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Epipyrone A is a unique C-galactosylated 4-hydroxy-2-pyrone derivative with an antifungal potential from the fungus Epicoccum nigrum. We elucidated its biosynthesis via heterologous expression and characterized an unprecedented membrane-bound pyrone C-glycosyltransferase biochemically. Molecular docking and mutagenesis experiments suggested a possible mechanism for the heterocyclic C-glycosylation and the importance of a transmembrane helix for its catalysis. These results expand the repertoire of C-glycosyltransferases and provide new insights into the formation of C-glycosides in fungi.

A novel composite down-shifting nanomaterial composed of YVO4:Eu3+, Bi3+nanoparticles (NPs) and Ag NPs was developed and introduced as a sunscreen on industrial SHJ solar cells to reduce the UV-induced degradation.

Metal ions have various important biological roles in proteins, including structural maintenance, molecular recognition and catalysis. Previous methods of predicting metal-binding sites in proteomes were based on either sequence or structural motifs. Here we developed a co-evolution-based pipeline named ‘MetalNetʼ to systematically predict metal-binding sites in proteomes. We applied MetalNet to proteomes of four representative prokaryotic species and predicted 4,849 potential metalloproteins, which substantially expands the currently annotated metalloproteomes. We biochemically and structurally validated previously unannotated metal-binding sites in several proteins, including apo-citrate lyase phosphoribosyl-dephospho-CoA transferase citX, an Escherichia colienzyme lacking structural or sequence homology to any known metalloprotein (Protein Data Bank (PDB) codes: 7DCMand 7DCN). MetalNet also successfully recapitulated all known zinc-binding sites from the human spliceosome complex. The pipeline of MetalNet provides a unique and enabling tool for interrogating the hidden metalloproteome and studying metal biology.

A novel coronavirus (the SARS‐CoV‐2) has been identified in January 2020 as the causal pathogen for COVID‐19, a pandemic started near the end of 2019. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS‐CoV as a receptor was found to facilitate the infection of SARS‐CoV‐2, initiated by the binding of the spike protein to human ACE2. Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure and dynamics of the ACE2 in complex with the receptor binding domain (RBD) of the SARS‐CoV‐2 spike protein. The predicted model is highly consistent with the experimentally determined structures, validating the homology modeling results. Besides the binding interface reported in the crystal structures, novel binding poses are revealed from all‐atom MD simulations. The simulation data are used to identify critical residues at the complex interface and provide more details about the interactions between the SARS‐CoV‐2 RBD and human ACE2. Simulations reveal that RBD binds to both open and closed state of ACE2. Two human ACE2 mutants and rat ACE2 are modeled to study the mutation effects on RBD binding to ACE2. The simulations show that the N‐terminal helix and the K353 are very important for the tight binding of the complex, the mutants are found to alter the binding modes of the CoV2‐RBD to ACE2. Author summary:The first step of SARS‐CoV‐2 virus infection is the binding of spike protein to a receptor protein called ACE2 on the surface of host cell. The detailed interactions between virus spike protein at the receptor binding domain (RBD) and ACE2 were computationally modelled and the binding stability was assessed using molecular dynamics simulations. Using the same method, the study reveals that the mutations mimicking rat ACE2 protein lead to reduced binding to the RBD of spike protein. Simulations reveals persistent presence of water molecules at the interface, indicating their roles in the binding between ACE2 and spike protein.

Neoadjuvant immunotherapy with chemotherapy improves outcomes in patients with resectable non-small-cell lung cancer (NSCLC). Given its immunomodulating effect, we investigated whether stereotactic body radiotherapy (SBRT) enhances the effect of immunochemotherapy.

ABSTRACTMethylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the folate cycle and its genetic variations have been associated with various human diseases. Previously we identified that MTHFR is phosphorylated by cyclin-dependent kinase 1 (CDK1) at T34 and MTHFR underlies heterochromatin maintenance marked by H3K9me3 levels. Herein we demonstrate that pT34 creates a binding motif that docks MTHFR to the polo-binding domain (PBD) of polo-like kinase 1 (PLK1), a fundamental kinase that orchestrates many cell cycle events. We show that PLK1 phosphorylates MTHFR at T549 in vitro and in vivo. Further, we uncovered a role of MTHFR in replication. First, MTHFR depletion increased the fraction of cells in S phase. This defect could not be rescued by siRNA resistant plasmids harboring T549A, but could be restored by overproduction of Suv4–20H2, the H4K20 methyltransferase. Moreover, siMTHFRattenuated H4K20me3 levels, which could be rescued by Suv4–20H2 overproduction. More importantly, we also investigated MTHFR-E429A, the protein product of an MTHFR single nucleotide variant. MTHFR-E429A overexpression also increased S phase cells and decreased H4K20me3 levels, and it is linked to a poor glioma prognosis in the Chinese population. Collectively, we have unveiled a vital role of PLK1-dependent phosphorylation of MTHFR in replication via histone methylation, and implicate folate metabolism with glioma.

Jasmonates (JAs) are essential plant hormones that play important roles in the regulation of plant growth and the response to environmental stress. In the JA signaling pathway, the core transcription factors are a class of basic helix-loop-helix (bHLH) proteins, including MYC2, MYC3, and MYC4, that have different regulatory capacities. Here, we report the 2.7 Å crystal structure of the MYC2 bHLH domain complexed with G-box DNA, showing a cis-tetrameric structure. Biochemical assays confirmed that full-length MYC2 forms a stable homo-tetramer both in solution and in DNA-bound states, whereas MYC3 forms only a homodimer. Isothermal titration calorimetry (ITC) assays demonstrated that tetramerization enhanced DNA binding affinity, and fluorescence resonance energy transfer (FRET) assay indicated DNA looping potential of tetrameric MYC2. Luciferase assay further confirmed the importance of tetramerization in transcriptional regulation. Our studies provide a mechanistic explanation for the regulatory differences of MYC transcription factors.

A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia niis an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.

In living systems, a complex network of protein-protein interactions (PPIs) underlies most biochemical events. The human protein-protein interactome has been surveyed using yeast two-hybrid (Y2H)- and mass spectrometry (MS)-based approaches such as affinity purification coupled to MS (AP-MS). Despite decades of systematic investigations and collaborative multi-disciplinary efforts, there is no “gold standard” for documenting PPIs. A surprisingly large fraction of the human interactome remains uncharted, which we refer to as the “dark interactome.” In this review, we highlight the complexity of the human interactome and discuss the current status of the human reference interactome maps. We discuss why a large proportion of the human interactome has remained refractory to traditional approaches. We propose an experimental model that can enable the identification of the dark interactome in a cell-type-specific manner. We also propose a framework to implement when embarking on studies designed to rigorously identify and characterize protein interactions.

Green petals pose a challenge for pollinators to distinguish flowers from leaves, but they are valuable as a specialty flower trait. However, little is understood about the molecular mechanisms that underlie the development of green petals. Here, we report that CINCINNATA (CIN)-like TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) proteins play key roles in the control of petal color. The septuple tcp2/3/4/5/10/13/17mutant produced flowers with green petals due to chlorophyll accumulation. Expression of TCP4complemented the petal phenotype of tcp2/3/4/5/10/13/17. We found that chloroplasts were converted into leucoplasts in the distal parts of wild-type petals but not in the proximal parts during flower development, whereas plastid conversion was compromised in the distal parts of tcp2/3/4/5/10/13/17petals. TCP4and most CIN-like TCPswere predominantly expressed in distal petal regions, consistent with the green–white pattern in wild-type petals and the petal greening observed in the distal parts of tcp2/3/4/5/10/13/17petals. RNA-sequencing data revealed that most chlorophyll biosynthesis genes were downregulated in the white distal parts of wild-type petals, but these genes had elevated expression in the distal green parts of tcp2/3/4/5/10/13/17petals and the green proximal parts of wild-type petals. We revealed that TCP4 represses chlorophyll biosynthesis by directly binding to the promoters of PROTOCHLOROPHYLLIDE REDUCTASE(PORB), DIVINYL REDUCTASE(DVR), and SUPPRESSOR OF OVEREXPRESSION OF CO 1(SOC1), which are known to promote petal greening. We found that the conversion of chloroplasts to leucoplasts and the green coloration in the proximal parts of petals appeared to be conserved among plant species. Our findings uncover a major molecular mechanism that underpins the formation of petal color patterns and provide a foundation for the breeding of plants with green flowers.

We report the refined structure of an iron-free form of cytochrome cperoxidase (CcP) at 2.3 Å resolution. The backbone comparison between native CcP and iron-free CcP shows that the two structures have the same protein fold within experimental error. The only difference noted is in the heme pocket where the distance between the proximal histidine and the center of the protoporphyrin has increased. The results show that the iron-free CcP should be a good substitute for native CcP in fluorescence studies and thus also validate previous studies using iron-free CcPs as efficient fluorescent probes in electron transfer studies.

We report the refined structure of an iron‐free form of cytochrome cperoxidase (CcP) at 2.3 Å resolution. The backbone comparison between native CcP and iron‐free CcP shows that the two structures have the same protein fold within experimental error. The only difference noted is in the heme pocket where the distance between the proximal histidine and the center of the protoporphyrin has increased. The results show that the iron‐free CcP should be a good substitute for native CcP in fluorescence studies and thus also validate previous studies using iron‐free CcPs as efficient fluorescent probes in electron transfer studies.

In present paper the influence of nano-SiC particles on the flux pinning of YBa2Cu3O7—y has been studied. The investigation reveals that nano-SiC does not change the superconducting transition and the microstructure of the superconductors. At a temperature of 5 K the addition of 0.5 and 1 wt% nano-SiC can lead to an additional flux pinning effect and improve evidently the critical current density Jc. For composites with 1 wt% nano-SiC Jc is two times larger than the reference sample. Meanwhile, at temperature of 20 and 50 K, the fishtail effect is observed for composites YBa2Cu3O7—y/nano-SiC. A reasonable interpretation of the enhanced flux pinning and the occurrence of the fishtail effect is given.

The N-acetylglutamate kinase from Streptococcus mutans was expressed in Escherichia coli in soluble form and purified to homogeneity. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method and diffracted to 2.06 Å. The crystal belonged to space group P21212, with unit cell parameters a 57.19 Å, b 94.76 Å, c 47.58 Å. The gel filtration and initial phasing results showed that the enzyme exists as a monomer, which is different from previously reported N-acetylglutamate kinases.

The thrC gene of Streptococcus mutans encodes threonine synthase, which is a potential target for drug design. To study the structure and function of the enzyme, the thrC gene was amplified from Streptococcus mutans genomic DNA and cloned into the expression vector pET28. The protein was expressed in Escherichia coli in soluble form and purified to homogeneity. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method. The crystal diffracted to 2.5 Å and belonged to space group P31 or P32, with unit cell parameters ab60.39 Å, c118.62 Å.

DNA binding allosterically activates the cytosolic DNA sensor cGAS (cyclic GMP-AMP [cGAMP] synthase) to synthesize 2′3′-cGAMP, using Mg2+as the metal cofactor that catalyzes two nucleotidyl-transferring reactions. We previously found that Mn2+potentiates cGAS activation, but the underlying mechanism remains unclear. Here, we report that Mn2+directly activates cGAS. Structural analysis reveals that Mn2+-activated cGAS undergoes globally similar conformational changes to DNA-activated cGAS but forms a unique η1 helix to widen the catalytic pocket, allowing substrate entry and cGAMP synthesis. Strikingly, in Mn2+-activated cGAS, the linear intermediates pppGpG and pGpA take an inverted orientation in the active pocket, suggesting a noncanonical but accelerated cGAMP cyclization without substrate flip-over. Moreover, unlike the octahedral coordination around Mg2+, the two catalytic Mn2+are coordinated by triphosphate moiety of the inverted substrate, independent of the catalytic triad residues. Our findings thus uncover Mn2+as a cGAS activator that initiates noncanonical 2′3′-cGAMP synthesis.

B. subtilis dihydroorotase is an important enzyme in de novo pyrimidine biosynthesis pathway and encoded by pyrC gene in pyr operon. pyrC was amplified from B. subtilis genomic DNA and cloned into expression vector pET21- DEST. Dihydroorotase was expressed soluble form in E. coli and purified. The protein was crystallized and diffracted to 2.2 Å. The crystal belongs to P212121 space-group, with unit cell parameters a=48.864Å, b=84.99Å, c=203.05Å. There are 2 molecules per asymmetry unit.

The smu.134 gene encodes a putative transcriptional regulator of 217 residues in Streptococcus mutans, a major pathogen for human dental caries. The gene was cloned into expression vector pET28?? and expressed in soluble form in E. coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.134 was purified to homogeneity in a two step procedure of Ni2 chelating and size exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method and diffracted to 2.6 Å. The crystal belonged to space group P212121, with unit-cell parameters a55.03 Å, b80.84 Å, c107.96 Å.

Smu.1392c is a protein with 158 residues of uncharacterized function. Bioinformatics studies predict it is a putative acetyltransferase. In order to identify its exact function via structural studies, Smu.1392c gene was amplified from Streptococcus mutans genomic DNA and cloned into expression vector PET28a. Smu.1392c was crystallized and diffracted to a resolution of 3 Å in-house. The crystal belongs to R32 space group, with unit cell parameters a b 229.10, c 63.49 Å. There are 2 or 3 molecules in the asymmetric unit.

B. subtilis YjbK is a protein with 190 residues of uncharacterized function, it has been annotated by Pfam database as a member of adenylate cyclase family (EC: 4.6.1.1). In order to identify its exact function via structural studies, yjbK gene was amplified from B. subtilis genomic DNA and cloned into expression vector pET21-DEST. The protein was expressed in a soluble form in E. coli and purified to homogeneity. YjbK was crystallized and diffracted to a resolution of 2.0 Å in-house. The crystals belong to P1 space group, with unit cell parameters a=32.38 Å, b=34.69 Å, c=46.02 Å, =96.560 , =99.683° , =111.333° . There is one molecule per asymmetric unit.

Single crystals of a low molecular weight phosphotyrosine protein phosphatase from bovine liver have been grown. The crystals belong to space group P212121, have cell dimensions a= 46.3 Å, b‐ 62.2 Å, c= 62.7 Å and diffract to better than 2.0 Å resolution. The crystals are well suited for high resolution X‐ray studies.

In present study the influence of several factors on the texture of Bi-based superconductor prepared by powder in tube (PIT) method has been studied. The grain growth of 2212 and 2223 phases during the heat-treatment process leads to obvious improvement of their texture degrees. The texture of mother phase 2212 is well beneficial to improving these texture degrees. These results are analyzed based on the mechanism of the minimum interface energy texture formation that was raised in the early works.