Investigating Epidermal Growth Factor Receptor Trafficking with High-Resolution Enzymatic Protein-Tagging and Transmission Electron Microscopy.

Enzymatic ascorbate peroxidase (APEX) tagging allows for high-resolution, three-dimensional protein distribution analyses in cells and tissues. This chapter describes the application of APEX-tagging to visualize the trafficking of the epidermal growth factor receptor (EGFR) during epidermal growth factor-mediated receptor activation. Here, we describe the preparation of cells, methods to validate the stimulation of the EGFR, and visualization of the APEX-resolved distribution of the EGFR in the transmission electron microscope.

Novel insight into functions of ascorbate peroxidase in higher plants: More than a simple antioxidant enzyme.

As plants are sessile organisms, they are inevitably exposed to a variety of environmental stimuli that trigger rapid changes in the generation and disposal of reactive oxygen species such as hydrogen peroxide (H2O2). A major H2O2 scavenging system in plant cells is the ascorbate-glutathione cycle, in which ascorbate peroxidase (APX) catalyzes the conversion of H2O2 into water employing ascorbate as specific electron donor. In higher plants, distinct APX isoforms can occur in multiple subcellular compartments, including chloroplasts, mitochondria, and peroxisomes and the cytosol, to modulate organellar and cellular levels of H2O2. It is well established that APX plays crucial roles in protecting plant cells against diverse environmental stresses, as well as in plant growth and development. Apart from ascorbate, recently, APXs have been found to have a broader substrate specificity and possess chaperone activity, hence participating various biological processes. In this review, we describe the antioxidant properties of APXs and highlight their novel roles beyond 'ascorbate peroxidases'.

Proteomic Mapping by APEX2-Catalyzed Proximity Labeling in Saccharomyces cerevisiae Semipermeabilized Cells.

Enzyme-catalyzed proximity labeling (PL) has proven to be a valuable resource for proteomic mapping of subcellular compartments and protein networks in living cells. We have used engineered ascorbate peroxidase (APEX2) to develop a PL approach for budding yeast. It is based on semipermeabilized cells to overcome poor cellular permeability of the APEX2 substrate biotin-phenol and difficulties in its delivery into the cell. The use of semipermeabilized cells has several advantages, in particular the avoidance of generating fragile spheroplasts and the opportunity of employing cells from a glucose-containing medium for APEX2 tagging. In this protocol we describe how to perform a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach that allows to map an open cellular compartment like the yeast nucleus. In particular, we focus on the proteomic sample preparation and provide instructions to achieve high-resolution mapping of a subcellular yeast proteome.

APEX Proximity Labeling of Stress Granule Proteins.

Ascorbate peroxidase (APEX)-catalyzed proximity labeling has been recently established as a robust approach to uncover localized protein environments and transient protein-protein interactions occurring across mammalian cells. This molecular tool enables improved identification of individual proteins localized to and involved in specific cellular and subcellular pathways and functions. Engineering of an APEX2 fusion protein into the endogenous loci of proteins of interest enables directed biotinylation of neighboring polypeptides and mRNAs. This results in identification of subcellular and context-dependent proteomes or transcriptomes via quantitative mass spectrometry or RNA sequencing, respectively. Here, we describe the utility of APEX-mediated proximity labeling to recover components of stress granules (SGs) by endogenous tagging of well-established SG-associated proteins.

Interrogating Kinase-Substrate Relationships with Proximity Labeling and Phosphorylation Enrichment.

Kinases govern many cellular responses through the reversible transfer of a phosphate moiety to their substrates. However, pairing a substrate with a kinase is challenging. In proximity labeling experiments, proteins proximal to a target protein are marked by biotinylation, and mass spectrometry can be used for their identification. Here, we combine ascorbate peroxidase (APEX) proximity labeling and a phosphorylation enrichment-based workflow, Phospho-APEX (pAPEX), to rapidly identify phosphorylated and biotinylated neighbor proteins which can be considered for candidate substrates. The pAPEX strategy enriches and quantifies differences in proximity for proteins and phosphorylation sites proximal to an APEX2-tagged kinase under the kinase "ON" and kinase "OFF" conditions. As a proof of concept, we identified candidate substrates of MAPK1 in HEK293T and HCT116 cells and candidate substrates of PKA in HEK293T cells. In addition to many known substrates, C15orf39 was identified and confirmed as a novel MAPK1 substrate. In all, we adapted the proximity labeling-based platform to accommodate phosphorylation analysis for kinase substrate identification.

Atomically Dispersed Cu Nanozyme with Intensive Ascorbate Peroxidase Mimic Activity Capable of Alleviating ROS-Mediated Oxidation Damage.

Ascorbate peroxidase (APX) as a crucial antioxidant enzyme has drawn attentions for its utilization in preventing cells from oxidative stress responses by efficiently scavenging H2 O2 in plants. For eliminating the specific inactivation of natural APXs and regulating the catalytic activity, single-atom nanozymes are considered as promising classes of alternatives with similar active sites and maximal atomic utilization efficiency to natural APXs. Herein, graphitic carbon nitride (g-C3 N4 ) anchored with isolated single copper atoms (Cu SAs/CN) is designed as an efficient nanozyme with intrinsic APX mimetic behavior. The engineered Cu SAs/CN exhibits comparable specific activity and kinetics to the natural APXs. Based on the density functional theory (DFT), Cu-N4 moieties in the active center of Cu SAs/CN are determined to exert such favorable APX catalytic performance, in which the electron transfer between Cu and coordinated N atoms facilitates the activation and cleavage of the adsorbed H2 O2 molecules and results in fast kinetics. The constructed Cu SAs/CN nanozyme with superior APX-like performance and high biocompatibility can be applied for effectively protecting the H2 O2 -treated cells against oxidative injury in vitro. These findings report the single-atom nanozymes as a successful paradigm for guiding nanozymes to implement APX mimetic performance for reactive oxygen species-related biotherapeutic.

NRVS investigation of ascorbate peroxidase compound II: Observation of Iron(IV)oxo stretching.

The protonation state of ascorbate peroxidase compound II (APX-II) has been a subject of debate. A combined X-ray/neutron crystallographic study reported that APX-II is best described as an iron(IV)hydroxide species with an FeO distance of 1.88 Å (Kwon, et al. Nat Commun2016, 7, 13,445), while X-ray absorption spectroscopy (XAS) experiments (utilizing extended X-ray absorption fine structure (EXAFS) and pre-edge analyses) indicate APX-II is an authentic iron(IV)oxo species with an FeO distance 1.68 Å (Ledray, et al. Journal of the American Chemical Society2020,142, 20,419). Previous debates concerning ferryl protonation states have been resolved through the application of Badger's rule, which correlates FeO bond distances with FeO vibrational frequencies. To obtain the required vibrational data, we have collected Nuclear Resonance Vibrational Spectroscopy (NRVS) data for APX-II. We observe a broad vibrational feature in the range associated with iron(IV)oxo stretching (700-800 cm-1). This feature appears to have two peaks at 732 cm-1 and 770 cm-1, corresponding to FeO distances of 1.69 and 1.67 Å, respectively. The broad vibrational envelope and the presence of multiple resonances could reflect a distribution of hydrogen bonding interactions within the active-site pocket.

Carboxymethyl cellulose coating delays chilling injury development and maintains eating quality of 'Kinnow' mandarin fruits during low temperature storage.

The application of edible coatings is an efficient way to reduce mass loss and to conserve the quality of a coated fresh produce during postharvest storage. In the present research, the impact of carboxymethyl cellulose [CMC (1%] coating was studied on 'Kinnow' mandarins during cold storage at 5 ± 1 °C for 30 days. Results showed that CMC treatment substantially suppressed chilling injury symptoms, disease incidence, fresh weight loss, malondialdehyde content, hydrogen peroxide and electrolyte leakage compared with control. The CMC coated 'Kinnow' mandarins showed markedly higher ascorbate peroxidase, peroxidase, superoxide dismutase and catalase enzyme activities compared to control. The treatment of 'Kinnow' mandarins with CMC also suppressed the increase in total soluble solids, ripening index and showed substantially higher titratable acidity, ascorbic acid, total phenolics content, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity along with better sensory quality in contrast with uncoated fruits. In conclusion, CMC coating could be an effective approach for the chilling injury reduction and quality maintenance of harvested 'Kinnow' mandarin fruits during cold storage.

New Heterocyclic Compounds: Synthesis, Antioxidant Activity and Computational Insights of Nano-Antioxidant as Ascorbate Peroxidase Inhibitor by Various Cyclodextrins as Drug Delivery Systems.

AIM: The synthesis of seven new antioxidant agents based on the combination of thiazole, pyridine, triazole and pyrazole moieties. The studies of their antioxidant activity using DPPH reduction method. The DFT analysis of the 7 ligands. The docking study was also investigated. The better binding affinity with α-cyclodextrin as best drug delivery system. BACKGROUND: The screening of new antioxidant compounds and find the good mechanism for binding sites, with correlating between experience and computer theory. OBJECTIVES: The DFT analysis of the 7 synthesized ligands.The docking study was also investigated by using the amino acids Ala167 and Arg172. The better binding affinity with α-cyclodextrin as best drug delivery system. METHODS: The studies of their antioxidant activity using DPPH reduction method. RESULTS: Chemistry: synthesis of 7 ligands by condensation reaction with 89% yield. Antioxidant activities using DPPH reduction with a good value IC50=13.05 ± 3.73 µg/ml. Using DFT (EHOMO and ELUMO) and Docking APX with the amino acids Ala167 and Arg172 compared to the ascorbic acid. Correlation between all these properties. α-cyclodextrin as best drug delivery system (better binding affinity than caffeic acid). CONCLUSION: For the drug delivery study, The ACD is best system for all the compounds due to the smallest cavity size which makes the binding affinities favorable and possible to prepare prospective nano-antioxidants.

Tragacanth gum coating modulates oxidative stress and maintains quality of harvested apricot fruits.

Tragacanth gum is a polysaccharide-based complex with a good coating property. However, its use in postharvest storage of fresh fruits and vegetables is very limited. In the current work, the effect of tragacanth gum (1%) was investigated on postharvest quality of apricot fruits during storage at 20 ± 1 °C for 8 days. Apricot fruits coated with tragacanth gum coating showed significantly reduced weight loss, decay and electrolyte leakage, hydrogen peroxide and malondialdehyde production. Tragacanth gum coating suppressed increase in total soluble solids and showed higher titratable acidity compared with control. The coated fruits had higher total phenolics and ascorbic acid along with greater 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in contrast with control. Postharvest application of tragacanth gum coating maintained markedly higher activities of ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) enzymes activities as compared with control. In addition, tragacanth gum application suppressed polygalacturonase (PG), pectin methylesterase (PME), and cellulase (CX) enzymes activities, thereby suppressed softening of apricot fruits. Sensory quality attributes such as taste, juiciness, aroma, appearance, and overall acceptability were also significantly higher in tragacanth gum coated fruits compared with control. In conclusion, tragacanth gum coating could be considered suitable for postharvest quality conservation of apricot fruits.

Proximity-CLIP Provides a Snapshot of Protein-Occupied RNA Elements at Subcellular Resolution and Transcriptome-Wide Scale.

The distribution of messenger RNAs (mRNAs) to specific subcellular locations has been studied for the past two decades. Technically, studies of RNA localization are lagging those related to protein localization. Here we provide a detailed protocol for Proximity-CLIP, a method recently developed by our group, that combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA cross-linking to simultaneously profile the proteome including RNA-binding proteins (RBPs) and the RBP-bound transcriptome in any given subcellular compartment. The approach is fractionation independent and also enables studying localized RNA-processing intermediates, as well as the identification of regulatory cis-acting elements on RNAs occupied by proteins in a cellular compartment-specific manner.

Molecular characterization of ascorbate peroxidase (APX) and APX-related (APX-R) genes in Triticum aestivum L.

Ascorbate peroxidases (APXs) are heme-dependent H2O2 scavenging enzymes involved in myriad biological processes. Herein, a total of 21 TaAPX and six TaAPX-R genes were identified from the A, B and D sub-genomes of Triticum aestivum L. The occurrence of three paralogous gene pairs with unequal evolutionary rate suggested functional divergence. The phylogenetic analysis formed four distinct clades having conserved gene and protein architecture, and sub-cellular localization. The tertiary structure analysis revealed the presence of helices and coils and residues involved in ligand binding. Transcriptional profiling of each TaAPX and TaAPX-R gene suggested their specific role during development and stress response. Modulated transcript expression and APX enzyme activity during various stress conditions indicated their role in stress response. Interaction analyses suggested their association with other genes, miRNAs and various legends. The present study reported numerous features of these genes, and may provide a platform for their detailed functional characterization in future studies.

A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast.

The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

Visualizing the protons in a metalloenzyme electron proton transfer pathway.

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling.

Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.

APEX2-mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells.

Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.

APEX, a Master Key To Resolve Membrane Topology in Live Cells.

Determining the topology of the membrane proteome is fundamental for understanding its function at the membrane. However, conventional methods involving test tube reactions often lead to unreliable results, which do not accurately reflect membrane topology under physiological conditions, as perturbations occur during lysis. In this Perspective, we introduce a new method using engineered ascorbate peroxidase (APEX) for revealing membrane topological information in live cells without performing complicated sample preparation. We also discuss several examples of clearly resolved membrane topologies of various important mitochondrial proteins (e.g., LETM1, NDUFB10, MCU, SFXN1, and EXD2) and endoplasmic reticulum proteins (e.g., HMOX1) determined by using APEX-based methods.

APEX Proximity Labeling as a Versatile Tool for Biological Research.

Most proteins are specifically localized in membrane-encapsulated organelles or non-membrane-bound compartments. The subcellular localization of proteins facilitates their functions and integration into functional networks; therefore, protein localization is tightly regulated in diverse biological contexts. However, protein localization has been mainly analyzed through immunohistochemistry or the fractionation of subcellular compartments, each of which has major drawbacks. Immunohistochemistry can examine only a handful of proteins at a time, and fractionation inevitably relies on the lysis of cells, which disrupts native cellular conditions. Recently, an engineered ascorbate peroxidase (APEX)-based proximity labeling technique combined with mass spectrometry was developed, which allows for temporally and spatially resolved proteomic mapping. In the presence of H2O2, engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are subsequently enriched by streptavidin and identified by mass spectrometry. This permits the sensitive and efficient labeling of proximal proteins around locally expressed APEX. Through the targeted expression of APEX in the subcellular region of interest, proteomic profiling of submitochondrial spaces, the outer mitochondrial membrane, the endoplasmic reticulum (ER)-mitochondrial contact, and the ER membrane has been performed. Furthermore, this method has been modified to define interaction networks in the vicinity of target proteins and has also been applied to analyze the spatial transcriptome. In this Perspective, we provide an outline of this newly developed technique and discuss its potential applications to address diverse biological questions.

Comparative analysis of ascorbate peroxidases (APXs) from selected plants with a special focus on Oryza sativa employing public databases.

Reactive oxygen species (ROS) are produced by plants. Hydrogen peroxide (H2O2) is one important component of ROS and able to modulate plant growth and development at low level and damage plant cells at high concentrations. Ascorbate peroxidase (APX) shows high affinity towards H2O2 and plays vital roles in H2O2-scavenging. In order to explore the differences of APXs from selected plant species, bioinformatics methods and public databases were used to evaluate the physicochemical properties, conserved motifs, potential modifications and cis-elements in all the APXs, and protein-protein network and expression profiles of rice APXs. The results suggested that APXs in the selected plant species showed high evolutionary conservation and were able to divide into seven groups, group I to VII. Members in the groups contained abundant phosphorylation sites. Interestingly, group I and VII had only PKC site. Additionally, promoters of the APXs contained abundant stress-related cis-elements. APXs in rice plant were able to interact with dehydroascorbate reductase 2. The eight APXs expressed differently in root, leaf, panicle, anther, pistil and seed. Drought, Pi-free, Cd and Xanthomonas oryzae pv. oryzicola B8-12 treatments were able to significantly alter the expression profiles of rice APXs. This study increases our knowledge to further explore functions and mechanisms of APXs and also guides their applications.

Isolation, purification and characterization of an ascorbate peroxidase from celery and overexpression of the AgAPX1 gene enhanced ascorbate content and drought tolerance in Arabidopsis.

BACKGROUND: Celery is a widely cultivated vegetable abundant in ascorbate (AsA), a natural plant antioxidant capable of scavenging free radicals generated by abiotic stress in plants. Ascorbate peroxidase (APX) is a plant antioxidant enzyme that is important in the synthesis of AsA and scavenging of excess hydrogen peroxide. However, the characteristics and functions of APX in celery remain unclear to date. RESULTS: In this study, a gene encoding APX was cloned from celery and named AgAPX1. The transcription level of the AgAPX1 gene was significantly upregulated under drought stress. AgAPX1 was expressed in Escherichia coli BL21 (DE3) and purified. The predicted molecular mass of rAgAPX1 was 33.16 kDa, which was verified by SDS-PAGE assay. The optimum pH and temperature for rAgAPX1 were 7.0 and 55 °C, respectively. Transgenic Arabidopsis hosting the AgAPX1 gene showed elevated AsA content, antioxidant capacity and drought resistance. Less decrease in net photosynthetic rate, chlorophyll content, and relative water content contributed to the high survival rate of transgenic Arabidopsis lines after drought. CONCLUSIONS: The characteristics of APX in celery were different from that in other species. The enhanced drought resistance of overexpressing AgAPX1 in Arabidopsis may be achieved by increasing the accumulation of AsA, enhancing the activities of various antioxidant enzymes, and promoting stomatal closure. Our work provides new evidence to understand APX and its response mechanisms to drought stress in celery.