RESUMO
Glycogen synthase kinase 3 (GSK3) is an evolutionarily conserved serine/threonine protein kinase in eukaryotes. In plants, the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) functions as a central signaling node through which hormonal and environmental signals are integrated to regulate plant development and stress adaptation. BIN2 plays a major regulatory role in brassinosteroid (BR) signaling and is critical for phosphorylating/inactivating BRASSINAZOLE-RESISTANT 1 (BZR1), also known as BRZ-INSENSITIVE-LONG HYPOCOTYL 1 (BIL1), a master transcription factor of BR signaling, but the detailed regulatory mechanism of BIN2 action has not been fully revealed. In this study, we identified BIL8 as a positive regulator of BR signaling and plant growth in Arabidopsis (Arabidopsis thaliana). Genetic and biochemical analyses showed that BIL8 is downstream of the BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and promotes the dephosphorylation of BIL1/BZR1. BIL8 interacts with and inhibits the activity of the BIN2 kinase, leading to the accumulation of dephosphorylated BIL1/BZR1. BIL8 suppresses the cytoplasmic localization of BIL1/BZR1, which is induced via BIN2-mediated phosphorylation. Our study reveals a regulatory factor, BIL8, that positively regulates BR signaling by inhibiting BIN2 activity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Brassinosteroides , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Brassinosteroides/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
Senescent cells promote cancer development and progression through chronic inflammation caused by a senescence-associated secretory phenotype (SASP). Although various senotherapeutic strategies targeting senescent cells have been developed for the prevention and treatment of cancers, technology for the in vivo detection and evaluation of senescent cell accumulation has not yet been established. Here, we identified activatable fluorescent probes targeting dipeptidylpeptidase-4 (DPP4) as an effective probe for detecting senescent cells through an enzymatic activity-based screening of fluorescent probes. We also determined that these probes were highly, selectively, and rapidly activated in senescent cells during live cell imaging. Furthermore, we successfully visualized senescent cells in the organs of mice using DPP4-targeted probes. These results are expected to lead to the development of a diagnostic technology for noninvasively detecting senescent cells in vivo and could play a role in the application of DPP4 prodrugs for senotherapy.
Assuntos
Senescência Celular , Dipeptidil Peptidase 4 , Corantes Fluorescentes , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidase 4/análise , Animais , Camundongos , HumanosRESUMO
Drought stress is a major threat leading to global plant and crop losses in the context of the climate change crisis. Brassinosteroids (BRs) are plant steroid hormones, and the BR signaling mechanism in plant development has been well elucidated. Nevertheless, the specific mechanisms of BR signaling in drought stress are still unclear. Here, we identify a novel Arabidopsis gene, BRZ INSENSITIVE LONG HYPOCOTYL 9 (BIL9), which promotes plant growth via BR signaling. Overexpression of BIL9 enhances drought and mannitol stress resistance and increases the expression of drought-responsive genes. BIL9 protein is induced by dehydration and interacts with the HD-Zip IV transcription factor HOMEODOMAIN GLABROUS 11 (HDG11), which is known to promote plant resistance to drought stress, in vitro and in vivo. BIL9 enhanced the transcriptional activity of HDG11 for drought-stress-resistant genes. BIL9 is a novel BR signaling factor that enhances both plant growth and plant drought resistance.
RESUMO
9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging.
Assuntos
Análise Espectral Raman , Estrutura Molecular , Vibração , Nitrilas/química , Nitrilas/síntese químicaRESUMO
Iron-sulfur clusters have been reported to catalyze various redox transformations, including the multielectron reduction of CO2 to hydrocarbons. Herein, we report the design and assembly of an artificial [Fe4S4]-containing Fischer-Tropschase relying on the biotin-streptavidin technology. For this purpose, we synthesized a bis-biotinylated [Fe4S4] cofactor with marked aqueous stability and incorporated it in streptavidin. The effect of the second coordination sphere provided by the protein environment was scrutinized by cyclic voltammetry, highlighting the accessibility of the doubly reduced [Fe4S4] cluster. The Fischer-Tropschase activity was improved by chemo-genetic means for the reduction of CO2 to hydrocarbons with up to 14 turnovers.
Assuntos
Proteínas Ferro-Enxofre , Metaloproteínas , Estreptavidina/metabolismo , Dióxido de Carbono/metabolismo , Metaloproteínas/metabolismo , Ferro/metabolismo , Oxirredução , Hidrocarbonetos , Proteínas Ferro-Enxofre/metabolismoRESUMO
Enantioselective C-H amidation offers attractive means to assemble C-N bonds to synthesize high-added value, nitrogen-containing molecules. In recent decades, complementary enzymatic and homogeneous-catalytic strategies for C-H amidation have been reported. Herein, we report on an artificial metalloenzyme (ArM) resulting from anchoring a biotinylated Ir-complex within streptavidin (Sav). The resulting ArM catalyzes the enantioselective amidation of unactivated C(sp3)-H bonds. Chemogenetic optimization of the Ir cofactor and Sav led to significant improvement in both the activity and enantioselectivity. Up to >700 TON and 92% ee for the amidation of unactivated C(sp3)-H bonds was achieved. The single crystal X-ray analysis of the artificial nitrene insertase (ANIase) combined with quantum mechanics-molecular mechanics (QM-MM) calculations sheds light on critical second coordination sphere contacts leading to improved catalytic performance.
RESUMO
By anchoring a metal cofactor within a host protein, so-called artificial metalloenzymes can be generated. Such hybrid catalysts combine the versatility of transition metals in catalyzing new-to-nature reactions with the power of genetic-engineering to evolve proteins. With the aim of gaining better control over second coordination-sphere interactions between a streptavidin host-protein (Sav) and a biotinylated cofactor, we engineered a hydrophobic dimerization domain, borrowed from superoxide dismutase C (SOD), on Sav's biotin-binding vestibule. The influence of the SOD dimerization domain (DD) on the performance of an asymmetric transfer hydrogenase (ATHase) resulting from anchoring a biotinylated Cp*Ir-cofactor - [Cp*Ir(biot-p-L)Cl] (1-Cl) - within Sav-SOD is reported herein. We show that, depending on the nature of the residue at position Sav S112, the introduction of the SOD DD on the biotin-binding vestibule leads to an inversion of configuration of the reduction product, as well as a fivefold increase in catalytic efficiency. The findings are rationalized by QM/MM calculations, combined with X-ray crystallography.
Assuntos
Biotina , Superóxidos , Estreptavidina/química , Estreptavidina/metabolismo , Biotina/química , Biotina/metabolismo , Domínio Catalítico , Hidrogenação , Superóxido Dismutase/metabolismoRESUMO
Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology. By incorporating bio-mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo-genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional-group tolerance. According to QM/MM calculations and X-ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base-meal cofactors for enantioselective organic synthesis.
Assuntos
Hidrogenase , Metaloproteínas , Biotina/química , Estreptavidina/química , Hidrogenase/química , Manganês , Metaloproteínas/química , CatáliseRESUMO
The selective functionalization of sp3 C-H bonds is a versatile tool for the diversification of organic compounds. Combining attractive features of homogeneous and enzymatic catalysts, artificial metalloenzymes offer an ideal means to selectively modify these inert motifs. Herein, we report on a copper(I) heteroscorpionate complex embedded within streptavidin that catalyzes the intramolecular insertion of a carbene into sp3 C-H bonds. Target residues for genetic optimization of the artificial metalloenzyme were identified by quantum mechanics/molecular mechanics simulations. Double-saturation mutagenesis yielded detailed insight on the contribution of individual amino acids on the activity and the selectivity of the artificial metalloenzyme. Mutagenesis at a third position afforded a set of artificial metalloenzymes that catalyze the enantio- and regioselective formation of ß- and γ-lactams with high turnovers and promising enantioselectivities.
Assuntos
Cobre , Metaloproteínas , Catálise , Cobre/química , Metaloproteínas/química , Metano/análogos & derivados , Metano/químicaRESUMO
Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Clorofila/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Virtual reality (VR) is a new methodology for behavioral studies. In such studies, the millisecond accuracy and precision of stimulus presentation are critical for data replicability. Recently, Python, which is a widely used programming language for scientific research, has contributed to reliable accuracy and precision in experimental control. However, little is known about whether modern VR environments have millisecond accuracy and precision for stimulus presentation, since most standard methods in laboratory studies are not optimized for VR environments. The purpose of this study was to systematically evaluate the accuracy and precision of visual and auditory stimuli generated in modern VR head-mounted displays (HMDs) from HTC and Oculus using Python 2 and 3. We used the newest Python tools for VR and Black Box Toolkit to measure the actual time lag and jitter. The results showed that there was an 18-ms time lag for visual stimulus in both HMDs. For the auditory stimulus, the time lag varied between 40 and 60 ms, depending on the HMD. The jitters of those time lags were 1 ms for visual stimulus and 4 ms for auditory stimulus, which are sufficiently low for general experiments. These time lags were robustly equal, even when auditory and visual stimuli were presented simultaneously. Interestingly, all results were perfectly consistent in both Python 2 and 3 environments. Thus, the present study will help establish a more reliable stimulus control for psychological and neuroscientific research controlled by Python environments.
Assuntos
Realidade Virtual , HumanosRESUMO
Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.
RESUMO
Chemical inducers that can control target-protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the retention using selective hook or "RUSH" system for reversible localization change of proteins of interest by addition/washout of small-molecule artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS was restricted by limited solubility in water. Here, we overcame this problem by X-ray crystal structure-guided design of a more soluble ALiS derivative (ALiS-3), which retains sufficient streptavidin-binding affinity for use in the RUSH system. The ALiS-3-streptavidin interaction was characterized in detail. ALiS-3 is a convenient and effective tool for dynamic control of α-mannosidaseâ II localization between ER and Golgi in living cells.
Assuntos
Ligantes , Modelos Moleculares , Ácidos Ftálicos/química , Transporte Proteico/fisiologia , Proteínas/metabolismo , Piridonas/química , Estreptavidina/química , Sulfonamidas/química , Sítios de Ligação , Cristalização , Humanos , Morfolinas/química , Morfolinas/metabolismo , Ácidos Ftálicos/farmacologia , Ligação Proteica , Proteínas/química , Piridonas/metabolismo , Piridonas/farmacologia , Siloxanas/química , Siloxanas/metabolismo , Solubilidade , Estreptavidina/metabolismo , Sulfonamidas/metabolismoRESUMO
Tailored enzymes are crucial for the transition to a sustainable bioeconomy. However, enzyme engineering is laborious and failure-prone due to its reliance on serendipity. The efficiency and success rates of engineering campaigns may be improved by applying machine learning to map the sequence-activity landscape based on small experimental data sets. Yet, it often proves challenging to reliably model large sequence spaces while keeping the experimental effort tractable. To address this challenge, we present an integrated pipeline combining large-scale screening with active machine learning, which we applied to engineer an artificial metalloenzyme (ArM) catalyzing a new-to-nature hydroamination reaction. Combining lab automation and next-generation sequencing, we acquired sequence-activity data for several thousand ArM variants. We then used Gaussian process regression to model the activity landscape and guide further screening rounds. Critical characteristics of our pipeline include the cost-effective generation of information-rich data sets, the integration of an explorative round to improve the model's performance, and the inclusion of experimental noise. Our approach led to an order-of-magnitude boost in the hit rate while making efficient use of experimental resources. Search strategies like this should find broad utility in enzyme engineering and accelerate the development of novel biocatalysts.
RESUMO
Single-molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single-molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water-soluble thiol-reacting probes based on phosphonate-substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single-molecule level, and it is shown that the dissecting alterations of single-molecule esterase activities can serve as an informative platform for activity-based diagnosis.
Assuntos
Esterases , Esterases/análise , Esterases/químicaRESUMO
While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.
RESUMO
Chloroplast development adapts to the environment for performing suitable photosynthesis. Brassinosteroids (BRs), plant steroid hormones, have crucial effects on not only plant growth but also chloroplast development. However, the detailed molecular mechanisms of BR signaling in chloroplast development remain unclear. Here, we identify a regulator of chloroplast development, BPG4, involved in light and BR signaling. BPG4 interacts with GOLDEN2-LIKE (GLK) transcription factors that promote the expression of photosynthesis-associated nuclear genes (PhANGs), and suppresses their activities, thereby causing a decrease in the amounts of chlorophylls and the size of light-harvesting complexes. BPG4 expression is induced by BR deficiency and light, and is regulated by the circadian rhythm. BPG4 deficiency causes increased reactive oxygen species (ROS) generation and damage to photosynthetic activity under excessive high-light conditions. Our findings suggest that BPG4 acts as a chloroplast homeostasis factor by fine-tuning the expression of PhANGs, optimizing chloroplast development, and avoiding ROS generation.
Assuntos
Brassinosteroides , Cloroplastos , Espécies Reativas de Oxigênio , Reguladores de Crescimento de Plantas , Homeostase , Fatores de Transcrição/genéticaRESUMO
Design of experiments (DoE) plays an important role in optimizing the catalytic performance of chemical reactions. The most commonly used DoE relies on the response surface methodology (RSM) to model the variable space of experimental conditions with the fewest number of experiments. However, the RSM leads to an exponential increase in the number of required experiments as the number of variables increases. Herein we describe a Bayesian optimization algorithm (BOA) to optimize the continuous parameters (e.g., temperature, reaction time, reactant and enzyme concentrations, etc.) of enzyme-catalyzed reactions with the aim of maximizing performance. Compared to existing Bayesian optimization methods, we propose an improved algorithm that leads to better results under limited resources and time for experiments. To validate the versatility of the BOA, we benchmarked its performance with biocatalytic C-C bond formation and amination for the optimization of the turnover number. Gratifyingly, up to 80% improvement compared to RSM and up to 360% improvement vs previous Bayesian optimization algorithms were obtained. Importantly, this strategy enabled simultaneous optimization of both the enzyme's activity and selectivity for cross-benzoin condensation.
RESUMO
Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.
Assuntos
Microscopia , Vibração , Animais , Microscopia/métodos , Análise Espectral Raman/métodos , MamíferosRESUMO
Fluorogenic probes are essential tools for real-time visualization of dynamic intracellular processes in living cells, but so far, their design has been largely dependent on trial-and-error methods. Here we propose a quantum chemical calculation-based method for rational prediction of the fluorescence properties of hydroxymethyl rhodamine (HMR)-based fluorogenic probes. Our computational analysis of the intramolecular spirocyclization reaction, which switches the fluorescence properties of HMR derivatives, reveals that consideration of the explicit water molecules is essential for accurate estimation of the free energy difference between the open (fluorescent) and closed (non-fluorescent) forms. We show that this approach can predict the open-closed equilibrium (pKcycl values) of unknown HMR derivatives in aqueous media. We validate this pKcycl prediction methodology by designing red and yellow fluorogenic peptidase probes that are highly activated by γ-glutamyltranspeptidase, without the need for prior synthesis of multiple candidates.