Basic Region Research Articles

Regulation of c-Fos gene transcription by stimulus-responsive protein kinases

The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is regulated by numerous extracellular signaling molecules including mitogens, metabolites, and ligands for receptor tyrosine kinases, G protein-coupled receptors, and cytokine receptors. Here, we analyzed the effects of the stimulus-responsive MAP kinases ERK1/2 (extracellular signal-regulated protein kinase), JNK (c-Jun N-terminal protein kinase) and p38 protein kinase on transcription of the c-Fos gene. We used chromatin-integrated c-Fos promoter-luciferase reporter genes containing inactivating point mutations of DNA binding sites for distinct transcription factors. ERK1/2, JNK, and p38 protein kinases were specifically activated following expression of either a mutant of B-Raf, a truncated version of mitogen-activated/extracellular signal responsive kinase kinase kinase-1 (MEKK1), or a mutant of MAP kinase kinase-6 (MKK6), respectively. The results show that the DNA binding sites for serum response factor (SRF) and for the ternary complex factor (TCF) are of major importance for stimulating c-Fos promoter activity by MAP kinases. ERK1/2 and p38-induced stimulation of the c-Fos promoter additionally required the DNA binding site for the transcription factor AP-1. Mutation of the DNA binding site for STAT had no or only a small effect on c-Fos promoter activity. We conclude that MAP kinases do not activate distinct transcription factors involving distinct genetic elements. Rather, these kinases mainly target SRF and TCF proteins, leading to an activation of transcription of the c-Fos gene via the serum response element.

The binding specificity of bZIP/DNA from a knob-socket analysis

The basic leucine zipper motif (bZIP) is a simple DNA-binding domain recognizing consensus sequences through interactions between α-helices and DNA. This α-helical domain consists of an N-terminal DNA-binding basic and C-terminal leucine zipper region forming a coiled-coil. As a transcription factor, bZIPs exhibit a high degree of variance in the DNA binding region. This study applies a knob-socket analysis to characterize the multibody determinants of the variation in specificity between individual bZIP monomers and their respective four-base half-site.

The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α-SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1-/- [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, SH2B1β and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1.

Phosphomimetic mutations in the C-terminal basic region of troponin T increases actin filament activity

The last 14-16 residues in the C-terminal domain of cardiac and skeletal troponin T (TnT) are essential to achieve full inhibition at low calcium levels (Baxley, Johnson, Pinto and Chalovich, 2017). Those C-terminal residues also prevent Ca2+ from fully activating the regulatory system. Full activation occurs by binding force producing crossbridges to actin. Because the inhibitory region in the C-terminal region of TnT is highly basic, it is possible that phosphorylation of Ser and Thr within that region might also attenuate the limitation on Ca2+ activation.

Phytochrome interacting factor MdPIF7 modulates anthocyanin biosynthesis and hypocotyl growth in apple.

Light affects many physiological and developmental processes of plants by regulating the expression and activity of light-responsive proteins. Among them, phytochrome interacting factors (PIFs) play pivotal roles in the regulation of anthocyanin accumulation and hypocotyl growth. However, the molecular mechanism is not well understood, especially in woody plants, such as apple (Malus × domestica). In this study, we identified a light-responsive PIF protein, MdPIF7, in apple and investigated the molecular mechanism of its regulation of anthocyanin biosynthesis and hypocotyl growth. We found that overexpression of MdPIF7 decreased anthocyanin accumulation in transgenic apple materials and promoted hypocotyl elongation in ectopically expressed Arabidopsis (Arabidopsis thaliana). Further investigation showed that MdPIF7 functioned by interacting with B-box 23 (MdBBX23), a positive regulator of anthocyanin biosynthesis in apple and hypocotyl growth inhibition in ectopically expressed Arabidopsis, and attenuating the transcriptional activation of MdBBX23 on LONG HYPOCOTYL 5 (MdHY5). In addition, MdPIF7 interacted with basic region leucine zipper 44 (MdbZIP44) and ethylene response factor 38 (MdERF38), two positive regulators of anthocyanin biosynthesis, and it negatively regulated MdbZIP44- and MdERF38-promoted anthocyanin accumulation by interfering with the interaction between MdbZIP44/MdERF38 and MdMYB1. Taken together, our results reveal that MdPIF7 regulates anthocyanin biosynthesis in apple and hypocotyl growth in ectopically expressed Arabidopsis through MdPIF7-MdBBX23-MdHY5 and MdPIF7-MdbZIP44/MdERF38-MdMYB1 modules. Our findings enrich the functional studies of PIF proteins and provide insights into the molecular mechanism of PIF-mediated anthocyanin biosynthesis and hypocotyl growth.

Micro-CT Study of Mongolian Gerbil Humeral Bone After Prolonged Spaceflight Based on a New Algorithm for Delimitation of Long-Bone Regions.

The Mongolian gerbil displays unique physiological and anatomical features that make this species an attractive object for biological experiments in space. However, until recently, the Mongolian gerbil has remained a novel, mostly unstudied animal model in investigating bone loss in weightlessness (G0). After 12 days of orbital Foton-M3 mission, the humerus of Mongolian gerbils has been studied here via micro-computed tomography (micro-CT) to quantify bone morphometric parameters. The samples from the flight group, delayed synchronous ground-control group, and basal control group were investigated, and main morphometric parameters were reported in the article. The accurate selection of a region of interest is an essential step for a correct assessment of bone parameters. We proposed a new, easy and efficient method for delimiting the bone’s basic regions in the humerus. It is based on quantitative estimation of X-ray attenuation in the cortical bone as a function of humerus bone length. The micro-CT analysis of the basic bone regions revealed a difference in bone morphometric parameters between the flight and control gerbils. The most significant bone loss was observed in the cortical part of the proximal humeral zone in the flight group. No statistically significant changes of volume fraction in the cancellous tissue of proximal and distal epiphyses and metaphyses were observed. A statistically significant increase in both cancellous bone volume and bone X-ray attenuation in the flight group was detected in the proximal part of the diaphyses. We assume that enhanced calcium deposition in the diaphyseal cancellous tissue occurred due to a bone response to G0 conditions.

Molecular Determinants in tRNA D-arm Required for Inhibition of HIV-1 Gag Membrane Binding

Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P2 and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P2-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNAPro, but not that of tRNALys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNALys3 showed greater inhibition of MA membrane binding than full-length tRNAPro. While transplantation of the D loop sequence of tRNALys3 into tRNAPro resulted in a modest increase in the inhibitory effect relative to WT tRNAPro, replacing the entire D arm sequence with that of tRNALys3 was necessary to confer the full inhibitory effects upon tRNAPro. Together, these results demonstrate that the D arm of tRNALys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.

CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil’s major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε27 but independent of sumoylation of the RDI core “VKEEP” sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein–protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.

The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study.

The calcium release-activated calcium channel, composed of the Orai channel and the STIM protein, plays a crucial role in maintaining the Ca2+ concentration in cells. Previous studies showed that the L138F mutation in the human Orai1 creates a constitutively open channel independent of STIM, causing severe myopathy, but how the L138F mutation activates Orai1 is still unclear. Here, based on the crystal structure of Drosophila melanogaster Orai (dOrai), molecular dynamics simulations for the wild-type (WT) and the L210F (corresponding to L138F in the human Orai1) mutant were conducted to investigate their structural and dynamical properties. The results showed that the L210F dOrai mutant tends to have a more hydrated hydrophobic region (V174 to F171), as well as more dilated basic region (K163 to R155) and selectivity filter (E178). Sodium ions were located deeper in the mutant than in the wild-type. Further analysis revealed two local but essential conformational changes that may be the key to the activation. A rotation of F210, a previously unobserved feature, was found to result in the opening of the K163 gate through hydrophobic interactions. At the same time, a counter-clockwise rotation of F171 occurred more frequently in the mutant, resulting in a wider hydrophobic gate with more hydration. Ultimately, the opening of the two gates may facilitate the opening of the Orai channel independent of STIM.

Antibacterial and Hemocompatible pH-Responsive Hydrogel for Skin Wound Healing Application: In Vitro Drug Release.

The treatment of successive skin wounds necessitates meticulous medical procedures. In the care and treatment of skin wounds, hydrogels produced from natural polymers with controlled drug release play a crucial role. Arabinoxylan is a well-known and widely available biological macromolecule. We produced various formulations of blended composite hydrogels (BCHs) from arabinoxylan (ARX), carrageenan (CG), and reduced graphene oxide (rGO) using and cross-linked them with an optimal amount of tetraethyl orthosilicate (TEOS). The structural, morphological, and mechanical behavior of the BCHs samples were determined using Fourier-transform infrared spectroscopy (FT-IR), Scanning electron microscope (SEM), mechanical testing, and wetting, respectively. The swelling and degradation assays were performed in phosphate-buffered saline (PBS) solution and aqueous media. Maximum swelling was observed at pH 7 and the least swelling in basic pH regions. All composite hydrogels were found to be hemocompatible. In vitro, silver sulfadiazine release profile in PBS solution was analyzed via the Franz diffusion method, and maximum drug release (87.9%) was observed in 48 h. The drug release kinetics was studied against different mathematical models (zero-order, first-order, Higuchi, Hixson–Crowell, Korsmeyer–Peppas, and Baker–Lonsdale models) and compared their regression coefficient (R2) values. It was observed that drug release follows the Baker–Lonsdale model, as it has the highest value (0.989) of R2. Hence, the obtained results indicated that, due to optimized swelling, wetting, and degradation, the blended composite hydrogel BCH-3 could be an essential wound dressing biomaterial for sustained drug release for skin wound care and treatment.

Effects of pH and Electrolytes on Castor Oil Emulsions with Various Stabilisers Using Khaya senegalensis Gum as an Emulsifier.

Dispersed systems such as emulsions are easily destabilised during processing and storage since they are thermodynamically unstable systems. It is for this reason emulsifiers/stabilisers are frequently employed in pharmaceutical emulsion formulations to increase their short- and long-term kinetic stability. This current study seeks to investigate the potential emulsifying property of gums obtained from Khaya senegalensis (family: Meliaceae) trees. Gums were collected, authenticated, oven-dried, milled, filtered, and purified using 96% ethanol. The microbial quality of the gum was assessed following the BP (2013) specifications. The purified gum was free from some selected pathogenic microorganisms, rendering the gum safe for consumption. The emulsifying property was investigated by formulating emulsions using castor oil and employing the dry gum method. The ratios of oil-to-water-to-gum for the formulation of a stable emulsion were determined. The stability of the emulsion was evaluated, and an effort was made to improve the stability by incorporating Tween 80, hydroxypropyl methylcellulose, and xanthan gum. From the results, it can be inferred that Tween 80 (0.5%) was able to stabilise the emulsion. Addition of xanthan gum worsened the creaming. The effects of pH (4.0, 5.5, 7.2, 9.0, and 11.0) and electrolytes (0.1 M of NaCl, KCl, and CaCl2) on the physical stability of oil-in-water emulsions were studied during 12 weeks of storage. Percentage creaming volume and whether there was phase inversion were the criteria used as the evaluation parameter. From the percentage creaming volume data, emulsions formulated with both gums showed the lowest creaming volumes at pH of 7.2, followed by the acidic regions (pH 4.0, 5.5), with the basic regions (pH 9.0, 11.0) recording the highest creaming volumes. The effects of the various electrolytes at a constant concentration of 0.1 M on the o/w emulsions were found in this order NaCl < KCl < CaCl2. This study proves that Khaya senegalensis gum can successfully be employed as an emulsifying agent in pharmaceutical formulations.

Characterization and functional study of nuclear factor erythroid 2-related factor 2 (Nrf2) in black tiger shrimp (Penaeus monodon)

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of the Cap'n′collar basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants and chemopreventive agents. The full-length cDNA of Nrf2 from Penaeus monodon (PmNrf2; 2024 bp long with 729 bp coding region, GenBank accession no. MW390830) was cloned. The 242-amino-acid polypeptide encoded by this gene had a predicted molecular mass of 27.80 kDa. Sequence homology and phylogenetic analysis showed that PmNrf2 was similar to the insect Cap'n′Collar (CNC) transcription factor and mammalian Nrf2. Tissue expression profile analyzed by quantitative real-time RT-PCR (qRT-PCR) demonstrated that PmNrf2 was constitutively expressed in all examined tissues, with the highest expression observed in the intestines and the weakest expression observed in the hemocyte. PmNrf2 expression profiles were detected in the hepatopancreas of shrimp after bacterial challenge. The results suggested that PmNrf2 was involved in the responses to bacterial challenge, but the temporal expression pattern trend of PmNrf2 differed between the gram-negative and gram-positive bacterial challenges in the shrimp hepatopancreas. The recombinant PmNrf2 protein was expressed and purified through affinity chromatography. Furthermore, an anti-PmNrf2 polyclonal antibody was obtained, which was able to clearly detect PmNrf2 protein expression in the hepatopancreas of shrimp. Knockdown of PmNrf2 by RNA interference (RNAi) resulted in a reduction in the expression of PmGPx gene. Taken together, the results of our study indicated that PmNrf2 played a role in regulation the transcription of PmGPx antioxidant enzyme genes.

An Overview of the Nrf2/ARE Pathway and Its Role in Neurodegenerative Diseases.

Nrf2 is a basic region leucine-zipper transcription factor that plays a pivotal role in the coordinated gene expression of antioxidant and detoxifying enzymes, promoting cell survival in adverse environmental or defective metabolic conditions. After synthesis, Nrf2 is arrested in the cytoplasm by the Kelch-like ECH-associated protein 1 suppressor (Keap1) leading Nrf2 to ubiquitin-dependent degradation. One Nrf2 activation mechanism relies on disconnection from the Keap1 homodimer through the oxidation of cysteine at specific sites of Keap1. Free Nrf2 enters the nucleus, dimerizes with small musculoaponeurotic fibrosarcoma proteins (sMafs), and binds to the antioxidant response element (ARE) sequence of the target genes. Since oxidative stress, next to neuroinflammation and mitochondrial dysfunction, is one of the hallmarks of neurodegenerative pathologies, a molecular intervention into Nrf2/ARE signaling and the enhancement of the transcriptional activity of particular genes are targets for prevention or delaying the onset of age-related and inherited neurogenerative diseases. In this study, we review evidence for the Nrf2/ARE-driven pathway dysfunctions leading to various neurological pathologies, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, as well as amyotrophic lateral sclerosis, and the beneficial role of natural and synthetic molecules that are able to interact with Nrf2 to enhance its protective efficacy.

Regulatory mechanisms of heme regulatory protein BACH1: a potential therapeutic target for cancer.

A limited number of overexpressed transcription factors are associated with cancer progression in many types of cancer. BTB and CNC homology 1 (BACH1) is the first mammalian heme-binding transcription factor that belongs to the basic region leucine zipper (bZIP) family and a member of CNC (cap 'n' collar). It forms heterodimers with the small musculoaponeurotic fibrosarcoma (MAF) proteins and stimulates or suppresses the expression of target genes under a very low intracellular heme concentration. It possesses a significant regulatory role in heme homeostasis, oxidative stress, cell cycle, apoptosis, angiogenesis, and cancer metastasis progression. This review discusses the current knowledge about how BACH1 regulates cancer metastasis in various types of cancer and other carcinogenic associated factors such as oxidative stress, cell cycle regulation, apoptosis, and angiogenesis. Overall, from the reported studies and outcomes, it could be realized that BACH1 is a potential pharmacological target for discovering new therapeutic anticancer drugs.

High efficiency orientated milling parameter optimization with tool wear monitoring in roughing operation

High efficiency milling is one of the main goals of CNC machining. Although roughing operation has low requirement for machining accuracy, the milling parameter selection usually need to consider the cutting force, milling stability, spindle torque and power. Besides, the tool wear in difficult-to-cut material machining is another very prominent problem. Therefore, how to achieve the high efficient milling parameters under the multi-constraint condition has been a severe challenge in the field of CNC machining. In this paper, a milling parameter optimization method for efficient rough machining is proposed by combining the off-line optimization and real-time monitoring. Firstly, a mathematical model of the optimization problem is established, which takes the machining efficiency as the objective, the spindle speed, radial and axial depth of cuts as the variables, and considers the multi-constraint, such as the basic parameter feasible region, cutting force, stability, spindle torque and power. Secondly, the quantitative relationship between the spindle three-phase current and milling force is established to realize the real-time estimation of cutting force coefficients. Furthermore, a numerical optimization method based on the random vector search is proposed, and the overall optimization procedure is given. Finally, a series of experimental verifications on the titanium alloy Ti5Al5Mo5VCrFe machining has been carried out, and the case study results show that the proposed parameter optimization method can greatly improve the machining efficiency, which proves the validity of the method.

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering.

ABSTRACTDuring replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

Ectopic expression of Id1 or Id3 inhibits transcription of the GATA-4 gene in P19CL6 cells under differentiation condition.

Inhibitor of DNA binding (Id) is a dominant negative form of the E-box binding basic-helix-loop-helix (bHLH) transcription factor since it is devoid of the basic region required for DNA binding and forms an inactive hetero dimer with bHLH proteins. The E-box sequence located in the promoter region of the GATA-binding protein 4 (GATA-4) gene is essential for transcriptional activation in P19CL6 cells. These cells differentiate into cardiomyocytes and start to express GATA-4, which further triggers cardiac-specific gene expression. In this study, expression plasmids for Ids tagged with human influenza hemagglutinin (HA)-FLAG were constructed and introduced into P19CL6 cells. The stable clones expressing the recombinant Id proteins (Id1 or Id3) were isolated. The GATA-4 gene expression in these clones under differentiation condition in the presence of 1% dimethyl sulfoxide (DMSO) was repressed, with concomitant abolishment of the transcription of α-myosin heavy chain (α-MHC), which is a component of cardiac myofibrils. Thus, the increased expression of Id protein could affect GATA-4 gene expression and negatively regulate the differentiation of P19CL6 cells.

Genome-wide comparative analyses of GATA transcription factors among seven Populus genomes

GATA transcription factors (TFs) are widespread eukaryotic regulators whose DNA-binding domain is a class IV zinc finger motif (CX2CX17–20CX2C) followed by a basic region. We identified 262 GATA genes (389 GATA TFs) from seven Populus genomes using the pipeline of GATA-TFDB. Alternative splicing forms of Populus GATA genes exhibit dynamics of GATA gene structures including partial or full loss of GATA domain and additional domains. Subfamily III of Populus GATA genes display lack CCT and/or TIFY domains. 21 Populus GATA gene clusters (PCs) were defined in the phylogenetic tree of GATA domains, suggesting the possibility of subfunctionalization and neofunctionalization. Expression analysis of Populus GATA genes identified the five PCs displaying tissue-specific expression, providing the clues of their biological functions. Amino acid patterns of Populus GATA motifs display well conserved manner of Populus GATA genes. The five Populus GATA genes were predicted as membrane-bound GATA TFs. Biased chromosomal distributions of GATA genes of three Populus species. Our comparative analysis approaches of the Populus GATA genes will be a cornerstone to understand various plant TF characteristics including evolutionary insights.

Heterologous expression of CYP81A6 from rice (Oryza sativa) in Escherichia coli and structural analyses of bensulfuron‐methyl metabolites

AbstractHerbicides are often selective and kill only weeds without affecting crops. In rice, CYP81A6 is responsible for tolerance toward acetolactate synthase herbicides such as bensulfuron‐methyl (BSM). CYP81A6 is thought to metabolize BSM into a nontoxic metabolite, that is, O‐demethylated BSM. However, direct evidences for this reaction have not been reported, likely due to difficulties in the heterologous expression of plant P450s in bacterial expression systems. In this study, we established CYP81A6 expression in Escherichia coli via modification of its N‐terminal basic region coding sequence, optimization of cultivation temperature, and selection of E. coli strains. Heterologous production of CYP81A6 was evaluated by the conversion of BSM in autoinduction medium after the culture, compared to that with an empty vector control. The optimized heterologous production condition of rice CYP81A6 in E. coli using 48‐well plates involved replacement of the N‐terminal basic region with that of CYP2C3, the use of E. coli BL21(DE3) as an expression host, and culture at 34°C. Further, co‐expression of gene encoding glucose dehydrogenase enhanced the conversion of 0.5 mM BSM from 58.7 to 73.1% in the optimized condition. In the optimization process, we detected two metabolites, M1 and M2. Mass spectrometry and 1H nuclear magnetic resonance analyses indicated that M1 was O‐demethylated BSM. Although we could not isolate M2 because of its low abundance in the medium, mass spectrometry analysis suggested that it was the monooxygenated (+O) form of BSM. Overall, these results confirm that CYP81A6 catalyzes the formation of O‐demethylated BSM from BSM.

Structural insight into host plasma membrane association and assembly of HIV-1 matrix protein

Oligomerization of Pr55Gag is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55Gag. However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55Gag to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18–33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles’ membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain’s 2D layers during assembly and budding.