Biology subjects

Goto, Y.

Publications and source records attributed to Goto, Y..

65 records · Page 4Linked to original sources

bioRxiv2020

The phagocytosis oxidase/Bem1p (PB1) domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity

O_LIPerception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). ROS have direct antimicrobial properties but also serve as signaling molecules to activate additional defense responses such as stomatal closure. RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. C_LIO_LITo better understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD coupled with mass spectrometry analysis to identify RBOHD-associated proteins. C_LIO_LIAmong RBOHD-associated proteins, we identified PHAGOCYTOSIS OXIDASE/ BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). We found that PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP directly interacts with RBOHD in vitro, and PAMP treatment increases the interaction in vivo. PB1CP is localized at the cell periphery and in cytoplasm, but PAMP treatment induces PB1CP relocalization to small endomembrane compartments. PB1CP overexpression reduces plasma membrane localization of RBOHD, suggesting that PB1CP down-regulates RBOHD function by relocalizing it away from the plasma membrane. C_LIO_LIWe reveal a novel negative regulation mechanism of ROS production through the relocalization of RBOHD by PB1CP. C_LI

plant biology↗

bioRxiv2020

Soaring styles of extinct giant birds and pterosaurs

The largest extinct volant birds (Pelagornis sandersi and Argentavis magnificens) and pterosaurs (Pteranodon and Quetzalcoatlus) are thought to have used wind-dependent soaring flight, similar to modern large birds. There are two types of soaring: thermal soaring, used by condors and frigatebirds, which involves the use of updrafts to ascend and then glide horizontally over the land or the sea; and dynamic soaring, used by albatrosses, which involves the use of wind speed differences with height above the sea surface. Previous studies have suggested that Pelagornis sandersi used dynamic soaring, while Argenthavis magnificens, Pteranodon, and Quetzalcoatlus used thermal soaring. However, the performance and wind speed requirements of dynamic and thermal soaring for these species have not yet been quantified comprehensively. We quantified these values using aerodynamic models and compared them with that of extant birds. For dynamic soaring, we quantified maximum flight speeds and maximum upwind flight speeds. For thermal soaring, we quantified the animals sinking speed circling at a given radius and how far it could glide losing a given height. Our results confirmed those from previous studies that Pteranodon and Argentavis magnificens used thermal soaring. Conversely, the results for Pelagornis sandersi and Quetzalcoatlus were contrary to those from previous studies. Pelagornis sandersi used thermal soaring, and Quetzalcoatlus had a poor ability both in dynamic and thermal soaring. Our results demonstrate the need for comprehensive assessments of performance and required wind conditions when estimating soaring styles of extinct flying species.

animal behavior and cognition↗

bioRxiv2020

Human trans-editing enzyme displays tRNA acceptor stem specificity and relaxed amino acid selectivity

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase (ProRS). Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, while tRNAPro is selected via recognition of tRNA acceptor stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family and provides the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

biochemistry↗

bioRxiv2020

Multistep changes in amyloid structure that are induced by cross-seeding on a rugged energy landscape

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures depending on species, and each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new-type amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin fibrils. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition (SVD) analysis of the UV-Vis absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

biochemistry↗

bioRxiv2020

Development of optogenetic tools to manipulate cell cycle checkpoints

In order to understand the systematic regulation of the cell cycle, we need more precise tools for cell-cycle perturbation. Optogenetics is a powerful technique for precisely controlling cellular signaling at higher spatial and temporal resolution. Here, we report optogenetic tools for the rapid and reversible control of cell-cycle checkpoints with a red/far-red light photoreceptor, phytochrome B (PhyB). We established fission yeast cells producing phycocyanobilin as a chromophore of PhyB, and demonstrated light-dependent protein recruitment to the plasma membrane, nucleus, and kinetochore. Using this system, we developed optogenetic manipulation of the cell cycle in two ways: the Opto-G2/M checkpoint triggered G2/M cell cycle arrest in response to red light, and Opto-SAC induced a spindle assembly checkpoint (SAC) in response to red light and then quickly released the SAC by far-red light.

cell biology↗

bioRxiv2020

A genetically encoded red fluorescence dopamine biosensor enables dual imaging of dopamine and norepinephrine

Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in finely controlling output functions is currently challenging, primarily due to a lack of techniques to visualize spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed R-GenGAR-DA. More specifically, a circular permutated red fluorescent protein (cpmApple) was inserted into the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated reasonable dynamic range ({Delta}F/F0 = -50%) and DA affinity (EC50 = 0.7 {micro}M) as well as the highest selectivity for DA over NE (143-fold) amongst available DA biosensors. Due to its high selectivity, R-GenGAR-DA1.2 allowed dual-color fluorescence live imaging for monitoring DA and NE, combined with the existing green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (>350-fold) in HeLa cells and hippocampal neurons grown from primary culture. By enabling precise measurement of DA, as well as simultaneous visualization of DA and NE, the red-DA biosensor R-GenGAR-DA1.2 is promising in advancing our understanding of the interplay between DA and NE in organizing key brain functions. Significance StatementThe neuromodulators dopamine and norepinephrine modulate a broad range of brain functions, often in concert. One current challenge is to measure dopamine and norepinephrine dynamics simultaneously with high spatial and temporal resolution. We therefore developed a red-dopamine biosensor that has 143-fold higher selectivity for dopamine over norepinephrine. Taking advantage of its high selectivity for dopamine over norepinephrine, this red-dopamine biosensor allowed dual-color fluorescence live imaging for monitoring dopamine and norepinephrine in both HeLa cells and hippocampal neurons in vitro combined with the existing green-norepinephrine biosensor that has 350-fold selectivity for norepinephrine over dopamine. Thus, this approach can provide new opportunities to advance our understanding of high spatial and temporal dynamics of dopamine and norepinephrine in normal and abnormal brain functions.

cell biology↗

bioRxiv2020

Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Enzymes involved in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, and yet usually, the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level, and supplement this study with substrate scope analysis for participating enzymes. Combined, our results reveal a dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined enzymatic action, and the overall substrate-level cooperation between the enzymes. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

biochemistry↗

bioRxiv2020

Improvement of phycocyanobilin synthesis for genetically encoded phytochrome-based optogenetics.

Optogenetics is a powerful technique using photoresponsive proteins, and light-inducible dimerization (LID) systems, an optogenetic tool, allow to manipulate intracellular signaling pathways. One of the red/far-red responsive LID system, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phytochromobilin or phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (synPCB), and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated PhyB-PIF and synPCB into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

synthetic biology↗

bioRxiv2019

Minimal lactazole scaffold for in vitro production of pseudo-natural thiopeptides

Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we established a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis revealed remarkable substrate tolerance of the biosynthetic enzymes, and led to the development of the \"minimal lactazole scaffold\", a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz enabled access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and up to 18 amino acid-long tail regions. Moreover, utilizing genetic code reprogramming, we demonstrated synthesis of pseudo-natural lactazoles containing 4 non-proteinogenic amino acids. This work opens possibilities in exploring novel sequence space of pseudo-natural thiopeptides.

biochemistry↗

bioRxiv2019

Novel syngeneic animal model of tobacco-associated oral cancer reveals the activity of in situ anti-CTLA-4

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity ([≤]20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately reflect the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report the first mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 ([≤]20%) immunotherapy as human HNSCCs. Remarkably, we found that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform for the development of novel immunotherapeutic options for HNSCC.

cancer biology↗

bioRxiv2018

Multiple interfacial hydration of dihydro-sphingomyelin bilayer reported by the Laurdan fluorescence

The hydration properties of the lipid bilayer interface are important for determining membrane characteristics. The hydration properties of different lipid bilayer species were evaluated using the solvent sensitive fluorescence probe, 6-lauroyl-2-dimethylamino naphthalene (Laurdan). Sphingolipids, D-erythro-N-palmitoyl-sphingosylphosphorylcholine (PSM) and D-erythro-N-palmitoyl-dihydrosphingomyelin (DHPSM) showed specific, interfacial hydration properties stemming from their intra- and intermolecular hydrogen bonds. As control, the bilayers of glycerophospholipids, such as 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine (DOPC), were also evaluated. The fluorescence properties of Laurdan in sphingolipids indicated multiple excited states according to the results obtained from the emission spectra, fluorescence anisotropy, and the center of mass spectra during the decay time. Deconvolution of the Laurdan emission spectra into four components enabled us to identify the variety of hydration and the configurational states derived from intermolecular hydrogen bonding in sphingolipids. Particularly, the Laurdan in DHPSM revealed more hydrated properties compared to the case in PSM, even though DHPSM has a higher Tm than PSM. Since DHPSM forms hydrogen bonds with water molecules (in 2NH configurational functional groups) and the different flexibility among the head groups compared with PSM, which could modulate space to retain a high amount of water molecules. The careful analysis of Laurdan such as the deconvolution of emission spectra into four components performed in this study gives the important view for understanding the membrane hydration property.

biophysics↗