Human Olfactory Receptor Sensor for Odor Reconstitution.

Among the five human senses, light, sound, and force perceived by the eye, ear, and skin, respectively are physical phenomena, and therefore can be easily measured and expressed as objective, univocal, and simple digital data with physical quantity. However, as taste and odor molecules perceived by the tongue and nose are chemical phenomena, it has been difficult to express them as objective and univocal digital data, since no reference chemicals can be defined. Therefore, while the recording, saving, transmitting to remote locations, and replaying of human visual, auditory, and tactile information as digital data in digital devices have been realized (this series of data flow is defined as DX (digital transformation) in this review), the DX of human taste and odor information is not yet in the realization stage. Particularly, since there are at least 400,000 types of odor molecules and an infinite number of complex odors that are mixtures of these molecules, it has been considered extremely difficult to realize "human olfactory DX" by converting all odors perceived by human olfaction into digital data. In this review, we discuss the current status and future prospects of the development of "human olfactory DX", which we believe can be realized by utilizing odor sensors that employ the olfactory receptors (ORs) that support human olfaction as sensing molecules (i.e., human OR sensor).

Effects of 3-octen-2-one on human olfactory receptor responses to vanilla flavor.

Most of the odors that humans perceive daily are complex odors. It is believed that the modulation, enhancement, and suppression of overall complex odors are caused by interactions between odor molecules. In this study, to understand the interaction between odor molecules at the level of human olfactory receptor responses, the effects of 3-octen-2-one, which has been shown to modulate vanilla flavors, were analyzed using a human olfactory receptor sensor that uses all human olfactory receptors (388 types) as sensing molecules. As a result, the response intensity of 1 common receptor (OR1D2) was synergistically enhanced in vanilla flavor with 3-octen-2-one compared with vanilla flavor, and the response of 1 receptor (OR5K1) to vanilla flavor was completely suppressed. These results strongly suggested that the response of human olfactory receptors to complex odors is enhanced or suppressed by relatively few other odor molecules.

Combining machine learning and nanopore construction creates an artificial intelligence nanopore for coronavirus detection.

High-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.

Digital Pathology Platform for Respiratory Tract Infection Diagnosis via Multiplex Single-Particle Detections.

The variability of bioparticles remains a key barrier to realizing the competent potential of nanoscale detection into a digital diagnosis of an extraneous object that causes an infectious disease. Here, we report label-free virus identification based on machine-learning classification. Single virus particles were detected using nanopores, and resistive-pulse waveforms were analyzed multilaterally using artificial intelligence. In the discrimination, over 99% accuracy for five different virus species was demonstrated. This advance is accessed through the classification of virus-derived ionic current signal patterns reflecting their intrinsic physical properties in a high-dimensional feature space. Moreover, consideration of viral similarity based on the accuracies indicates the contributing factors in the recognitions. The present findings offer the prospect of a novel surveillance system applicable to detection of multiple viruses including new strains.

Construction of a Macrophage-Targeting Bio-nanocapsule-Based Nanocarrier.

The construction protocol of bio-nanocapsule (BNC)-based nanocarriers, named GL-BNC and GL-virosome, for targeted drug delivery to macrophages is described here. First, genes encoding the Streptococcus sp. protein G-derived C2 domain (binds to IgG Fc) and Finegoldia magna protein L-derived B1 domain (binds to Igκ light chain) are prepared by PCR amplification. Subsequently, the genes encoding hepatic cell-specific binding domain of hepatitis B virus envelope L protein are replaced by these PCR products. The expression plasmid for this fused gene (encoding GL-fused L protein) can be used to transform Saccharomyces cerevisiae AH22R- cells. To obtain GL-BNC, the transformed yeast cells are disrupted with glass beads, treated with heat, and then subjected to IgG affinity column chromatography followed by size exclusion column chromatography. In addition, GL-BNCs can be fused with liposomes to form GL-virosome. The targeted delivery of GL-BNC and GL-virosome to macrophages can be confirmed by in vitro phagocytosis assays using the murine macrophage cell line RAW264.7.

High-throughput single nanoparticle detection using a feed-through channel-integrated nanopore.

The outstanding sensitivity of solid-state nanopore sensors comes at a price of low detection efficiency due to the lack of active means to transfer objects into the nanoscale sensing zone. Here we report on a key technology for high-throughput single-nanoparticle detection which exploits mutual effects of microfluidics control and multipore electrophoresis in nanopore-in-channel units integrated on a thin Si3N4 membrane. Using this novel nanostructure, we demonstrated a proof-of-concept for influenza viruses via hydropressure regulation of mass transport in the fluidic channel for continuous feeding of biosamples into the effective electric field extending out from the nanopores, wherein the feed-through mechanism allowed us to selectively detect charged objects in physiological media such as human saliva. With the versatility of nanopore sensing technologies applicable to analytes of virtually any size from cells to polynucleotides, the present integration strategy may open new avenues for practical ultrasensitive bioanalytical tools.

A hepatitis B virus-derived human hepatic cell-specific heparin-binding peptide: identification and application to a drug delivery system.

Viruses are naturally evolved nanocarriers that can evade host immune systems, attach specifically to the surfaces of target cells, enter the cells through endocytosis, escape from endosomes efficiently, and then transfer their genomes to host cells. Hepatitis B virus (HBV) is a ∼42 nm enveloped DNA virus that can specifically infect human hepatic cells. To utilize the HBV-derived early infection machinery in synthetic nanocarriers, the human hepatic cell-binding site (i.e., the sodium taurocholate co-transporting polypeptide (NTCP)-binding site, with myristoylated pre-S1(2-47)) and the low pH-dependent fusogenic domain (pre-S1(9-24)) are indispensable for targeting and endosomal escape, respectively. However, cell-surface NTCP has recently been shown not to be involved in the initial attachment of HBV. In this study, we identified a novel heparin-binding site (pre-S1(30-42)) in the N-terminal half of the pre-S1 region, which presumably interacts with cell-surface heparan sulfate proteoglycan (HSPG) and plays a pivotal role in the initial attachment of HBV to human hepatic cells. The evolutionarily conserved amino acid residues Asp-31, Trp-32, and Asp-33 are indispensable for the heparin-binding activity. Liposomes (LPs) displaying the peptide were endocytosed by human hepatic cells in a cell-surface heparin-dependent manner and delivered doxorubicin to human hepatic cells more efficiently than myristoylated pre-S1(2-47)-displaying LPs. These results demonstrated that the pre-S1(30-42) peptide is the most promising HBV-derived targeting peptide for synthetic nanocarriers, and that this peptide exhibits high specificity for human hepatic cells and efficiently induces endocytosis.

Automated Single-Cell Analysis and Isolation System: A Paradigm Shift in Cell Screening Methods for Bio-medicines.

We have developed an automated robot that facilitates non-invasive isolation of a single cell with the most favorable properties from arrays containing >105 cells, thus allowing the establishment of new cell screening methods for bio-medicines. In this chapter, an outline of the proposed automated single-cell analysis and isolation system (hereafter called 'single-cell robot') is reviewed by comparison with a conventional fluorescence-activated cell sorter (FACS). The single-cell robot could perform high-throughput screening for both mammalian cells secreting the highest amount of bio-medicines (e.g. Chinese hamster ovary (CHO) cells or hybridomas), and stem cells with the highest pluripotency (e.g., embryonic stem (ES) cells), from huge number of cell libraries based on the recently proposed concept of "single cell-based breeding". The rational screening method for the de novo agonist design could also be performed using yeast cells expressing functional mammalian cytokine receptors (e.g., epidermal growth factor receptor (EGFR), somatostatin G protein-coupled receptor (SSTR5), and interleukin 5 receptor (IL5R)). Furthermore, the single-cell robot could comprehensively analyze the reaction between olfactory sensory neurons and specific odorants, which will shed light on how odorants are recognized by olfactory receptors. Taken together, these unique features of the proposed single-cell robot will contribute to the high-throughput development of forthcoming bio-medicines.

Development of a macrophage-targeting and phagocytosis-inducing bio-nanocapsule-based nanocarrier for drug delivery.

Macrophage hyperfunction or dysfunction is tightly associated with various diseases, such as osteoporosis, inflammatory disorder, and cancers. However, nearly all conventional drug delivery system (DDS) nanocarriers utilize endocytosis for entering target cells; thus, the development of macrophage-targeting and phagocytosis-inducing DDS nanocarriers for treating these diseases is required. In this study, we developed a hepatitis B virus (HBV) envelope L particle (i.e., bio-nanocapsule (BNC)) outwardly displaying a tandem form of protein G-derived IgG Fc-binding domain and protein L-derived IgG Fab-binding domain (GL-BNC). When conjugated with the macrophage-targeting ligand, mouse IgG2a (mIgG2a), the GL-BNC itself, and the liposome-fused GL-BNC (i.e., GL-virosome) spontaneously initiated aggregation by bridging between the Fc-binding domain and Fab-binding domain with mIgG2a. The aggregates were efficiently taken up by macrophages, whereas this was inhibited by latrunculin B, a phagocytosis-specific inhibitor. The mIgG2a-GL-virosome containing doxorubicin exhibited higher cytotoxicity toward macrophages than conventional liposomes and other BNC-based virosomes. Thus, GL-BNCs and GL-virosomes may constitute promising macrophage-targeting and phagocytosis-inducing DDS nanocarriers. STATEMENT OF SIGNIFICANCE: We have developed a novel macrophage-targeting and phagocytosis-inducing bio-nanocapsule (BNC)-based nanocarrier named GL-BNC, which comprises a hepatitis B virus envelope L particle outwardly displaying protein G-derived IgG Fc- and protein L-derived IgG Fab-binding domains in tandem. The GL-BNC alone or liposome-fused form (GL-virosomes) could spontaneously aggregate when conjugated with macrophage-targeting IgGs, inducing phagocytosis by the interaction between IgG Fc of aggregates and FcγR on phagocytes. Thereby these aggregates were efficiently taken up by macrophages. GL-virosomes containing doxorubicin exhibited higher cytotoxicity towards macrophages than ZZ-virosomes and liposomes. Our results suggested that GL-BNCs and GL-virosomes would serve as promising drug delivery system nanocarriers for targeting delivery to macrophages.

Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications.

Cellular uptake of hepatitis B virus envelope L particles is independent of sodium taurocholate cotransporting polypeptide, but dependent on heparan sulfate proteoglycan.

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.

Mutational analysis of hepatitis B virus pre-S1 (9-24) fusogenic peptide.

A hollow nanoparticle known as a bio-nanocapsule (BNC) consisting of hepatitis B virus (HBV) envelope L protein and liposome (LP) can encapsulate drugs and genes and thereby deliver them in vitro and in vivo to human hepatic tissues, specifically by utilizing the HBV-derived infection machinery. Recently, we identified a low pH-dependent fusogenic domain at the N-terminal part of the pre-S1 region of the HBV L protein (amino acid residues 9 to 24; NPLGFFPDHQLDPAFG), which shows membrane destabilizing activity (i.e., membrane fusion, membrane disruption, and payload release) upon interaction with target LPs. In this study, instead of BNC and HBV, we generated LPs displaying a mutated form of the pre-S1 (9-24) peptide, and performed a membrane disruption assay using target LPs containing pyranine (fluorophore) and p-xylene-bis (N-pyridinium bromide) (DPX) as a quencher. The membrane disruption activity was found to correlate with the hydrophobicity of the whole structure, while the peptide retained a random-coil structure even under low pH condition. One large hydrophobic cluster (I) and one small hydrophobic cluster (II) residing in the peptide would be connected by the protonation of residues D16 and D20, and thereby exhibit strong membrane disruption activity in a low pH-dependent manner. Furthermore, the introduction of a positively charged residue enhanced the activity significantly, suggesting that a sole positively charged residue (H17) may be important for the interaction with target LPs by electrostatic interaction. Collectively, these results suggest that the pre-S1 (9-24) peptide may be involved in the endosomal escape of the BNC's payloads, as well as in the HBV uncoating process.

Cytokine-dependent activation of JAK-STAT pathway in Saccharomyces cerevisiae.

Protein phosphorylation is an important post-translational modification for intracellular signaling molecules, mostly found in serine and threonine residues. Tyrosine phosphorylations are very few events (less than 0.1% to phosphorylated serine/threonine residues), but capable of governing cell fate decisions involved in proliferation, differentiation, apoptosis, and oncogenic transformation. Hence, it is important for drug discovery and system biology to measure the intracellular level of phosphotyrosine. Although mammalian cells have been conventionally utilized for this purpose, accurate determination of phosphotyrosine level often suffers from high background due to the unexpected crosstalk among endogenous signaling molecules. This situation led us firstly to establish the ligand-induced activation of homomeric receptor tyrosine kinase (i.e., epidermal growth factor receptor) in Saccharomyces cerevisiae, a lower eukaryote possessing organelles similar to higher eukaryote but not showing substantial level of tyrosine kinase activity. In this study, we expressed heteromeric receptor tyrosine kinase (i.e., a complex of interleukin-5 receptor (IL5R) α chain, common ß chain, and JAK2 tyrosine kinase) in yeast. When coexpressed with a cell wall-anchored form of IL5, the yeast exerted the autophosphorylation of JAK2, followed by the phosphorylation of transcription factor STAT5a and subsequent nuclear accumulation of phosphorylated STAT5a. Taken together, yeast could be an ideal host for sensitive detection of phosphotyrosine generated by a wide variety of tyrosine kinases. Biotechnol. Bioeng. 2016;113: 1796-1804. © 2016 Wiley Periodicals, Inc.

Bio-nanocapsules displaying various immunoglobulins as an active targeting-based drug delivery system.

The bio-nanocapsule (BNC) is an approximately 30-nm particle comprising the hepatitis B virus (HBV) envelope L protein and a lipid bilayer. The L protein harbors the HBV-derived infection machinery; therefore, BNC can encapsulate payloads such as drugs, nucleic acids, and proteins and deliver them into human hepatocytes specifically in vitro and in vivo. To diversify the possible functions of BNC, we generated ZZ-BNC by replacing the domain indispensable for the human hepatotrophic property of BNC (N-terminal region of L protein) with the tandem form of the IgG Fc-binding Z domain of Staphylococcus aureus protein A. Thus, the ZZ-BNC is an active targeting-based drug delivery system (DDS) nanocarrier that depends on the specificity of the IgGs displayed. However, the Z domain limits the animal species and subtypes of IgGs that can be displayed on ZZ-BNC. In this study, we introduced into BNC an Ig κ light chain-binding B1 domain of Finegoldia magna protein L (protein-L B1 domain) and an Ig Fc-binding C2 domain of Streptococcus species protein G (protein-G C2 domain) to produce LG-BNC. The LL-BNC was constructed in a similar way using a tandem form of the protein-L B1 domain. Both LG-BNC and LL-BNC could display rat IgGs, mouse IgG1, human IgG3, and human IgM, all of which not binding to ZZ-BNC, and accumulate in target cells in an antibody specificity-dependent manner. Thus, these BNCs could display a broad spectrum of Igs, significantly improving the prospects for BNCs as active targeting-based DDS nanocarriers. STATEMENT OF SIGNIFICANCE: We previously reported that ZZ-BNC, bio-nanocapsule deploying the IgG-binding Z domain of protein A, could display cell-specific antibody in an oriented immobilization manner, and act as an active targeting-based DDS nanocarrier. Since the Z domain can only bind to limited types of Igs, we generated BNCs deploying other Ig-binding domains: LL-BNC harboring the tandem form of Ig-binding domain of protein L, and LG-BNC harboring the Ig binding domains of protein L and protein G sequentially. Both BNCs could display a broader spectrum of Igs than does the ZZ-BNC. When these BNCs displayed anti-CD11c IgG or anti-EGFR IgG, both of which cannot bind to Z domain, they could bind to and then enter their respective target cells.

High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.

An automated system for high-throughput single cell-based breeding.

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae.

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.

LIM domains regulate protein kinase C activity: a novel molecular function.

Enigma homolog protein 1 (ENH1) acts as a scaffold that selectively associates protein kinases and transcription factors with cytoskeletal elements. ENH1 comprises an N-terminal PDZ domain and three C-terminal LIM domains. Through the LIM domains ENH1 interacts with the N-terminal region of protein kinase C ßI (PKCßI). Here, we show that when ENH1 is co-expressed, PKCßI is translocated from the cytoplasm to the plasma membrane in the absence of any other stimulation. Moreover expression of ENH1 markedly increases PKCßI activity in the absence of PKC activators. A similar activation of PKCßI was observed with co-expression of Cypher1 or Enigma, but not other LIM proteins. The region including the three LIM domains of ENH1 (residues 415-591) appears to be sufficient for this PKCßI activation. Finally, interaction with ENH1 also increases the activity of PKCα and PKCγ, whereas it reduces PKCζ activity. These findings provide strong evidence that ENH1 activates conventional PKCs by directly binding through its LIM domains. Thus, LIM domains have a novel molecular function: the regulation of PKC activities in a PKC isoform-specific manner.

Accumulation of polyubiquitinated proteins by overexpression of RBCC protein interacting with protein kinase C2, a splice variant of ubiquitin ligase RBCC protein interacting with protein kinase C1.

The nuclear-cytoplasmic shuttling protein RBCC protein interacting with protein kinase C1 (RBCK1) possesses transcriptional and ubiquitin ligase activities. We have recently reported that RBCC protein interacting with protein kinase C2 (RBCK2), a RING-in-between-RING fingers domain-lacking splice variant of RBCK1, lacks transcriptional activity, but rather represses the RBCK1-mediated transcriptional activity as a cytoplasmic tethering protein for RBCK1. In this study, we have found that RBCK2 overexpressed in human embryonic kidney 293 cells interacts with the polyubiquitin chain and the polyubiquitin-interacting subunit S5a, and significantly increases the intracellular amount of polyubiquitinated proteins. These results strongly suggested that RBCK2 functions as an adaptor protein for the polyubiquitinated protein and the S5a subunit in 26S proteasome through its novel zinc finger motif and ubiquitin-like domain, respectively, presumably delivering the polyubiquitinated proteins to the entrance of the 26S proteasome catalytic domain for degradation.