9 proteins of different specificity are linked by heterodimerization or translational fusion to create bivalent complexes able to link two separate DNA regions. After model-directed optimization, the reagents are validated using a quantitative DNA looping assay in E. coli. Looping efficiency is ~15% for a 4.7 kb loop, but is significantly improved by loop multiplexing with additional guides. Bivalent dCas9 complexes are also used to activate endogenous norVW genes by rewiring chromosomal DNA to bring distal enhancer elements to the gene promoters. Such reagents should allow manipulation of DNA looping in a variety of cell types, aiding understanding of endogenous loops and enabling creation of new regulatory connections.Nan Hao, Keith E. Shearwin, Ian B. Dod