KRAS G12D and G12V Mediated by CANDDY Technology Independent of Ubiquitination.

"Undruggable" targets such as KRAS are particularly challenging in the development of drugs. We devised a novel chemical knockdown strategy, CANDDY (Chemical knockdown with Affinity aNd Degradation DYnamics) technology, which promotes protein degradation using small molecules (CANDDY molecules) that are conjugated to a degradation tag (CANDDY tag) modified from proteasome inhibitors. We demonstrated that CANDDY tags allowed for direct proteasomal target degradation independent of ubiquitination.

Development of a novel conditional knockdown mouse based on YB-1 protein degradation.

To clarify the pathogenic mechanism of disease and establish effective therapies, animal disease models that can be dynamically analyzed are urgently required. Knockout mouse models and conditional genetically engineered mouse models were developed to analyze genes and proteins involved in disease. However, these methods have drawbacks, including embryonic lethality, side effects and low efficiency.

Cell-Free Technologies for Proteomics and Protein Engineering.

Cell-free translation systems facilitate rapid production of specific proteins and are particularly suited as high-throughput methods for whole-genome protein synthesis. Moreover, these systems do not rely on living cells, thereby allowing the synthesis of unstable or cytotoxic proteins in vitro. In this review, we describe the principles and potential applications of cell-free protein translation systems and the future prospects of proteomics approaches using next-generation sequencing and cell-free expression technologies.

Catalytic subunits of the phosphatase calcineurin interact with NF-κB-inducing kinase (NIK) and attenuate NIK-dependent gene expression.

Nuclear factor (NF)-κB-inducing kinase (NIK) is a serine/threonine kinase that activates NF-κB pathways, thereby regulating a wide variety of immune systems. Aberrant NIK activation causes tumor malignancy, suggesting a requirement for precise regulation of NIK activity. To explore novel interacting proteins of NIK, we performed in vitro virus screening and identified the catalytic subunit Aα isoform of serine/threonine phosphatase calcineurin (CnAα) as a novel NIK-interacting protein.

Next-generation technologies for multiomics approaches including interactome sequencing.

The development of high-speed analytical techniques such as next-generation sequencing and microarrays allows high-throughput analysis of biological information at a low cost. These techniques contribute to medical and bioscience advancements and provide new avenues for scientific research. Here, we outline a variety of new innovative techniques and discuss their use in omics research (e.

Next-generation sequencing coupled with a cell-free display technology for reliable interactome of translational factors.

Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem.

Analysis of transcription factor networks using IVV method.

We have developed a simple and totally in vitro selection procedure based on cell-free cotranslation using a highly stable and efficient in vitro virus (IVV). Cell-free cotranslation of tagged bait and prey proteins is advantageous for the formation of protein complexes and allows high-throughput analysis of protein-protein interactions (PPI) as a result of providing in vitro instead of in vivo preparation of bait proteins. The use of plural selection rounds and a two-step purification of the IVV selection, followed by in vitro post-selection, is advantageous for decreasing false positives.

Mitochondria-nucleus shuttling FK506-binding protein 51 interacts with TRAF proteins and facilitates the RIG-I-like receptor-mediated expression of type I IFN.

Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS.

Towards personalized medicine mediated by in vitro virus-based interactome approaches.

We have developed a simple in vitro virus (IVV) selection system based on cell-free co-translation, using a highly stable and efficient mRNA display method. The IVV system is applicable to the high-throughput and comprehensive analysis of proteins and protein-ligand interactions. Huge amounts of genomic sequence data have been generated over the last decade.

Efficiency of puromycin-based technologies mediated by release factors and a ribosome recycling factor.

Two puromycin-based techniques, in vitro virus (IVV) and C-terminal labelling of proteins, were developed based on the observation that puromycin binds the C-terminus of a protein. Puromycin technology is a useful tool for the detection of proteins and analysis of protein-protein interactions (PPIs); however, problems arise due to the existence of stop codons in the native mRNAs. Release factors (RFs) that enter the A-site of the ribosome at stop codons compete with puromycin.

Next-generation sequencing coupled with a cell-free display technology for high-throughput production of reliable interactome data.

Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem.

PRD: A protein-RNA interaction database.

Unlabelled: Although protein-RNA interactions (PRIs) are involved in various important cellular processes, compiled data on PRIs are still limited. This contrasts with protein-protein interactions, which have been intensively recorded in public databases and subjected to network level analysis. Here, we introduce PRD, an online database of PRIs, dispersed across several sources, including scientific literature.

IRView: a database and viewer for protein interacting regions.

Unlabelled: Protein-protein interactions (PPIs) are mediated through specific regions on proteins. Some proteins have two or more protein interacting regions (IRs) and some IRs are competitively used for interactions with different proteins. IRView currently contains data for 3417 IRs in human and mouse proteins.

Protein complex prediction via verifying and reconstructing the topology of domain-domain interactions.

Background: High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions.

A comprehensive resource of interacting protein regions for refining human transcription factor networks.

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.

mRNA display selection of a high-affinity, Bcl-X(L)-specific binding peptide.

Bcl-X(L), an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-X(L) from random and degenerate peptide libraries using mRNA display.

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments.

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor alpha ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity.

The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line.

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network.

Rapid antibody selection by mRNA display on a microfluidic chip.

In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10(6)- to 10(8)-fold per round.

Six classes of nuclear localization signals specific to different binding grooves of importin alpha.

The importin alpha/beta pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin alpha.

Toward functional analysis of protein interactome using "in vitro virus": in silico analyses of Fos/Jun interactors.

Our high-throughput in vitro virus (IVV) method for selection of protein-protein interactions (PPI) and complexes, based on a simple cell-free co-translation and selection followed by computational sequence data analysis, was previously used to identify 31 Fos and Jun interactors. Here, in silico analyses of biological function, localization and phenotype of these AP-1 (Fos/Jun) interactors were performed. The results suggest that Fos and Jun do not necessarily work together, but also interact separately with novel interactors, including products of disease-related genes.

In vitro evolution of single-chain antibodies using mRNA display.

Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations.

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy.

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS).

Affinity selection of DNA-binding protein complexes using mRNA display.

Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection.