Fenofibrate Recognition and Gq Protein Coupling Mechanisms of the Human Cannabinoid Receptor CB1.

The G-protein-coupled human cannabinoid receptor 1 (CB1) is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. The structures of CB1-Gi complexes in synthetic agonist-bound forms have been resolved to date. However, the commercial drug recognition and Gq coupling mechanisms of CB1 remain elusive. Herein, the cryo-electron microscopy (cryo-EM) structure of CB1-Gq complex, in fenofibrate-bound form, at near-atomic resolution, is reported. The structure elucidates the delicate mechanisms of the precise fenofibrate recognition and Gq protein coupling by CB1 and will facilitate future drug discovery and design.

Insights into human eNOS, nNOS and iNOS structures and medicinal indications from statistical analyses of their interactions with bound compounds.

83 Structures of human nNOS, 55 structures of human eNOS, 13 structures of iNOS, and about 126 reported NOS-bound compounds are summarized and analyzed. Structural and statistical analysis show that, at least one copy of each analyzed compound binds to the active site (the substrate arginine binding site) of human NOS. And binding features of the three isoforms show differences, but the binding preference of compounds is not in the way helpful for inhibitor design targeting nNOS and iNOS, or for activator design targeting eNOS. This research shows that there is a strong structural and functional similarity between oxygenase domains of human NOS isoforms, especially the architecture, residue composition, size, shape, and distribution profile of hydrophobicity, polarity and charge of the active site. The selectivity and efficacy of inhibitors over the rest of isoforms rely a lot on chance and randomness. Further increase of selectivity via rational improvement is uncertain, unpredictable and unreliable, therefore, to achieve high selectivity through targeting this site is complicated and requires combinative investigation. After analysis on the current two targeting sites in NOS, the highly conserved arginine binding pocket and H4B binding pocket, new potential drug-targeting sites are proposed based on structure and sequence profiling. This comprehensive analysis on the structure and interaction profiles of human NOS and bound compounds provides fresh insights for drug discovery and pharmacological research, and the new discovery here is practically applied to guide protein-structure based drug discovery.

Analysis on spatial structure characteristics and influencing factors of Beijing-Tianjin-Hebei Region MICE cities.

Industrial upgrading and transfer is one of the three key areas in the coordinated development of Beijing-Tianjin-Hebei Region. Meetings, incentives, conferences and exhibitions (MICE) are important means of promoting industrial upgrading. Urban MICE industry, as an important carrier of event activities, become an important gripper for coordinated development of Beijing-Tianjin-Hebei Region. City exhibition space structure plays an dominant role in regional economic development, and it will greatly promote smooth implementation of the coordinated development strategy of Beijing-Tianjin-Hebei Region. In this paper, 13 prefecture-level cities in Beijing, Tianjin and Hebei were selected as research objects, and the data from 2012 to 2018 were selected to establish a gravity model of the attractiveness of MICE cities. With the help of UCINET software, the network density, centrality, cohesive subgroup and core-edge of nodes researches were obtained to analyze the spatial structure characteristics of the attractiveness of MICE cities in Beijing-Tianjin-Hebei Region. The influencing factors of the spatial characteristics of MICE city attractiveness are analyzed by using geographically weighted regression model. The results show that: (1) Beijing, as the overall core area, act as a prominent role. Beijing strengthens the attraction to the superior resources of the surrounding areas, but the network of MICE cities tends to be unbalanced. (2) Overall space forms a subgroup from a single independent subgroup to a subgroup that is spatially separated from each other and acts as an intermediary channel to connect each other, and the core region decreases from 3 to 2. Langfang was removed from the list, leaving Beijing and Tianjin as the core. (3) The influence of supporting facilities, urban environment and population factors on the MICE city attractiveness of Beijing-Tianjin-Hebei Region is increasing gradually. The influence of tourism development level on the MICE city attractiveness of Beijing-Tianjin-Hebei Region is decreasing gradually. The influence of economic development level and Internet development level on the MICE city attractiveness of Beijing-Tianjin-Hebei Region remains unchanged.

Study and design of amino acid-based radical enzymes using unnatural amino acids.

Radical enzymes harness the power of reactive radical species by placing them in a protein scaffold, and they are capable of catalysing many important reactions. New native radical enzymes, especially those with amino acid-based radicals, in the category of non-heme iron enzymes (including ribonucleotide reductases), heme enzymes, copper enzymes, and FAD-radical enzymes have been discovered and characterized. We discussed recent research efforts to discover new native amino acid-based radical enzymes, and to study the roles of radicals in processes such as enzyme catalysis and electron transfer. Furthermore, design of radical enzymes in a small and simple scaffold not only allows us to study the radical in a well-controlled system and test our understanding of the native enzymes, but also allows us to create powerful enzymes. In the study and design of amino acid-based radical enzymes, the use of unnatural amino acids allows precise control of pKa values and reduction potentials of the residue, as well as probing the location of the radical through spectroscopic methods, making it a powerful research tool. Our understanding of amino acid-based radical enzymes will allow us to tailor them to create powerful catalysts and better therapeutics.

Repurposing Photosensitizer Proteins Through Genetic Code Expansion to Facilitate Photo-Biocatalysis.

Artificial photoenzymes with noncanonical photo-redox cofactors have paved the way for enzyme rational design and the creation of new-to-nature biocatalysts. Genetically encoded photo-redox cofactors endow photoenzymes with enhanced or novel activities that catalyze numerous transformations with high efficiency. Herein, we describe a protocol of repurposing photosensitizer proteins (PSP) through genetic code expansion to facilitate multiple photocatalytic conversions including photo-activated dehalogenation of aryl halides, CO2 to CO and CO2 to formic acid reduction. The methods for expression, purification, and characterization of the PSP are detailed. The installation of the catalytic modules and the utilization of PSP-based artificial photoenzymes for photoenzymatic CO2 reduction and dehalogenation are also described.

Simplified Construction of Engineered Bacillus subtilis Host for Improved Expression of Proteins Harboring Noncanonical Amino Acids.

The UAG-based genetic code expansion (GCE) enables site-specific incorporation of noncanonical amino acids (ncAAs) harboring novel chemical functionalities in specific target proteins. However, most GCE studies were done in several whole-genome engineered chassis cells whose hundreds of UAG stop codons were systematically edited to UAA to avoid readthrough in protein synthesis in the presence of GCE. The huge workload of removing all UAG limited the application of GCE in other microbial cell factories (MCF) such as Bacillus subtilis, which has 607 genes ended with UAG among its 4245 coding genes. Although the 257 essential genes count only 6.1% of the genes in B. subtilis, they transcribe 12.2% of the mRNAs and express 52.1% of the proteins under the exponential phase. Here, we engineered a strain named Bs-22 in which all 22 engineerable UAG stop codons in essential genes were edited to UAA via CRISPR/Cas9-mediated multiple-site engineering to minimize the negative effect of GCE on the expression of essential genes. Besides the process of constructing GCE-compatible B. subtilis was systematically optimized. Compared with wild-type B. subtilis (Bs-WT), the fluorescence signal of the eGFP expression could enhance 2.25-fold in Bs-22, and the production of protein tsPurple containing l-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) was increased 2.31-fold in Bs-22. We verified that all purified tsPurple proteins from Bs-22 contained Cou, indicating the excellent fidelity of the strategy. This proof-of-concept study reported efficient overexpression of ncAA-rich proteins in MCF with minimized engineering, shedding new light on solving the trade-off between efficiency and workload.

Genetically Encoded Phosphine Ligand for Metalloprotein Design.

Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.

Jiyuan oridonin A induces differentiation of acute myeloid leukemia cells including leukemic stem-like cells.

Acute myeloid leukemia (AML) is an aggressive form of hematological neoplasia characterized by failure of myeloid differentiation. AML is a leading cause of death from leukemia. Cytarabine chemotherapy resistance is a major source of refractory/relapsed AML. A major obstacle to the successful treatment of AML results from residual disease maintained by leukemic stem cells (LSCs), which are mostly resistant to conventional chemotherapy. Here, we determined the effect of a natural compound, Jiyuan oridonin A (JOA), on the differentiation blockade in the M2 subtype [particularly t (8;21)] of AML cells, M3 subtype of AML cells (APL cells), and leukemic stem-like cells both in vitro and in vivo. We found that JOA induced cell differentiation and suppressed the colony formation capacity in various AML cell lines (Kasumi-1, KG-1, MUTZ-8, NB4, and HL-60) without eliciting apoptosis. The mechanism of JOA-induced cell differentiation depends on the specificity of cell type. JOA mediated the differentiation of Kasumi-1 cells by activating the hematopoietic cell lineage signaling pathway, while inhibition of c-MYC was involved in the JOA-induced differentiation of NB4 cells. Moreover, JOA was identified to target leukemic stem-like cells by induced cell differentiation in vivo. These findings demonstrated that JOA could inhibit the proliferation of M2 and M3 subtypes of AML cells and leukemic stem-like cells by overcoming the differentiation blockade, which may offer a novel therapeutic strategy for AML to overcome relapse and drug resistance in patients with AML. Our findings highlight the possibility of using compounds like JOA as a promising differentiation-induced agent for the treatment of AML.

Histone Deacetylase Inhibitor I3 Induces the Differentiation of Acute Myeloid Leukemia Cells with t (8; 21) or MLL Gene Translocation and Leukemic Stem-Like Cells.

Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by the clonal expansion and differentiation arrest of leukemic cells in peripheral blood and bone marrow. Though the treatment using cytarabine-based protocol for AML patients with t (8; 21) translocation has improved the 5-year overall survival rate, drug resistance continues to be the principal limiting factor for the cure of the disease. In addition, very few AML patients with mixed lineage leukemia gene rearrangements (MLLr) have a desirable outcome. This study evaluated the cell differentiation effect of a potent HDAC (histone deacetylase) inhibitor, I3, and its possible mechanism on the AML cells with t (8; 21) translocation or MLLr and leukemic stem-like cells (Kasumi-1, KG-1, MOLM-13, and THP-1). I3 exhibited efficient anti-proliferative activity on these cells via promoting cell differentiation, accompanied by the cell cycle exit at G0/G1. Importantly, I3 showed the properties of HDAC inhibition, as assessed by the acetylation of histones H3 and H4, which resulted in blocking the activation of the VEGF (vascular endothelial growth factor)-MAPK (mitogen-activated protein kinase) signaling pathway in the Kasumi-1 cell line. These data demonstrate that I3 could be a potent chromatin-remodeling agent to surmount the differentiation block in AML patients, including those with t (8; 21) translocation or MLLr, and could be a potent and selective agent for AML treatment.

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria.

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay, which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

Excited-state intermediates in a designer protein encoding a phototrigger caught by an X-ray free-electron laser.

One of the primary objectives in chemistry research is to observe atomic motions during reactions in real time. Although X-ray free-electron lasers (XFELs) have facilitated the capture of reaction intermediates using time-resolved serial femtosecond crystallography (TR-SFX), only a few natural photoactive proteins have been investigated using this method, mostly due to the lack of suitable phototriggers. Here we report the genetic encoding of a xanthone amino acid (FXO), as an efficient phototrigger, into a rationally designed human liver fatty-acid binding protein mutant (termed XOM), which undergoes photo-induced C-H bond transformation with high selectivity and quantum efficiency. We solved the structures of XOM before and 10-300 ns after flash illumination, at 1.55-1.70 Å resolutions, and captured the elusive excited-state intermediates responsible for precise C-H bond activation. We expect that most redox enzymes can now be investigated by TR-SFX, using our method, to reveal reaction intermediates key for their efficiency and selectivity.

Genetic Incorporation of Fluorescent Amino Acid into Fatty Acid Binding Protein for Fatty Acid Detection.

Fatty acids play critical roles in biological processes, such as energy storage, metabolism, signal transduction, and immune regulation. Therefore, it is necessary to develop in-vitro fluorescent sensors to detect free fatty acids. By genetically incorporating a synthetic fluorescent amino acid (L-(7-hydroxycoumarin-4-yl) ethylglycine, Cou) into fatty acid-binding protein (FABP), we obtained a fluorescent sensor that has a turn-on signal in the presence of the fatty acids. Its response to medium-chain and long-chain fatty acids can be increased by 5.8-fold within several minutes, highlighting its potential applications in fatty acids-related biological processes. Our newly developed fatty acid detection system based on genetic expansion technology has extended the molecular toolboxes available for important biological molecular analysis.

Development of an RNA-protein crosslinker to capture protein interactions with diverse RNA structures in cells.

Characterization of RNA-protein interaction is fundamental for understanding the metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact, and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.

Implications for an imidazol-2-yl carbene intermediate in the rhodanase-catalyzed C-S bond formation reaction of anaerobic ergothioneine biosynthesis.

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain containing enzyme (EanB) activates tne hercynine's sp2 ε-C-H Dona ana replaces it with a C-S bond to produce ergothioneine. The key intermediate for this trans-sulfuration reaction is the Cys412 persulfide. Substitution of the EanB-Cys412 persulfide with a Cys412 perselenide does not yield the selenium analog of ergothioneine, selenoneine. However, in deuterated buffer, the perselenide-modified EanB catalyzes the deuterium exchange between hercynine's sp2 ε-C-H bond and D2O. Results from QM/MM calculations suggest that the reaction involves a carbene intermediate and that Tyr353 plays a key role. We hypothesize that modulating the pKa of Tyr353 will affect the deuterium-exchange rate. Indeed, the 3,5-difluoro tyrosine containing EanB catalyzes the deuterium exchange reaction with k ex of ~10-fold greater than the wild-type EanB (EanBWT). With regards to potential mechanisms, these results support the involvement of a carbene intermediate in EanB-catalysis, rendering EanB as one of the few carbene-intermediate involving enzymatic systems.

A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3'-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569's distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.

Correction: Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

[This corrects the article DOI: 10.1039/D1SC02653D.].

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and ß-arrestin-1 (ßarr1). Using this strategy, we monitored the subtle but functionally important conformational change of ßarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

Health-Related Quality of Life and Its Influencing Factors in Patients with Hepatitis B: A Cross-Sectional Assessment in Southeastern China.

Health-related quality of life (HRQoL) is an important aspect in the management of patients with hepatitis B (HB), which remains a serious health problem in China. There have been relatively few HRQoL studies involving Chinese patients with HB. The aim of this study was to analyze HRQoL in patients diagnosed with HB living in Zhejiang Province, China. A cross-sectional sample of 98 patients with chronic HB (CHB), 56 patients with advanced HB that have developed cirrhosis, and 48 healthy controls (HCs), all from Zhejiang Province, was used in this study. HRQoL was assessed using Short-Form 36 (SF-36) version 2, European quality of life questionnaire-5 dimensions (EQ-5D), and chronic liver disease questionnaire (CLDQ). Intergroup score differences were detected with U tests. Factors with a significant effect on HRQoL were identified with Spearman correlational analyses. Patients with HB (both groups) had lower SF-36 scores than HCs (p < 0.01), with the exception of general health subscores. Patients with HB cirrhosis had the lowest scores in the EQ-5D visual analog scale (VAS) component. Furthermore, patients with HB cirrhosis had lower (p < 0.01) CLDQ scores than patients with CHB. In our HB patient cohort, disease stage and income level were the factors most associated with HRQoL variables; age, education level, and marital status were, each, also significantly associated with some HRQoL variables in patients with HB in our study (p < 0.05 or p < 0.01). HRQoL is diminished in patients with HB in southeastern China. Disease stage and income emerged as key determinants of HRQoL scores. Augmenting social and medical supports for patients with HB, especially those with a socioeconomic status and an advanced disease stage, may help to enhance HRQoL.