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Yamada, R.

Publications and source records attributed to Yamada, R..

At least 19 recordsLinked to original sources

Disruption of CaMKII biomolecular condensation induces attention-deficit/hyperactivity disorder-like phenotypes

CaMKII is a multifunctional kinase essential for synaptic plasticity and memory formation. While its canonical role involves enzymatic phosphorylation, recent evidence suggests CaMKII also functions through liquid-liquid phase separation (LLPS) with substrate proteins, including GluN2B-containing NMDA receptors. However, the physiological significance remains unclear. Here, we generated CaMKII subtype knock-in (KI) mice carrying a point mutation (I205K) in the hydrophobic pocket, a key interface required for LLPS. These mice exhibited a complete loss of structural long-term potentiation (sLTP) despite normal spine morphology, marked hyperactivity and profound deficits in aversive memory formation. Atomoxetine, an approved attention-deficit/hyperactivity disorder (ADHD) treatment, ameliorated the hyperactive phenotype. Notably, we identified a patient carrying the I205N variant presenting with ADHD and mild intellectual disability, mirroring the behavioral features observed in I205K KI mice. Additional neurodevelopmental disorder-associated variants within the same hydrophobic pocket similarly disrupted LLPS in vitro. Molecular dynamics simulations revealed these variants destabilize the CaMKII-GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKII-mediated phase separation as critical for linking synaptic molecular assembly to cognitive function and provide a unifying molecular basis for synaptic disorganization, hyperactivity, and memory deficits associated with neurodevelopmental disorders. SignificanceCaMKII is essential for synaptic plasticity and memory, yet the contribution of its non-catalytic functions, including liquid-liquid phase separation (LLPS), to brain function remains unclear. Here, we show that disrupting CaMKII LLPS with its synaptic partners impairs synaptic localization, abolishes LTP, and causes profound memory deficits in vivo. A knock-in mouse carrying the I205K mutation showed hyperactivity and impaired learning. This recapitulate the clinical phenotypes of a human patients with the I205N variant, which is associated with ADHD and intellectual disability. Together, these findings establish LLPS as a critical mechanism underlying CaMKII function in the brain and provide a disease-relevant framework for understanding synaptic dysfunction in neurodevelopmental disorders.

neuroscience↗

Synthetic activation of gibberellin signaling reveals spatial coordination of root growth

Gibberellins (GAs) influence cell division and elongation, profoundly shaping plant architecture and yield. GA perception occurs when bioactive GAs bind the receptor GID1, promoting DELLA degradation and activating transcriptional programs. While GA signaling in the root endodermis is essential for promoting root elongation, functions of other layers in spatial control of GA responses have not been explored. Here, we developed a synthetic GA (sGA) that does not bind endogenous GID1, together with a modified GID1 (mGID1) engineered to selectively recognize sGA, enabling cell-specific activation of GA signaling in vivo. Using this system in Arabidopsis, we demonstrate that coordinated action of GA signaling in the endodermis, epidermis, and other layers is required for full root elongation. Moreover, cell type-specific expression of GA biosynthetic enzymes indicates the existence of intercellular GA transport. The sGA-mGID1 system provides a versatile platform for spatially precise reprogramming of hormone signaling, enabling synthetic control of developmental processes such as root-shoot growth balance, thereby advancing applications in plant synthetic biology and sustainable crop improvement.

plant biology↗

Synaptic mitochondrial oxidative stress drives individual variability in age-related cognitive decline in mice

Aging leads to cognitive decline with considerable individual variability, yet the biological mechanisms remain unclear. Here we performed ultrastructural and proteomic analyses of the medial prefrontal cortex (mPFC) alongside behavioral assessments of attentional set shifting in mice across age groups. Although reduced synaptic density did not consistently lead to cognitive decline, proteomic analyses of synaptosomes and whole tissue revealed that the molecular signatures associated with individual variability in cognitive decline were distinct from those associated with chronological aging, and that synaptic mitochondria and their proteins were more abundant in aged mice with greater cognitive decline. Moreover, treatment with the mitochondria-targeted antioxidant MitoQ reduced the abundance of synaptic mitochondrial proteins, including pro-apoptotic proteins, and mitigated age-related cognitive decline. These findings demonstrate that synaptic mitochondrial oxidative stress in the mPFC, distinct from chronological age-related processes, contributes to individual variability in age-related cognitive decline and offers a potential target for prevention and intervention.

animal behavior and cognition↗

Aging Converts Microglial Repopulation into Maladaptive Reprogramming that Exacerbates Cognitive Deficits

Background and PurposeAging has been associated with neuroinflammation and cognitive decline. Microglial repopulation after pharmacological depletion has been proposed as a strategy to alleviate microglia-driven neuropathology. However, the effects of microglial repopulation on age-related cognitive decline remain largely unexplored. In the present study, we examined how microglial repopulation affects the transcriptomic profiles of cortical microglia and the decline in prefrontal cortex-dependent cognitive function in aged mice. Experimental ApproachYoung and aged male C57BL/6J mice were used in this study. Microglial depletion was induced by treatment with PLX3397, a CSF1R inhibitor, followed by microglial repopulation after drug withdrawal. Microglia isolated from the entire cerebral cortex were subjected to bulk RNA-sequencing analysis. The visual discrimination test followed by the response direction test was conducted to assess sensory learning and attentional set shifting abilities, respectively. Key ResultsRepopulated cortical microglia in aged, but not young, mice exhibited aberrant gene expression patterns, including reduced expression of microglial identity genes, reprogramming of innate and adaptive immune-related gene expression, and derepression of neuronal gene expression. Furthermore, microglial repopulation selectively impaired visual discrimination learning and attentional set shifting in aged mice. Conclusion and ImplicationsThese findings demonstrate that aging converts microglial repopulation into maladaptive reprogramming that exacerbates cognitive decline, possibly through aberrant gene expression programs. Therefore, the therapeutic potential of this approach must be carefully evaluated with respect to specific behavioral domains and disease contexts, including aging.

neuroscience↗

Genotype-first approach revealed unrecognized breed-specific genetic diseases in dogs

Numerous dogs are affected by genetic diseases because of their unique breeding situation. The identification of pathogenic variants in dogs not only has veterinary benefits but also leads to the development of new human disease models. However, fewer pathogenic variants have been identified in dogs than in humans, and standard genetic approaches, such as large-scale case/control studies and family analyses, often fail to identify novel causative variants in dogs. To address this, we applied a genotype-first approach by developing a multiplex polymerase chain reaction (PCR)-based targeted sequencing method for dogs, analyzing 203 genes--most of which are related to human disease--from 6,333 dogs collected from a veterinary hospital, and characterizing pathogenic variant carriers. In total, 120 pathogenic variants in 83 genes were identified. Frequent pathogenic variants were enriched in specific dog breeds. Dogs homozygous for a pathogenic CHEK2 variant, frequently identified in French Bulldogs (5.74%, homozygous), showed a five-fold increased risk of cancer (P = 3.45 x 10-2). A pathogenic NOD2 variant was detected in half of the Italian Greyhounds. However, their medical records did not document the typical clinical symptoms observed in humans with NOD2 deficiency. Some pathogenic variants were present in three or fewer dogs, and 11.0% of these dogs exhibited symptoms potentially associated with gene defects. These rare variants could become common within a short period due to the canine breeding system to induce breed-specific genetic diseases. These findings suggest that the genotype-first approach successfully identified unrecognized breed-specific genetic diseases. Further characterization of dogs with pathogenic variants enable to define clinical diagnosis, providing an important spontaneous model for human genetic diseases.

genomics↗

Multiphase separation in postsynaptic density regulated by membrane geometry via interaction valency and volume

Biomolecular condensates are found at various cellular locations, nucleus, cytoplasm, and membrane. These condensates often contain multiple components and can separate into multiple phases with various morphologies such as core-shell droplets, implicating functional roles. Demixing and arrangements of condensates are determined by competitive interactions and their locations. Recent studies reported a puzzling multiphase morphology in postsynaptic density components: AMPA-receptor, NMDA-receptor, PSD-95, and CaMKII. The multiphase morphology appears reversed when transitioning from the solution to the membrane. Using this system as a model, we study the multiphase behavior of condensates in solution (3D) and domain formation on and beneath the membrane (2D) and elucidate molecular mechanisms behind the puzzle. Our simulations reproduce the core-shell structure in 3D in vitro solution, where AMPA-receptor/PSD-95 form the core and NMDA-receptor/CaMKII form the shell, triggered by CaMKII activation. Then, we obtain a reversed morphology on the membrane. This reversal is primarily driven by CaMKIIs high valency and large volume. We find that, in solution, CaMKIIs non-specific volume interaction dominate, while on the membrane, specific multivalent interactions overcome the excluded volume interaction of CaMKII. The layered structures of receptors and CaMKIIs reduce the excluded volume effects of CaMKII on receptors, making the multivalent interaction dominant. These findings highlight the differences between condensate formation in solution and membrane domain formation, modulated by their layered arrangement. SignificanceMany proteins form condensates in cells, which are often further separated into two or more phases, implicating functional roles. Recent experiments on postsynaptic density proteins provide an interesting puzzle on a multiphasic morphology; while they form core-shell droplets with AMPA-receptor at the core and NMDA receptor in the shell in soluble constructs, the multiphase morphology is reversed when they form nanodomains on the membrane. This study identifies the mechanism behind the puzzle by using computer simulations with a mesoscale model. While active CaMKII, an inducer of the multiphase morphology, has dominant non-specific volume interactions in solution, its specific domain-domain multivalent interaction overcomes the volume interaction under the membrane with their layered arrangement.

biophysics↗

Improvement of D-lactic acid production from methanol by metabolically engineered Komagataella phaffii via ultra-violet mutagenesis

Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. Transcriptome analysis revealed 167 differentially expressed genes in DLac_Mut2_221, comprising 104 upregulated and 63 downregulated genes. These results suggest that the improvement in D-lactic acid production from methanol in the mutant strain involves three important mechanisms: (1) avoiding excessive formaldehyde accumulation, (2) activating the glyoxylate pathway, and (3) reducing the expression of O-glycosylated proteins required for cell wall stability. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.

bioengineering↗

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

MyD88 plays a pivotal role in Toll-like receptor (TLR) and interleukin-1 family signaling through its oligomerization upon receptor activation, leading to downstream protein recruitment. The Toll/interleukin-1 receptor domain of MyD88 (TIRMyD88) is responsible for this receptor-mediated oligomerization, but the detailed mechanism involved remains elusive. We investigated the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy revealed that tandemly arrayed TIRMyD88 subunits formed an antiparallel double-stranded filament that could further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro was markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captured the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. Based on these results, a novel regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step was revealed.

biophysics↗

Co-utilization of microalgae and heterotrophic microorganisms improves wastewater treatment efficiency

Wastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms is expected to be useful under atmospheric dilute carbon dioxide conditions. In this study, we investigated the combination of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Furthermore, to elucidate the cause of the changes in wastewater treatment efficiency in the co-culture system, changes in gene expression were revealed through transcriptome analysis. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination of Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of TOC (Total organic carbon), PO43-, and NH4+ reached 80%, 93%, and 63%, respectively, after 18 h of treatment. Transcriptome analysis revealed that the combined wastewater treatment altered the expression of 1371 and 692 genes in C. reinhardtii and S. cerevisiae, respectively. The genes upregulated in C. reinhardtii included those related to molecular and ion transport. Genes upregulated in S. cerevisiae included those related to cell protection from various types of damage and stress. To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. As both the green alga C. reinhardtii and the yeast S. cerevisiae are highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant.

bioengineering↗

Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations.

Classical Anfinsen's dogma states that a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, recent advances have revealed that single amino acid sequences can fold into two or more conformations. Here, we propose a novel approach to design a protein that adopts interconvertible alternative functional conformations, termed "seesaw" protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal {beta}-strand and DHFR lacking the N-terminal {beta}-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analysis, including AFM imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. In addition, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a new direction for creating artificial proteins with on-off behaviors.

biochemistry↗

Development of a metabolic engineering technology to simultaneously suppress the expression of multiple genes in yeast and application in carotenoid production

In yeast metabolic engineering, there is a need for technologies that simultaneously suppress and regulate the expression of multiple genes and improve the production of target chemicals. In this study, we aimed to develop a novel technology that simultaneously suppresses the expression of multiple genes by combining RNA interference with global metabolic engineering technology (GMES). Furthermore, using {beta}-carotene as the target chemical, we attempted to improve its production by using the technology. First, we developed a technology to suppress the expression of the target genes with various strengths using RNA interference. Using this technology, total carotenoid production was successfully improved by suppressing the expression of a single gene out of 10 candidate genes. Then, using this technology, RNA interference strain targeting 10 candidate genes for simultaneous suppression was constructed. The total carotenoid production of the constructed RNA interference strain was 1.7 times compared with the parental strain. In the constructed strain, the expression of eight out of the 10 candidate genes was suppressed. We developed a novel technology that can simultaneously suppress the expression of multiple genes at various intensities and succeeded in improving carotenoid production in yeast. Because this technology can suppress the expression of any gene, even essential genes, using only gene sequence information, it is considered a useful technology that can suppress the formation of by-products during the production of various target chemicals by yeast.

bioengineering↗

Improvement of cell growth in green algae Chlamydomonas reinhardtii through co-cultivation with yeast Saccharomyces cerevisiae

Biological fixation methods have attracted considerable attention because they can be applied for the fixation of dilute CO2 in the atmosphere. Co-cultivation of certain microalgae with heterotrophic microorganisms can increase the growth potential of microalgae under dilute CO2 conditions. The objective of this study was to determine the culture conditions under which the growth potential of green algae Chlamydomonas reinhardtii is enhanced by co-culturing with the yeast Saccharomyces cerevisiae, and to identify the cause of the enhanced growth potential using transcriptome analysis. When C. reinhardtii and S. cerevisiae were co-cultured with an initial green algae to yeast inoculum ratio of 1:3, the cell concentration of C. reinhardtii reached 133 x 105 cells/mL on day 18 of culture, which was 1.5 times higher than that of the monoculture. Transcriptome analysis revealed that the expression levels of 363 green algae and 815 yeast genes were altered through co-cultivation. These include genes responsible for ammonium transport and CO2 enrichment mechanism in green algae and the genes responsible for glycolysis and stress responses in yeast. In conclusion, we identified the culture condition suitable for the co-cultivation of C. reinhardtii and S. cerevisiae. In addition, we discuss the cause of the increased growth potential of C. reinhardtii based on transcriptome analysis data. Although further studies are needed to elucidate the full impact of microbial interactions in C. reinhardtii and S. cerevisiae co-cultures, the findings of this study represent an important first step toward achieving this goal.

bioengineering↗

Relationship Between Domain and Function of the Yeast RNase T2, Rny1p, Which Mediates rRNA Degradation upon Starvation

RNase T2 is ubiquitous across diverse organisms, playing essential roles despite its simple enzymatic activity. In Saccharomyces cerevisiae, RNase T2, known as Rny1p, localizes in vacuoles and mediates rRNA degradation during autophagy of ribosomes. In this study, we elucidated novel aspects of ribosome degradation mechanisms and the function of Rny1p. First, we discovered that most ribosomes are degraded by selective autophagy, where Rsa1p is the specific receptor of ribosomes to be degraded. Complex structure prediction suggested that Rsa1p also interacts with Atg8p. Furthermore, we observed that the accumulation of rRNA in vacuoles, due to the lack of Rny1p, leads to a decrease in bulk autophagic activity. This decrease in autophagic activity may explain the inability of Rny1p-deficient strains to adapt to starvation conditions. Second, our structural prediction and biochemical analyses indicate that a C-terminal extension, characteristic in fungal RNase T2 including Rny1p, is not necessary for rRNA degradation but for anchoring to the cell wall. Together with molecular phylogenetic analysis, a species-specific role of RNase T2 conferred by the C-terminal extension is suggested.

molecular biology↗

Postsynaptic protein assembly in three- and two-dimensions studied by mesoscopic simulations

Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D) or beneath the membrane (2D), the underlying difference between the two has not been well clarified. To compare the phase behaviors in 3D and 2D, postsynaptic density (PSD) serves as a model system. PSD is a protein condensate located under the postsynaptic membrane that influences the localization of glutamate receptors and thus contributes to synaptic plasticity. Recent in vitro studies have revealed the formation of droplets of various soluble PSD proteins via liquid-liquid phase separation. However, it is unclear how these protein condensates are formed beneath the membrane and how they specifically affect the localization of glutamate receptors in the membrane. In this study, focusing on the mixture of a glutamate receptor complex, AMPAR-TARP, and a ubiquitous scaffolding protein, PSD-95, we constructed a mesoscopic model of protein-domain interactions in PSD and performed comparative molecular simulations. The results showed a sharp contrast in the phase behaviors of protein assemblies in 3D and those under the membrane (2D). A mixture of a soluble variant of the AMPAR-TARP complex and PSD-95 in the 3D system resulted in a phase-separated condensate, which was consistent with the experimental results. However, with identical domain interactions, AMPAR-TARP embedded in the membrane formed clusters with PSD-95, but did not form a stable separated phase. Thus, the cluster formation behaviors of PSD proteins in the 3D and 2D systems were distinct. The current study suggests that, more generally, stable phase separation can be more difficult to achieve in and beneath the membrane than in 3D systems. SIGNIFICANCESynaptic plasticity is a key factor in memory and learning. Upon learning, protein condensates that form beneath the postsynaptic membrane are known to change their nature. Recent studies have suggested that condensate formation is related to liquid-liquid phase separation based on in vitro experiments of soluble parts. However, the phase behavior can be strongly dependent on physical dimensions. The mechanism by which condensate grows beneath the membrane is not well characterized. Taking advantage of the ease of systematic comparison using computer simulations, we investigated the phase behaviors of postsynaptic protein assemblies in 3D and 2D systems. The results revealed that even when a 3D system exhibited clear phase separation, the corresponding 2D system did not exhibit it stably.

biophysics↗

Immunostimulatory effects of Bacillus coagulans SANK70258

Specific intestinal bacteria modulate immunoresponses through various pathways, and several probiotic bacteria have been identified as immunostimulants by screening. In the present study, we evaluated the immunomodulating effects of Bacillus coagulans SANK70258 (B. coagulans SANK70258), a spore-forming and lactic acid-producing bacterium usable as food supplement for human and animals. We found that treatment of mouse splenocytes with {gamma}-ray irradiated B. coagulans SANK70258 induced high amount of IFN-{gamma} in comparison with 7 kinds of typical lactic acid bacteria. Further analyses using splenocytes revealed that NK cell is a major source of IFN-{gamma}, and B. coagulans SANK70258-induced IFN-{gamma} production was inhibited by neutralization of IL-12 or IL-23, depletion of CD11c+ cells, and inhibition of NF{kappa}B. B. coagulans SANK70258 also induced release of IFN-{gamma} from activated CD8+ T cells, and increased expression of chemokine receptors in CD8+ T cells. B. coagulans SANK70258-treatment induced production of cytokines from bone marrow-derived dendritic cells, which is reduced by knockdown of Tlr2 and Nod2. B. coagulans SANK70258-treatment also induced IgA production from Peyers patch cells with high level among tested lactic bacteria. The oral intake of {gamma}-ray irradiated B. coagulans SANK70258 significantly increased intestinal IgA levels and IgA-expressing B cells in the Peyers patch of mice. Taken together, we conclude that B. coagulans SANK70258 possesses high activity as immunostimulant inducing production of IFN-{gamma} and IgA.

immunology↗

Circadian ribosome profiling reveals a role for the Period2 upstream opening reading frame in sleep

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered post-transcriptionally. We used ribosome profiling to examine post-transcriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA post-transcriptionally, which can impact physiological rhythms and sleep. Significance StatementPeriod (Per) is an iconic gene in the field of circadian rhythms since its discovery in 1971 by Seymour Benzer and Ronald Konopka in fruit flies. The inhibitory feedback loop of PER protein drives circadian rhythms. We show that Per2 is regulated by an upstream open reading frame (uORF) in the 5 untranslated region of Period2 mRNA. Mutation of the Per2 uORF altered the amplitude of luciferase reporter expression in well-characterized cell culture models. Per2 uORF mutant mice had significantly elevated Per2 mRNA levels and exhibited sleep loss, particularly during light-to-dark and dark-to-light transitions, which suggests a role for uORFs in modulating molecular and physiological circadian rhythms.

cell biology↗

Integrated Analysis of Cell Shape and Movement in Moving Frame

The cell movement and morphological change are two interrelated cellular processes. The integrated analysis is needed to explore the relationship between them. However, it has been challenging to investigate them as a whole. The cell trajectory can be described by its speed, curvature, and torsion. On the other hand, the 3-Dimensional (3D) cell shape can be studied by using a shape descriptor such as Spherical harmonic (SH) descriptor which is an extension of Fourier transform in 3D space. To integrate these shape-movement data, we propose a method to use a parallel-transport (PT) moving frame as the 3D-shape coordinate system. This moving frame is purely determined by the velocity vector. On this moving frame, the movement change will influence the coordinate system for shape analysis. By analyzing the change of the SH coefficients over time in the moving frame, we can observe the relationship between shape and movement. We illustrate the application of our approach using simulational and real datasets in this paper.

bioinformatics↗

A super sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

Auxin-Inducible Degron (AID) technology enables conditional depletion of targeted proteins. However, the applicability of the AID in vertebrate cells has been limited due to cytotoxicity caused by high auxin concentrations. Here, we establish an improved AID system using an engineered orthogonal auxin-TIR1 pair, which exhibits over 1,000 times stronger binding. With ~1,000-fold less auxin concentration, we achieved to generate the AID-based knockout cells in various human and mouse cell lines in a single transfection.

bioengineering↗