Biology subjects

Senoo, A.

Publications and source records attributed to Senoo, A..

8 recordsLinked to original sources

bioRxiv2026

Proximity directs microbial transglutaminase site selectivity in native antibody modification

Site-specific protein modification allows diverse functionalities to be introduced while minimizing perturbations to the protein structure and activity. Considerable efforts have been made to achieve site-specific modification of native proteins to overcome the heterogeneity resulting from conventional stochastic Lys or Cys modification. We have previously achieved the selective modification of Lys65 in a native immunoglobulin G1 (IgG1) antibody (trastuzumab) using EzMTG-pG(Fab), which is an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G [pG(Fab)]. However, this approach cannot be widely applied to different types of IgG antibodies. Here, we designed pG(Fab)-EzMTG by fusing pG(Fab) to the N-terminus of EzMTG. Notably, switching the fusion partners dramatically altered the IgG modification site from Lys65 to Lys225, which is located in the hinge site of native IgG1 antibodies. This Lys225-selective labeling was applicable to different IgG1 antibodies. As a functional application, the cytotoxic drug monomethyl auristatin E (MMAE) was conjugated to Lys225 of trastuzumab, and the resulting antibody-drug conjugate exhibited antigen-specific cytotoxicity. These findings demonstrate that fusion-protein architecture determines site selectivity in proximity-directed enzymatic modification, providing a strategy for the site-specific functionalization of native antibodies.

bioengineering↗

bioRxiv2026

Structural basis for the intestinal protocadherin-based intermicrovillar adhesion complex

The intestinal brush border (BB), composed of densely packed microvilli on enterocytes, is essential for nutrient absorption and host defense. Its organization relies on the intermicrovillar adhesion complex (IMAC), mediated by protocadherins CDHR2 and CDHR5. Despite their clinical relevance in inflammatory bowel disease and several carcinomas, structural details of IMAC assemblies have remained elusive. Herein, we report the Cryo-EM structure of the adhesive complex at 3.4 [A] resolution, revealing a heterotetrameric ensemble composed of a dimer of CDHR2 and a dimer of CDHR5. This assembly ensures uniform adhesive strength between neighboring microvilli, and facilitates hexagonal packing of microvilli. Biophysical analyses and molecular dynamics simulations revealed a kinked, Ca{superscript 2}-free linker between domains EC3 and EC4 of CDHR5 conferring the necessary flexibility to withstand the shear stress caused during intestinal peristalsis. Collectively, these findings provide a structural framework for understanding BB organization and suggest strategies for therapeutics targeting IMAC in intestinal disorders.

biophysics↗

bioRxiv2024

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 [A]-resolution X-ray structure that revealed a highly -helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

biochemistry↗

bioRxiv2024

Characterization of high affinity IgM and IgG monoclonal antibodies against norovirus variants GII.4 and GII.17

Human norovirus, a leading cause of viral gastroenteritis, results in significant global health and economic burden, requiring sensitive and accurate diagnosis and effective therapeutics and vaccines. In this study, we immunized mice with the virus like capsid particles of GII.4, a mainstream strain, and GII.17, a modern strain that began to circulate in 2014, and used hybridoma technology to generate hybridoma cells that produce norovirus-binding antibodies against GII.4 and GII.17, respectively. Selection of these hybridoma cells yielded monoclonal IgG and IgM antibodies against these strains. Characterization of these antibodies revealed that avidity effect by multivalent binding is necessary for IgM to bind to norovirus at high efficiency, while IgG achieve high affinity even by monovalent binding. Surface plasmon resonance and ELISA data suggest that the high density of antigen protrusion domain in the norovirus capsid, containing approximately protomers, facilitates IgM to bind to norovirus capsid with high efficiency.

biochemistry↗

bioRxiv2024

Key role of P230 in the hinge region on the IgG architecture and function

Immunoglobulin G (IgG) is a molecule that plays an important role in biological defense; IgG molecules have been applied as drugs due to their high specificity for antigens and their ability to activate immunity via effector molecules on immune cells. On the other hand, the flexibility of the hinge region makes it difficult to apply conventional structural biology approaches due to its dynamic conformational changes, and the mechanism of action of the molecule as a whole has not been elucidated. Here, we introduced a deletion amino acid mutation in the hinge region to elucidate the role of the hinge region and its effect on the structure and function of the IgG molecule. Deletion of Pro230 resulted in the formation of a half-molecular in which the interaction between heavy chains was lost. We elucidated the mechanism of half-IgG formation by structural analysis using nuclear magnetic resonance (NMR) measurements and by disulfide quantification using peptide mapping using LC-MS/MS. For this purpose, a new NMR stable isotope labeling method was introduced. Finally, cell assay revealed that the IgG half-molecules have specific Fc{gamma}RI-mediated activity. This report provides new insights into the higher-order structure formation of IgG molecules and is expected to contribute to the elucidation of the molecular basis of the Fc{gamma} receptor-mediated activation mechanism of the immune system.

biophysics↗

bioRxiv2021

Lipid nanodiscs facilitate the identification of a fragment compound inhibiting the enzymatic activity of the bacterial membrane protein MsbA.

Membrane proteins are critical elements of numerous therapeutic approaches ranging from cancer to bacterial infections. MsbA is a bacterial membrane protein that has received increasing attention as an antibacterial target for its role in the processing of Lipid A, a key precursor of lipopolysaccharide that is essential for bacterial growth. When employing nanodiscs it is possible to stabilize MsbA by providing a membrane-like environment that enhances its enzymatic activity. Taking advantage of this property we have carried out a fragment screening using the biophysical method of surface plasmon resonance. This approach identified several compounds that bind specifically to MsbA. In particular, one of these fragment molecules not only binds to the target, but also inhibits the ATPase activity of the MsbA protein. The similarity of this fragment to the adenine moiety of ATP points at a route to generate stronger and more potent inhibitors for MsbA and even other proteins of its family of ABC transporters. Collectively, our study reveals biophysical approaches that facilitate the identification of fragment candidates inhibiting the activity of membrane proteins.

biophysics↗

bioRxiv2020

Structural basis for binding mechanism of human serum albumin complexed with cyclic peptide dalbavancin

Cyclic peptides, with unique structural features, have emerged as new candidates for drug discovery; their association with human serum albumin (HSA; long blood half-life), is crucial to improve drug delivery and avoid renal clearance. Here, we present the crystal structure of HSA complexed with dalbavancin, a clinically used cyclic peptide. SAXS and ITC experiments showed that the HSA-dalbavancin complex exists in a monomeric state; dalbavancin is only bound to the subdomain IA of HSA in solution. Structural analysis and MD simulation revealed that the swing of Phe70 and movement of the helix near dalbavancin were necessary for binding. The flip of Leu251 promoted the formation of the binding pocket with an induced-fit mechanism; moreover, the movement of the loop region including Glu60 increased the number of non-covalent interactions with HSA. These findings may support the development of new cyclic peptides for clinical use, particularly the elucidation of their binding mechanism to HSA.

biochemistry↗

bioRxiv2020

On-rate modulation of cadherin interactions by chemical fragments

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the on-rate of X dimerization. Our findings will impact on a strategy for kinetic regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules.

biochemistry↗