A flavonoid metabolon: cytochrome b5 enhances B-ring trihydroxylated flavan-3-ols synthesis in tea plants.

Flavan-3-ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan-3-ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan-3-ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI-MSI method to reveal a distinct distribution pattern of B-ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3'H, and CsF3'5'H localizes endoplasmic reticulum. Protein-protein interaction studies demonstrated direct associations between CsC4H, CsF3'H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3'5'H, the enzyme for B-ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3'5'H. Our findings suggest the existence of distinct modules governing the synthesis of different B-ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B-ring trihydroxylated flavan-3-ols in tea plants.

Cellular forgetting, desensitisation, stress and ageing in signalling networks. When do cells refuse to learn more?

Recent findings show that single, non-neuronal cells are also able to learn signalling responses developing cellular memory. In cellular learning nodes of signalling networks strengthen their interactions e.g. by the conformational memory of intrinsically disordered proteins, protein translocation, miRNAs, lncRNAs, chromatin memory and signalling cascades. This can be described by a generalized, unicellular Hebbian learning process, where those signalling connections, which participate in learning, become stronger. Here we review those scenarios, where cellular signalling is not only repeated in a few times (when learning occurs), but becomes too frequent, too large, or too complex and overloads the cell. This leads to desensitisation of signalling networks by decoupling signalling components, receptor internalization, and consequent downregulation. These molecular processes are examples of anti-Hebbian learning and 'forgetting' of signalling networks. Stress can be perceived as signalling overload inducing the desensitisation of signalling pathways. Ageing occurs by the summative effects of cumulative stress downregulating signalling. We propose that cellular learning desensitisation, stress and ageing may be placed along the same axis of more and more intensive (prolonged or repeated) signalling. We discuss how cells might discriminate between repeated and unexpected signals, and highlight the Hebbian and anti-Hebbian mechanisms behind the fold-change detection in the NF-κB signalling pathway. We list drug design methods using Hebbian learning (such as chemically-induced proximity) and clinical treatment modalities inducing (cancer, drug allergies) desensitisation or avoiding drug-induced desensitisation. A better discrimination between cellular learning, desensitisation and stress may open novel directions in drug design, e.g. helping to overcome drug resistance.

Engineering a Self-Assembled Protein Cage for Targeted Dual Functionalization.

Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single protein cage remains challenging, resulting in diversity among the functionalized particles. Here, we present the engineering of a self-assembled protein cage, DTMi3ST, capable of independently recruiting two different POIs using SpyCatcher (SC)/SpyTag (ST) and DogCatcher (DC)/DogTag (DT) chemistries, thereby reducing variability between assemblies. Using fluorescent proteins as models, we demonstrate controlled targeting of two different POIs onto DTMi3ST protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the DTMi3ST protein cage with a membrane-targeting peptide and ß-galactosidase resulted in the construction of membrane-bound enzyme assemblies in Escherichia coli, leading to a 69.6% enhancement in substrate utilization across the membrane. This versatile protein cage platform provides dual functional nanotools for biological and biomedical applications.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Encapsulins: Nanotechnology's future in a shell.

Encapsulins, virus capsid-like bacterial nanocompartments have emerged as promising tools in medicine, imaging, and material sciences. Recent work has shown that these protein-bound icosahedral 'organelles' possess distinct properties that make them exceptionally usable for nanotechnology applications. A key factor contributing to their appeal is their ability to self-assemble, coupled with their capacity to encapsulate a wide range of cargos. Their genetic manipulability, stability, biocompatibility, and nano-size further enhance their utility, offering outstanding possibilities for practical biotechnology applications. In particular, their amenability to engineering has led to their extensive modification, including the packaging of non-native cargos and the utilization of the shell surface for displaying immunogenic or targeting proteins and peptides. This inherent versatility, combined with the ease of expressing encapsulins in heterologous hosts, promises to provide broad usability. Although mostly not yet commercialized, encapsulins have started to demonstrate their vast potential for biotechnology, from drug delivery to biofuel production and the synthesis of valuable inorganic materials. In this review, we will initially discuss the structure, function and diversity of encapsulins, which form the basis for these emerging applications, before reviewing ongoing practical uses and highlighting promising applications in medicine, engineering and environmental sciences.

Assessing the performance of a targeted absolute quantification isotope dilution liquid chromatograhy tandem mass spectrometry assay versus a commercial nontargeted relative quantification assay for detection of three major perfluoroalkyls in human blood.

Isotope dilution ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is commonly used for trace analysis of polyfluoroalkyl and perfluoroalkyl substances (PFAS) in difficult matrices. Commercial nontargeted analysis of major PFAS where relative concentrations are obtained cost effectively is rapidly emerging and is claimed to provide comparable results to that of absolute quantification using matrix matched calibration and isotope dilution UHPLC-MS/MS. However, this remains to be demonstrated on a large scale. We aimed to assess the performance of a targeted absolute quantification isotope dilution LC-MS/MS assay versus a commercial nontargeted relative quantification assay for detection of three major PFAS in human blood. We evaluated a population-based cohort of 503 individuals. Correlations were assessed using Spearman's rank correlation coefficients (rho). Precision and bias were assessed using Bland-Altman plots. For perfluorooctane sulfonic acid, the median concentrations were 5.10 ng/mL (interquartile range [IQR] 3.50-7.24 ng/mL), the two assays correlated with rho 0.83. For perfluorooctanoic acid, the median concentrations were 2.14 ng/mL (IQR 1.60-3.0 ng/mL), the two assays correlated with rho 0.92. For perfluorohexanesulfonate, the median concentrations were 5.5 ng/mL (IQR 2.50-11.61 ng/mL), the two assays correlated with rho 0.96. The Bland-Altman statistical test showed agreement of the mean difference for the majority of samples (97-98%) between the two assays. Absolute plasma concentrations of PFAS obtained using matrix matched calibration and isotope dilution UHPLC-MS/MS show agreement with relative plasma concentrations from a nontargeted commercial platform by Metabolon. We observed striking consistency between the two assays when examining the associations of the three PFAS with cholesterol, offering additional confidence in the validity of utilizing the nontargeted approach for correlations with various health phenotypes.

Is the TCA cycle malate dehydrogenase-citrate synthase metabolon an illusion?

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques.

Promising approaches for simultaneous enhancement of medicinally significant benzylisoquinoline alkaloids in opium poppy.

Benzylisoquinoline alkaloids (BIAs) produced in opium poppy have been evidenced to heal patients suffering from various diseases. They, therefore, hold an integral position in the herbal drug industry. Despite the adoption of several approaches for the large-scale production of BIAs, opium poppy remains the only platform in this purpose. The only disadvantage associated with producing BIAs in the plant is their small quantity. Thus, recruiting strategies that boost their levels is deemed necessary. All the methods which have been employed so far are just able to enhance a maximum of two BIAs. Thus, if these methods are utilized, a sizable amount of time and budget must be spent on the synthesis of all BIAs. Hence, the exploitation of strategies which increase the content of all BIAs at the same time is more commercially effective and time-saving, avoiding the laborious step of resolving the biosynthetic pathway of each compound. Exposure to biotic and abiotic elicitors, development of a synthetic auto-tetraploid, overexpression of a WRKY transcription factor, formation of an artificial metabolon, and suppression of a gene in the shikimate pathway and miRNA are strategies that turn opium poppy into a versatile bioreactor for the concurrent and massive production of BIAs. The last three strategies have never been applied for BIA biosynthetic pathways.

Acetylation, ADP-ribosylation and methylation of malate dehydrogenase.

Metabolism within an organism is regulated by various processes, including post-translational modifications (PTMs). These types of chemical modifications alter the molecular, biochemical, and cellular properties of proteins and allow the organism to respond quickly to different environments, energy states, and stresses. Malate dehydrogenase (MDH) is a metabolic enzyme that is conserved in all domains of life and is extensively modified post-translationally. Due to the central role of MDH, its modification can alter metabolic flux, including the Krebs cycle, glycolysis, and lipid and amino acid metabolism. Despite the importance of both MDH and its extensively post-translationally modified landscape, comprehensive characterization of MDH PTMs, and their effects on MDH structure, function, and metabolic flux remains underexplored. Here, we review three types of MDH PTMs - acetylation, ADP-ribosylation, and methylation - and explore what is known in the literature and how these PTMs potentially affect the 3D structure, enzymatic activity, and interactome of MDH. Finally, we briefly discuss the potential involvement of PTMs in the dynamics of metabolons that include MDH.

Soybean AROGENATE DEHYDRATASES (GmADTs): involvement in the cytosolic isoflavonoid metabolon or trans-organelle continuity?

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon.

Immunoprecipitation and FRET-FLIM to Determine Metabolons on the Plant ER.

Metabolons are protein complexes that contain all the enzymes necessary for a metabolic pathway but also scaffolding proteins. Such a structure allows efficient channeling of intermediate metabolites form one active site to the next and is highly advantageous for labile or toxic intermediates. Here we describe two methods currently used to identify metabolons via protein-protein interaction methodology: immunoprecipitations using GFP-Trap®_A beads to find novel interaction partners and potential metabolon components and FRET-FLIM to test for and quantify protein-protein interactions in planta.

Order wrapped in chaos: On the roles of intrinsically disordered proteins and RNAs in the arrangement of the mitochondrial enzymatic machines.

The analysis of cryo-electron tomography images of human and rat mitochondria revealed that the mitochondrial matrix is at least as crowded as the cytosol. To mitigate the crowding effects, metabolite transport in the mitochondria primarily occurs through the intermembrane space, which is significantly less crowded. The scientific literature largely ignores how enzyme systems and metabolite transport are organized in the crowded environment of the mitochondrial matrix. Under crowded conditions, multivalent interactions carried out by disordered protein regions (IDRs), may become extremely important. We analyzed the human mitochondrial proteome to determine the presence and physiological significance of IDRs. Despite mitochondrial proteins being generally more ordered than cytosolic or overall proteome proteins, disordered regions plays a significant role in certain mitochondrial compartments and processes. Even in highly ordered enzyme systems, there are proteins with long IDRs. Some IDRs act as binding elements between highly ordered subunits, while the roles of others are not yet established. Mitochondrial systems, like their bacterial ancestors, rely less on IDRs and more on RNA for LLPS compartmentalization. More evolutionarily advanced subsystems that enable mitochondria-cell interactions contain more IDRs. The study highlights the crucial and often overlooked role played by IDRs and non-coding RNAs in mitochondrial organization.