Publications by authors named "Shinya Honda"

74 Publications

Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells.

Cell Chem Biol 2021 Nov 3. Epub 2021 Nov 3.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address:

Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.
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http://dx.doi.org/10.1016/j.chembiol.2021.08.010DOI Listing
November 2021

Investigations into the Potential of Using Open Source CFD to Analyze the Differences in Hemodynamic Parameters for Aortic Dissections (Healthy versus Stanford Type A and B).

Ann Vasc Surg 2021 Oct 12. Epub 2021 Oct 12.

Department of Cardiac Surgery, Asahikawa Medical University, Asahikawa, Japan.

Background: The objective of this study was to develop a method to evaluate the effects of an aortic dissection on hemodynamic parameters by conducting a comparison with that of a healthy (nondissected) aorta. Open-source software will be implemented, no proprietary software/application will be used to ensure accessorily and repeatability, in all the data analysis and processing. Computed tomography (CT) images of aortic dissection are used for the model geometry segmentation. Boundary conditions from literature are implemented to computational fluid dynamics (CFD) to analyze the hemodynamic parameters.

Methods: A numerical simulation model was created by obtaining accurate 3-dimensional geometries of aortae from CT images. In this study, CT images of 8 cases of aortic dissection (Stanford type-A and type-B) and 3 cases of healthy aortae are used for the actual aorta model geometry segmentation. These models were exported into an open-source CFD software, OpenFOAM, where a simplified pulsating flow was simulated by controlling the flow pressure. Ten cycles of the pulsatile flow (0.50 sec/cycle) conditions, totaling 5 sec, were calculated.

Results: The pressure distribution, wall shear stress (WSS) and flow velocity streamlines within the aorta and the false lumen were calculated and visualized. It was found that the flow velocity and WSS had a high correlation in high WSS areas of the intermittent layer between the true and false lumen. Most of the Stanford type-A dissections in the study showed high WSS, over 38 Pa, at the systole phase. This indicates that the arterial walls in type-A dissections are more likely to be damaged with pulsatile flow.

Conclusions: Using CFD to estimate localized high WSS areas may help in deciding to treat a type-A or B dissection with a stent graft to prevent a potential rupture.
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http://dx.doi.org/10.1016/j.avsg.2021.08.007DOI Listing
October 2021

Nano-Microscopy of Therapeutic Antibody Aggregates in Solution.

Methods Mol Biol 2022 ;2313:219-239

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.
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http://dx.doi.org/10.1007/978-1-0716-1450-1_13DOI Listing
January 2022

Effect of backbone circularization on colloidal stability: Compaction of unfolded structures improves aggregation resistance of granulocyte colony-stimulating factor.

Int J Pharm 2021 Aug 9;605:120774. Epub 2021 Jun 9.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. Electronic address:

Aggregation of protein therapeutics can lead to immunogenicity and loss of function in vivo. Its effective prevention requires an understanding of the conformational and colloidal stability of protein and the improvement of both. Granulocyte colony-stimulating factor (G-CSF), which is one of the most widely used protein therapeutics, was previously shown to be conformationally stabilized by connecting its N- and C-termini with amide bonds (backbone circularization). In this study, we investigated whether circularization affects the colloidal stability of proteins. Colloidal stability was indirectly assessed by analyzing the aggregation behavior of G-CSF variants using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). Consequently, we found that the unfolded structure of circularized G-CSF was more compact than non-circularized G-CSF, and that backbone circularization improved its aggregation resistance against chemical denaturation by guanidine hydrochloride (GdnHCl). The improved aggregation resistance suggests that the expansion tolerance of circularized G-CSF in the unfolded state increased its colloidal stability. Thus, backbone circularization is an excellent method for enhancing the colloidal and the conformational stability of protein with minimal sequence changes. It is therefore expected to be effective in extending the storage stability of protein therapeutics, enhancing their biological stability.
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http://dx.doi.org/10.1016/j.ijpharm.2021.120774DOI Listing
August 2021

Local disorder of the C-terminal segment of the heavy chain as a common sign of stressed antibodies evidenced with a peptide affinity probe specific to non-native IgG.

Int J Biol Macromol 2021 Jul 25;182:1697-1703. Epub 2021 May 25.

Biomedical Research Institute, The National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address:

Therapeutic antibodies have many biopharmaceutical applications; however, characterization of their higher-order structures is a major concern in quality control. We have developed AF.2A1, an artificial protein, that specifically recognizes non-native, structured IgGs. We performed binding assays using various types of IgGs and fragments to investigate the mechanisms by which AF.2A1 interacts with the non-native IgG. AF.2A1 recognized the acid-stressed IgGs from human, mouse, and rat, but not rabbit. Binding assays using the human IgG1 fragments revealed that an interface emerged by deleting five C-terminal residues. We conclude that AF.2A1 recognizes an exposed hydrophobic core centered on the Trp417. Our results concur with those of the previous studies showing that C-terminal structural changes occur early during antibody denaturation and aggregation. Our findings explain the molecular rationale for using AF.2A1 in quality control of biopharmaceutical IgGs.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.05.137DOI Listing
July 2021

Polyploid engineering by increasing mutant gene dosage in yeasts.

Microb Biotechnol 2021 05 22;14(3):979-992. Epub 2020 Dec 22.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan.

The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best-understood biological systems. Diploid strains of S. cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole-genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth-based screening.
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http://dx.doi.org/10.1111/1751-7915.13731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085954PMC
May 2021

Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration.

Nat Commun 2020 10 20;11(1):5311. Epub 2020 Oct 20.

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.
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http://dx.doi.org/10.1038/s41467-020-18892-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576787PMC
October 2020

Autophagy involvement in oncogenesis.

Cancer Sci 2020 Nov 1;111(11):3993-3999. Epub 2020 Oct 1.

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Various clinical and experimental findings have revealed the causal relationship between autophagy failure and oncogenesis, and several mechanisms have been suggested to explain this relationship. We recently proposed two additional mechanisms: centrosome number dysregulation and the failure of autophagic cell death. Here, we detail the mechanical relationship between autophagy failure and oncogenesis.
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http://dx.doi.org/10.1111/cas.14646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648016PMC
November 2020

Identification of a phosphorylation site on Ulk1 required for genotoxic stress-induced alternative autophagy.

Nat Commun 2020 04 9;11(1):1754. Epub 2020 Apr 9.

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser, and loss of RIPK3 abolishes Ulk1 phosphorylation. These findings indicate that RIPK3-dependent Ulk1 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.
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http://dx.doi.org/10.1038/s41467-020-15577-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145817PMC
April 2020

Association Between Atg5-independent Alternative Autophagy and Neurodegenerative Diseases.

J Mol Biol 2020 04 21;432(8):2622-2632. Epub 2020 Jan 21.

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. Electronic address:

Autophagy is a cellular process that degrades intracellular components, including misfolded proteins and damaged organelles. Many neurodegenerative diseases are considered to progress via the accumulation of misfolded proteins and damaged organelles; therefore, autophagy functions in regulating disease severity. There are at least two types of autophagy (canonical autophagy and alternative autophagy), and canonical autophagy has been applied to therapeutic strategies against various types of neurodegenerative diseases. In contrast, the role of alternative autophagy has not yet been clarified, but it is speculated to be involved in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease.
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http://dx.doi.org/10.1016/j.jmb.2020.01.016DOI Listing
April 2020

Stabilization of backbone-circularized protein is attained by synergistic gains in enthalpy of folded structure and entropy of unfolded structure.

FEBS J 2020 04 27;287(8):1554-1575. Epub 2019 Oct 27.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.

Backbone circularization is an effective technique for protein stabilization. Here, we investigated the effect of a connector, an engineered segment that connects two protein termini, on the conformational stability of previously designed circularized variants of granulocyte colony-stimulating factor (G-CSF). Heat tolerance and chemical denaturation analyses revealed that aggregation resistance and thermodynamic stability of the circularized variants were superior to those of linear G-CSF. Crystal structure and molecular dynamics (MD) simulation of the most thermodynamically stable variant (C166) revealed a high number of intramolecular hydrogen bonds in both the connector region and Helix D adjacent to the connector region in the folded structure. MD simulations and theoretical calculations involving different force fields indicated a reduction in the main chain entropy of C166 in the unfolded state and increase in the intramolecular hydrogen bond energy of C166 in the folded structure. Although backbone circularization is usually considered to alter chain entropy of the unfolded state, the data indicated that it could also improve the conformational enthalpy of the folded state. Further structural examination of the connector region confirmed that protein design based on a statistical analysis of local structures is an effective approach for predicting an optimum connector length to improve the conformational stability of backbone-circularized proteins. Protein design using backbone circularization with an optimum connector length will be useful for the development of effective and safe protein therapeutics. DATABASE: Structural data are available in Protein Data Bank under the accession number 5ZO6.
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http://dx.doi.org/10.1111/febs.15092DOI Listing
April 2020

Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein-Protein Interaction.

ACS Chem Biol 2019 12 22;14(12):2729-2736. Epub 2019 Oct 22.

The National Institute of Advanced Industrial Science and Technology , 1-1-1, Higashi , Tsukuba 305-8566 , Japan.

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein-protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy-entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein-protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.
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http://dx.doi.org/10.1021/acschembio.9b00652DOI Listing
December 2019

pH-shift stress on antibodies.

Methods Enzymol 2019 12;622:329-345. Epub 2019 Mar 12.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan. Electronic address:

Antibody unfolding and aggregation have been gaining increasing concern as the need for pharmaceutical antibodies with high quality has increased. Assays for continuous application of severe stress to antibodies, which are typically employed to evaluate tolerance to aggregation or to understand the mechanism of aggregation, induce both unfolding and aggregation simultaneously. In this chapter, we describe the pH-shift stress on antibodies that uses a low pH solution to unfold the antibody and a neutralizing solution to induce folding and the aggregation. This pH-shift technique with light scattering analysis will be a useful assay to evaluate antibody aggregation at a neutral pH and ambient temperature without generation of unfolded molecules during aggregation.
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http://dx.doi.org/10.1016/bs.mie.2019.02.021DOI Listing
February 2020

Synthetic gene expression circuits regulating sexual reproduction.

Methods Enzymol 2019 12;621:17-30. Epub 2019 Mar 12.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

The budding yeast Saccharomyces cerevisiae has been widely utilized in fermentative production since ancient times. Several approaches for modification of yeast traits have been developed, including mutagenesis, protoplast fusion, and genetic modification. Crossbreeding provides an attractive means to improve and combine strain traits based on sexual reproduction. Common crossbreeding strategies require the isolation of MATa and MATα haploids via sporulation, as most of parental yeasts are MATa/α diploids and unable to mate directly. Unfortunately, many yeast strains used in industry exhibit low sporulation rates resulting in limited crossbreeding efficiency and numerous technical challenges. Here, we review the construction of synthetic gene expression circuits as a means to provide alternative methods for sporulation for yeast crossbreeding. These methods enable researchers to convert the sequence of the MAT locus and subsequently acquire crossbreds via mating of isolated yeast strains. The purpose of this chapter is to provide a basic guide for researchers who are attempting to expand the variety of yeast resources using the sexual reproduction machinery of yeast.
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http://dx.doi.org/10.1016/bs.mie.2019.02.036DOI Listing
February 2020

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins.

Biotechnol Prog 2019 07 22;35(4):e2820. Epub 2019 Apr 22.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. In this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.
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http://dx.doi.org/10.1002/btpr.2820DOI Listing
July 2019

In-Solution Microscopic Imaging of Fractal Aggregates of a Stressed Therapeutic Antibody.

Anal Chem 2019 04 19;91(7):4640-4648. Epub 2019 Mar 19.

Biomedical Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Higashi, Tsukuba , Ibaraki 305-8566 , Japan.

Aggregates of therapeutic proteins that can contaminate drug products during manufacture is a growing concern for the pharmaceutical industry because the aggregates are potentially immunogenic. Electron microscopy is a typical, indispensable method for imaging nanometer- to micrometer-sized structures. Nevertheless, it is not ideal because it must be performed with ex situ monitoring under high-vacuum conditions, where the samples could be altered by staining and drying. Here, we introduce a scanning electron-assisted dielectric microscopy (SE-ADM) technique for in-solution imaging of monoclonal immunoglobulin G (IgG) aggregates without staining and drying. Remarkably, SE-ADM allowed assessment of the size and morphology of the IgG aggregates in solution by completely excluding drying-induced artifacts. SE-ADM was also beneficial to study IgG aggregation caused by temporary acid exposure followed by neutralization, pH-shift stress. A box-counting analysis of the SE-ADM images provided fractal dimensions of the larger aggregates, which complemented the fractal dimensions of the smaller aggregates measured by light scattering. The scale-free or self-similarity nature of the fractal aggregates indicated that a common mechanism for antibody aggregation existed between the smaller and larger aggregates. Consequently, SE-ADM is a useful method for characterizing protein aggregates to bridge the gaps that occur among conventional analytical methods, such as those related to in situ/ ex situ techniques or size/morphology assessments.
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http://dx.doi.org/10.1021/acs.analchem.8b05979DOI Listing
April 2019

Beclin 1 regulates recycling endosome and is required for skin development in mice.

Commun Biol 2019 25;2:37. Epub 2019 Jan 25.

1Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 Japan.

Beclin 1 is a key regulator of autophagy and endocytosis. However, its autophagy-independent functions remain poorly understood. Here, we report that Beclin 1 regulates recycling endosome and is required for skin development in vivo. We first established keratinocyte-specific Beclin 1-knockout mice and found that these mutant mice died owing to severe impairment of epidermal barrier. Beclin 1 plays a role in autophagy and the endocytic pathway in cooperation with Atg14 and UVRAG, respectively, and keratinocyte-specific Atg14-knockout mice do not show any abnormal phenotypes, suggesting that Beclin 1 has a role in skin development via the endocytic pathway. Furthermore, we found that Beclin 1 deficiency causes mislocalization of integrins via a defect of recycling endosome, abnormal cell detachment of basal cells and their immature differentiation, and abnormal skin development. These results provide the first genetic evidence showing the roles of Beclin 1 in recycling endosome and skin development.
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http://dx.doi.org/10.1038/s42003-018-0279-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347619PMC
April 2020

Prediction of intracellular targets of a small compound by analyzing peptides presented on MHC class I.

Biochem Biophys Res Commun 2019 01 29;508(2):480-486. Epub 2018 Nov 29.

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. Electronic address:

In chemical biology, the elucidation of chemical target is crucial for successful drug development. Because MHC class I molecules present peptides from intracellular damaged proteins, it might be possible to identify targets of a chemical by analyzing peptide sequences on MHC class I. Therefore, we treated cells with the autophagy-inducing chemical TMD-457 and identified the peptides presented on MHC class I. Many of the peptides were derived from molecules involved in ER trafficking and ER stress, which were confirmed by morphological and biochemical analyses. Therefore, our results demonstrate that analyzing MHC class I peptides is useful for the detection of chemical targets.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.089DOI Listing
January 2019

C2 domain orientation of human immunoglobulin G in solution: Structural comparison of glycosylated and aglycosylated Fc regions using small-angle X-ray scattering.

MAbs 2019 04 12;11(3):453-462. Epub 2018 Dec 12.

a Biomedical Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Ibaraki , Japan.

The N-linked glycan in immunoglobulin G is critical for the stability and function of the crystallizable fragment (Fc) region. Alteration of these protein properties upon the removal of the N-linked glycan has often been explained by the alteration of the C2 domain orientation in the Fc region. To confirm this hypothesis, we examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution. Conformational characteristics of the C2 domain orientation were validated by comparison with SAXS profiles theoretically calculated from multiple crystal structures of the Fc region with different C2 domain orientations. The reduced chi-square values from the fitting analyses of gFc and aFc associated with the degree of openness or closure of each crystal structure, as determined from the first principal component that partially governed the variation of the C2 domain orientation extracted by a singular value decomposition analysis. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a "semi-closed" C2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the C2 domain orientation in solution. These findings will guide the development of methodology for the production of highly refined functional Fc variants.
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http://dx.doi.org/10.1080/19420862.2018.1546086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512918PMC
April 2019

Suppression of Aggregation of Therapeutic Monoclonal Antibodies during Storage by Removal of Aggregation Precursors Using a Specific Adsorbent of Non-Native IgG Conformers.

Bioconjug Chem 2018 10 28;29(10):3250-3261. Epub 2018 Sep 28.

Biomedical Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Higashi, Tsukuba , Ibaraki 305-8566 , Japan.

The quality of preparations of therapeutic IgG molecules, widely used for the treatment of various diseases, should be maintained during storage and administration. Nevertheless, recent studies demonstrate that IgG aggregation is one of the most critical immunogenicity risk factors that compromises safety and efficacy of therapeutic IgG molecules in the clinical setting. During the IgG manufacturing process, 0.22-μm membrane filters are commonly used to remove aggregates. However, particles with a diameter below 0.22 μm (small aggregates) are not removed from the final product. The residual species may grow into large aggregates during the storage period. In the current study, we devised a strategy to suppress IgG aggregate growth by removing aggregation precursors using the artificial protein AF.2A1. This protein efficiently binds the Fc region of non-native IgG conformers generated under chemical and physical stresses. Magnetic beads conjugated with AF.2A1 were used to remove non-native monomers and aggregates from solutions of native IgG and from native IgG solutions spiked with stressed IgG. The time-dependent growth of aggregates after the removal treatment was monitored. The removal of aggregation precursors, i.e., non-native monomers and nanometer aggregates (<100 nm), suppressed the aggregate growth. The presented findings demonstrate that a removal treatment with a specific adsorbent of non-native IgG conformers enables long-term stable storage of therapeutic IgG molecules and will facilitate mitigation of the immunogenicity of IgG preparations.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00360DOI Listing
October 2018

Generation of ubiquitin-based binder with an inserted active peptide.

Biochem Biophys Res Commun 2018 09 23;503(4):3162-3166. Epub 2018 Aug 23.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8562, Japan. Electronic address:

The grafting of active peptides onto structurally stable scaffold proteins is effective for the generation of functional proteins. In this study, we aimed to develop a grafting method using ubiquitin as a scaffold protein. Ubiquitin is a small protein consisting of 76 amino acid residues that is highly stable against heat and pH stress, which are desirable characteristics for a scaffold protein. Moreover, its structure is maintained even if it is split or additional residues are inserted. Therefore, we assumed that grafting of an active peptide into ubiquitin would result in a functional protein. As a proof of concept, we developed the ubiquitin-based binder (UbB), into which the p53 (17-28) peptide was inserted between Ile36 and Pro37. The p53 (17-28) peptide, utilized as a model active peptide in this work, is known to bind to mouse double minute 2 homolog (Mdm2). Size exclusion chromatography and circular dichroism indicated that UbB maintained a similar structure to that of ubiquitin. The affinity for Mdm2 measured by surface plasmon resonance was 292 times greater than that of the p53 (17-28) peptide. These observations indicate that ubiquitin is a robust scaffold for peptide grafting. We hope that this study will aid further development of ubiquitin-based protein engineering.
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http://dx.doi.org/10.1016/j.bbrc.2018.08.110DOI Listing
September 2018

Artificial Mating-Type Conversion and Repetitive Mating for Polyploid Generation.

ACS Synth Biol 2018 05 17;7(5):1413-1423. Epub 2018 Apr 17.

Biomedical Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Higashi 1-1-1 , Tsukuba, Ibaraki 305-8566 , Japan.

The yeast Saccharomyces cerevisiae is one of the best-understood biological systems and can produce numerous useful compounds. Sexual hybridization (mating) can drive dramatic evolution of yeasts by the inheritance of half of the parental genomic information from each cell. Unfortunately, half of the parental genomic information is lost in individual cells in the next generation. Additionally, recombination of homologous chromosomes during meiosis gives rise to diversity in the next generation; hence, it is commonly employed to identify targets from diverse cell populations, based on the mating machinery. Here, we established a system for generating polyploids that inherit all genetic information from the parental strains via artificial mating-type conversion and repetitive mating. We prepared α-type haploid strains whose chromosomes were tagged with genes encoding fluorescent proteins or transcriptional factors. Only the mating-type locus was successfully converted from α-type to a-type sequence by the endonuclease Ho, and the resultant a-type cells mated with each α-type haploid to yield an a/α-type diploid strain with all genetic information from both parental strains. Importantly, we repeatedly converted the mating-type of polyploid cells to obtain a-type cells capable of mating with α-type cells. This approach can potentially facilitate yeast-strain development with unparalleled versatility, utilizing vast available resources.
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http://dx.doi.org/10.1021/acssynbio.8b00020DOI Listing
May 2018

Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector.

Biochem Biophys Res Commun 2018 06 17;500(2):224-228. Epub 2018 Apr 17.

Biomedical Research Institute, The National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. Electronic address:

Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 3 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology.
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http://dx.doi.org/10.1016/j.bbrc.2018.04.045DOI Listing
June 2018

Dram1 regulates DNA damage-induced alternative autophagy.

Cell Stress 2018 Mar 7;2(3):55-65. Epub 2018 Mar 7.

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

Autophagy is an evolutionarily conserved process that degrades subcellular constituents. Mammalian cells undergo two types of autophagy; Atg5-dependent conventional autophagy and Atg5-independent alternative autophagy, and the molecules required for the latter type of autophagy are largely unknown. In this study, we analyzed the molecular mechanisms of genotoxic stress-induced alternative autophagy, and identified the essential role of p53 and damage-regulated autophagy modulator (Dram1). Dram1 was sufficient to induce alternative autophagy. In the mechanism of alternative autophagy, Dram1 functions in the closure of isolation membranes downstream of p53. These findings indicate that Dram1 plays a pivotal role in genotoxic stress-induced alternative autophagy.
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http://dx.doi.org/10.15698/cst2018.03.127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558930PMC
March 2018

AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

Sci Rep 2017 09 29;7(1):12466. Epub 2017 Sep 29.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.

Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.
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http://dx.doi.org/10.1038/s41598-017-12693-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622108PMC
September 2017

Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor.

ACS Chem Biol 2017 10 27;12(10):2690-2696. Epub 2017 Sep 27.

Biomedical Research Institute, The National Institute of Advanced Industrial Science and Technology , Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

Backbone circularization of protein is a powerful method to improve its structural stability. In this paper, we presumed that a tight connection leads to much higher stability. Therefore, we designed circularized variants of a granulocyte-colony stimulating factor (G-CSF) with a structurally optimized terminal connection. To estimate the appropriate length of the connection, we surveyed the Protein Data Bank to find local structures as a model for the connecting segment. We set the library of local structures composed of "helix-loop-helix," subsequently selected entries similar to the G-CSF terminus, and finally sorted the hit structures according to the loop length. Two, five, or nine loop residues were frequently observed; thus, three circularized variants (C163, C166, and C170) were constructed, prepared, and evaluated. All circularized variants demonstrated a higher thermal stability than linear G-CSF (L175). In particular, C166 that retained five connecting residues demonstrated apparent T values of 69.4 °C, which is 8.7 °C higher than that of the circularized variant with no truncation (C177), indicating that the optimization of the connecting segment is effective for enhancing the overall structural stability. C166 also showed higher proteolytic stability against both endoprotease and exopeptidase than L175. We anticipate that the present study will contribute to the improvement in the general design of circularized protein and development of G-CSF biobetters.
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http://dx.doi.org/10.1021/acschembio.7b00776DOI Listing
October 2017

Fate of a Stressed Therapeutic Antibody Tracked by Fluorescence Correlation Spectroscopy: Folded Monomers Survive Aggregation.

J Phys Chem B 2017 08 21;121(34):8085-8093. Epub 2017 Aug 21.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology , 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

Antibodies are therapeutic proteins that are becoming indispensable for the treatment of serious diseases. Their efficacies depend on their folded structures, the loss of which, through unfolding or aggregation, should be closely monitored during their manufacture, storage, and dosing for safe usage. In downstream processes, exposure of the crude antibody solution to acid, followed by neutralization, is an established standard protocol for antibody purification and inactivation of viruses in antibody preparations. Nevertheless, this treatment also triggers an unwanted side reaction, i.e., antibody aggregation. The aggregates continuously evolve at neutral pH. It remains unclear whether the aggregates progressively incorporate the native, folded, monomers into themselves, enabling aggregate growth as seen in amyloid fibrils. In the present study, the diffusion of fluorescently labeled monoclonal humanized immunoglobulin G1 monomers in aggregates produced by pH-shift stress was tracked by fluorescence correlation spectroscopy. This method was used in addition to monitoring aggregate formation by dynamic light scattering. The diffusing velocities of the monomers indicated that the folded monomers were not involved in aggregate formation, in contrast with unfolded monomers. On the basis of the results, we propose a bifurcated pathway for the refolding and aggregation of antibodies triggered by a pH shift from acidic to neutral. In the scheme, the refolded monomers survive until their unfolding. The insights in this study will contribute to the manufacture of aggregation-resistant therapeutic antibodies.
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http://dx.doi.org/10.1021/acs.jpcb.7b05603DOI Listing
August 2017

Molecular mechanisms and physiological roles of Atg5/Atg7-independent alternative autophagy.

Proc Jpn Acad Ser B Phys Biol Sci 2017 ;93(6):378-385

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University.

ATG5 and ATG7 are considered to be essential molecules for the induction of autophagy. However, we found that cells lacking ATG5 or ATG7 can still form autophagosomes/autolysosomes and perform autophagic protein degradation when subjected to certain types of stress. Although the lipidation of LC3 is accepted as a good indicator of autophagy, this did not occur during ATG5/ATG7-independent alternative autophagy. Unlike conventional autophagy, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of the phagophores with vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway.
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http://dx.doi.org/10.2183/pjab.93.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709538PMC
July 2017

Friability Testing as a New Stress-Stability Assay for Biopharmaceuticals.

J Pharm Sci 2017 10 8;106(10):2966-2978. Epub 2017 Jun 8.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. Electronic address:

A cycle of dropping and shaking a vial containing antibody solution was reported to induce aggregation. In this study, antibody solutions in glass prefillable syringes with or without silicone oil lubrication were subjected to the combined stresses of dropping and shaking, using a friability testing apparatus. Larger numbers of subvisible particles were generated, regardless of silicone oil lubrication, upon combination stress exposure than that with shaking stress alone. Nucleation of antibody molecules upon perturbation by an impact of dropping and adsorption of antibody molecules to the syringe surface followed by film formation and antibody film desorption were considered key steps in the particle formation promoted by combination stress. A larger number of silicone oil droplets was released when silicone oil-lubricated glass syringes containing phosphate buffer saline were exposed to combination stress than that observed with shaking stress alone. Polysorbate 20, a non-ionic surfactant, effectively reduced the number of protein particles, but failed to prevent silicone oil release upon combination stress exposure. This study indicates that stress-stability assays using the friability testing apparatus are effective for assessing the stability of biopharmaceuticals under the combined stresses of dropping and shaking, which have not been tested in conventional stress-stability assays.
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http://dx.doi.org/10.1016/j.xphs.2017.05.035DOI Listing
October 2017

Positive Detection of GPCR Antagonists Using a System for Inverted Expression of a Fluorescent Reporter Gene.

ACS Synth Biol 2017 08 18;6(8):1554-1562. Epub 2017 May 18.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) , Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.

The yeast Saccharomyces cerevisiae is a useful eukaryotic host organism for studying GPCRs as monomolecular models. Fluorescent reporter gene assays for GPCRs provide a convenient assay for measuring receptor activity using fluorometric instruments. Generally, these assays detect receptor activation by agonistic ligands as the induction of fluorescent reporter expression, whereas antagonistic activities are detected by competition with agonistic ligands, resulting in decreases in fluorescence intensity. In the current study, we established a system for inverted expression of a fluorescent reporter by incorporating a PEST-tag and finding out a promoter inhibited by activation of the GPCR signaling pathway from yeast endogenous promoters. Because agonists prevent fluorescent reporter expression in this system, antagonists compete with agonists and yield increased fluorescence intensity. We used the yeast endogenous pheromone receptor as a model GPCR to demonstrate the feasibility of our system for positive detection targeted at antagonists. Compared to results when only agonists were added to yeast cells, more than 10-fold higher fluorescence intensity was observed when antagonists were added in combination with agonists. The approach described here has the potential to markedly accelerate the identification of GPCR antagonists by providing rapid and straightforward responses.
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http://dx.doi.org/10.1021/acssynbio.7b00056DOI Listing
August 2017
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