A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID.

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

Cullin-3/KCTD10 complex is essential for K27-polyubiquitination of EIF3D in human hepatocellular carcinoma HepG2 cells.

Eukaryotic translation initiation factor 3 subunit D (EIF3D) binds to the 5'-cap of specific mRNAs, initiating their translation into polypeptides. From a pathological standpoint, EIF3D has been observed to be essential for cell growth in various cancer types, and cancer patients with high EIF3D mRNA levels exhibit poor prognosis, indicating involvement of EIF3D in oncogenesis. In this study, we found, by mass spectrometry, that Cullin-3 (CUL3)/KCTD10 ubiquitin (Ub) ligase forms a complex with EIF3D. We also demonstrated that EIF3D is K27-polyubiquitinated at the lysine 153 and 275 residues in a KCTD10-dependent manner in human hepatocellular carcinoma HepG2 cells. Similar to other cancers, high expression of EIF3D significantly correlated with poor prognosis in hepatocellular carcinoma patients, and depletion of EIF3D drastically suppressed HepG2 cell proliferation. These results indicate that EIF3D is a novel substrate of CUL3/KCTD10 Ub ligase and suggest involvement of K27-polyubiquitinated EIF3D in the development of hepatocellular carcinoma.

DANFIN functions as an inhibitor of transcription factor NF-κB and potentiates the antitumor effect of bortezomib in multiple myeloma.

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N'-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.

A proximity biotinylation-based approach to identify protein-E3 ligase interactions induced by PROTACs and molecular glues.

Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identify biotinylated peptides of well-known neo-substrates by mass spectrometry with high specificity and selectivity. Additional analyses reveal ZMYM2 and ZMYM2-FGFR1 fusion protein-responsible for the 8p11 syndrome involved in acute myeloid leukaemia-as CRBN neo-substrates. Furthermore, AirID-DCAF15 and AirID-CRBN biotinylate neo-substrates targeted by indisulam and PROTACs, respectively, suggesting that this approach has the potential to serve as a general strategy for characterizing drug-inducible protein-protein interactions in cells.

AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.

Proteins in a cell need to interact with each other to perform the many tasks required for organisms to thrive. A technique called proximity biotinylation helps scientists to pinpoint the identity of the proteins that partner together. It relies on attaching an enzyme (either BioID or TurboID) to a protein of interest; when a partner protein comes in close contact with this construct, the enzyme can attach a chemical tag called biotin to it. The tagged proteins can then be identified, revealing which molecules interact with the protein of interest. Although BioID and TurboID are useful tools, they have some limitations. Experiments using BioID take more than 16 hours to complete and require high levels of biotin to be added to the cells. TurboID is more active than BioID and is able to label proteins within ten minutes. However, under certain conditions, it is also more likely to be toxic for the cell, or to make mistakes and tag proteins that do not interact with the protein of interest. To address these issues, Kido et al. developed AirID, a new enzyme for proximity biotinylation. Experiments were then conducted to test how well AirID would perform, using proteins of interest whose partners were already known. These confirm that AirID was able to label partner proteins in human cells; compared with TurboID, it was also less likely to mistakenly tag non-partners or to kill the cells, even over long periods. The results by Kido et al. demonstrate that AirID is suitable for proximity biotinylation experiments in cells. Unlike BioID and TurboID, the enzyme may also have the potential to be used for long-lasting experiments in living organisms, since it is less toxic for cells over time.

A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors.

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins.

Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein.

Cell-Free Synthesis of Plant Receptor Kinases.

The wheat germ cell-free protein synthesis system has been used as a eukaryotic protein production system since it was first reported in 1964. Although initially the productivity of this system was not very high, it has now become one of the most versatile protein production systems, thanks to the enhancements made by several groups. In this chapter, we report a protein production method for plant receptor kinases using the wheat cell-free system. We describe a method for the preparation of a cell-free extract from wheat germ, the split-primer PCR method for preparation of transcription templates, and the bilayer cell-free protein synthesis method.

Autophosphorylation Assays Using Plant Receptor Kinases Synthesized in Cell-Free Systems.

The wheat germ cell-free protein synthesis system has a significant advantage for high-throughput production of eukaryotic multidomain proteins in a folded state. In this chapter, we describe two kinds of methods for performing autophosphorylation assay of plant receptor kinases (PRKs) by using the wheat cell-free system. One is an in vitro kinase assay performed using biotin-streptavidin affinity purification technology, and the other is a luminescence-based high-throughput assay for autophosphorylation analysis. We anticipate that our cell-free-based methods might facilitate the characterization of plant PRKs.

CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag.

There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.