An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol

Signaling of Human Frizzled Receptors to the Mating Pathway in Yeast

Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Gα protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors

A guide to efficient keyword, sequence and classification search strategies for biopharmaceutical drug-centric patent landscape searches - A human recombinant insulin patent landscape case study

Searching biopharmaceutical drug-related patent information is generally considered to be challenging. In particular, setting up efficient search strategies for comprehensive retrieval of high amounts of patent documents related to processes and methods of use, that achieve a reasonable level of precision, but still remain within a particular search scope. While it is generally accepted that patent information cannot be searched using standardized approaches, it is desirable to have a basic rule set for successful biopharmaceutical drug-related patent information retrieval, particularly facing a steady flow of patent expirations for prominent biologic drugs. The present human recombinant insulin case study shows an assessment of keyword, sequence and classification search strategies for establishing biopharmaceutical drug-centric patent landscapes. The search results of both crude and sophisticated keyword search strategies, as well as of a sequence search strategy, were compared in terms of the key information retrieval quality indicators; the recall and the precision. Through analyses of the relevant retrieved documents, a quality assessment of keyword choice is provided, as well as determining focused IPC and Derwent Manual classification codes and terminology from original patent and Derwent documentation abstract titles. All of which can be used for setting up more efficient search strategies and facilitated document categorization.Biopharmaceutical drug Biosimilar Patent landscape search case study Search strategy optimization Keyword searching Sequence searching BLAST Classification searching IPC Derwent manual classification codes Recall and precision analysis

The Tyrosine Kinase Receptor RET Interacts in Vivo with Aryl Hydrocarbon Receptor-Interacting Protein to Alter Survivin Availability

Context: RET is a tyrosine kinase transmembrane receptor expressed in two main alternative isoforms: RET9 and RET51. RET transduces a positive signal leading to survival, differentiation, or migration in the presence of its ligand glial cell line-derived neurotrophic factor, whereas in its absence a proapoptotic fragment that initiates a negative signaling for apoptosis is generated. The signal transduction mechanisms leading to apoptosis are still unclear. Objective: To shed light on the mechanisms of RET-induced apoptosis, we searched for novel interactors of RET51. Design: The "split ubiquitin yeast two-hybrid system" was used with RET51 as bait against a human brain expression library. Results: We identified aryl hydrocarbon receptor-interacting protein (AIP), a cochaperone recently found mutated in pituitary adenoma patients, as a novel interactor of RET. We showed that RET interacts specifically with AIP both in mammalian cell lines and in vivo in the pituitary gland, regardless of the presence of pituitary adenoma-specific mutations. AIP and RET genes were sequenced in 28 pituitary adenoma, but no relevant mutations were found. In addition, we identified the proapoptotic domain of RET as responsible for the interaction with AIP. Finally, we demonstrated that the AIP-RET interaction does not require RET kinase activity or kinase-dependent signal transduction and that it prevents the formation of the AIP-survivin complex. Conclusions: The identification of the AIP-RET complex represents a starting point to study key cellular processes involved in RET-induced apoptosis