Biology subjects

Numata, T.

Publications and source records attributed to Numata, T..

4 recordsLinked to original sources

bioRxiv2026

Polymorphic Structures of an Artificial Nucleocapsid Evolved from a Circularly Permuted Bacterial Protein Cage

Molecular self-assembly provides a route to nanoscale structures with defined sizes and shapes. Protein cages assembled from repeated subunits form compartments that can package molecular cargoes. Although amino acid substitutions can alter intersubunit interactions and cage architecture, how protein backbone connectivity relates to the cage architectures accessible during laboratory evolution remains unclear. Here we evolved an artificial nucleocapsid from a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS). Laboratory evolution increased the yield of purified nucleocapsids relative to the progenitor, and the evolved variant, NC119-3, packaged its encoding mRNA. Cryo-electron microscopy revealed a heterogeneous population of cages, within which we resolved 180- and 210-subunit cage families composed of 36 and 42 pentamers, respectively. Within each resolved family, particles with the same subunit stoichiometry differed in the arrangement and orientation of their pentameric building blocks. A 2.90 [A] reconstruction of a tetrahedral 180-subunit cage further revealed that sequence-identical subunits adopt distinct conformations depending on their positions within the assembly. These pentamer-based architectures differ from the domain-swapped T = 4 cage previously reported for nucleocapsids evolved from an AaLS variant with a different circular permutation site. Together with previous work, these findings show that RNA-packaging nucleocapsids evolved from scaffolds with different backbone connectivity can adopt distinct cage architectures.

synthetic biology↗

bioRxiv2024

Msp1 and Pex19-Pex3 cooperate to achieve correct localization of Pex15 to peroxisomes

Yeast Msp1 is a dual-localized AAA-ATPase on the mitochondrial outer membrane (OM) and peroxisomal membrane. We previously showed that Msp1 transfers mistargeted tail-anchored (TA) proteins from mitochondria to the endoplasmic reticulum (ER) for degradation or delivery to their original destinations. However, the mechanism by which Msp1 in mitochondria and peroxisomes handles authentic peroxisomal TA proteins remains unclear. We show that newly synthesized Pex15 is targeted to peroxisomes primarily via the Pex19- and Pex3-dependent pathway. Mistargeted Pex15 on the mitochondrial OM is extracted by mitochondrial Msp1, and transferred to the ER via the guided-entry of TA proteins pathway for degradation or to peroxisomes via the Pex19-Pex3 pathway. Intriguingly, endogenous Pex15 localized in peroxisomes is also extracted from the membranes by peroxisomal Msp1, but returns to peroxisomes via the Pex19-Pex3 pathway. These results suggest that correct Pex15 localization to peroxisomes relies on not only the initial targeting by Pex19-Pex3 but also the constant re-routing by Msp1 and Pex19-Pex3.

cell biology↗

bioRxiv2024

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

biochemistry↗

bioRxiv2021

Actin isovariant ACT7 regulates root meristem development in Arabidopsis through modulating auxin and ethylene responses

Meristem is the most functionally dynamic part in a plant body. The shaping of the meristem requires constant cell division and cell elongation, which are influenced by hormones and cell cytoskeletal component, actin. Although the roles of hormones in modulating meristem development have been extensively studied, the role of actin in this process is still elusive. Using the single and double mutants of the vegetative class actin, we demonstrate that actin isovariant ACT7 plays an important role in root meristem development. In absence of ACT7, but not ACT8 and ACT2, depolymerization of actin was observed. Consistently, act7 mutant showed reduced cell division, cell elongation, and meristem length. Intracellular distribution and trafficking of auxin transport proteins in the actin mutants revealed that ACT7 specifically functions in root meristem to facilitate the trafficking of auxin efflux carriers PIN1 and PIN2, and consequently the transport of auxin. Compared with act7, act7act8 double mutant shows slightly enhanced phenotypic response and altered intracellular trafficking. The altered distribution of auxin in act7 and act7act8 affects the response of the roots to ethylene but not to cytokinin. Collectively, our results suggest that ACT7 dependent auxin-ethylene response plays a key role in controlling Arabidopsis root meristem development.

plant biology↗