High-compatibility properties of Aronia melanocarpa extracts cross-linked chitosan/polyvinyl alcohol composite film for intelligent food packaging.

Although the active and intelligent properties of rich in anthocyanin extracts added to films have been extensively studied, there remains a sparsity of research pertaining to the miscibility of blended films. This work focused on the miscibility of the chitosan/polyvinyl alcohol (CP) film caused by the addition of Aronia melanocarpa extracts (AME), which are rich anthocyanins and phenolic acids, and its effect on physicochemical and functional properties. AME facilitated the amidation reaction and ionic interaction of chitosan in CP films, leading to loss of the crystallinity degree of chitosan. Furthermore, the crystal disruption promoted the formation of hydrogen bonds with polyvinyl alcohol (PVA) with the promoted miscibility. CP film incorporated with 8 % AME possessed the highest tensile strength (26.79 MPa), and elongation at break (66.38 %) as well as excellent ultraviolet-visible (UV-vis) light barrier property, water vapor barrier properties, due to its high miscibility degree. Moreover, this film also showed excellent antioxidant, antibacterial activity, and pH response function, which could be used to monitor the storage of highly perishable shrimp. Hence, the AME provided extra functionality and improved miscibility between chitosan and PVA, which showed great potential for the preparation of high-performance bioactive-fortified and intelligent food packaging films.

A structural perspective of liver X receptors.

Liver X receptors α and ß are members of the nuclear receptor family, which comprise a flexible N-terminal domain, a DNA binding domain, a hinge linker, and a ligand binding domain. Liver X receptors are important regulators of cholesterol and lipid homeostasis by controlling the transcription of numerous genes. Key to their transcriptional role is synergetic interaction among the domains. DNA binding domain binds on DNA; ligand binding domain is a crucial switch to control the transcription activity through conformational change caused by ligand binding. The Liver X receptors form heterodimers with retinoid X receptor and then the liganded heterodimer may recruit other necessary transcription components to form an active transcription complex.

Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells.

Gasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. Its cytolytic activity is sequestered by the intramolecular autoinhibitory mechanism. Upon caspase-1/11 mediated cleavage of GSDMD, the N-terminal pore-forming domain (GD-NT) is released to mediate pyroptosis. However, it remains unclear how GD-NT is regulated once it is generated. In the current study, we developed a TetOn system in which GD-NT was selectively induced in tumor cells to explore how the cytolytic activity of GD-NT is regulated. We found that the cytolytic activity of GD-NT was negatively regulated by the AMP-activated protein kinase (AMPK) and AMPK activation rendered tumor cells resistant to GD-NT-mediated pyroptosis. Mechanistically, AMPK phosphorylated GD-NT at the serine 46 (pS46-GD), which altered GD-NT oligomerization and subsequently eliminated its pore-forming ability. In our in vivo tumor model, AMPK-mediated phosphorylation abolished GD-NT-induced anti-tumor activity and resulted in an aggressive tumor growth. Thus, our data demonstrate the critical role of AMPK in negatively regulating the cytolytic activity of GD-NT. Our data also highlight an unexpected link between GSDMD-mediated pyroptosis and the AMPK signaling pathway in certain tumor cells.

TRIM56 coiled-coil domain structure provides insights into its E3 ligase functions.

Protein ubiquitination is a post-translation modification mediated by E3 ubiquitin ligases. The RING domain E3 ligases are the largest family of E3 ubiquitin ligases, they act as a scaffold, bringing the E2-ubiquitin complex and its substrate together to facilitate direct ubiquitin transfer. However, the quaternary structures of RING E3 ligases that perform ubiquitin transfer remain poorly understood. In this study, we solved the crystal structure of TRIM56, a member of the RING E3 ligase. The structure of the coiled-coil domain indicated that the two anti-parallel dimers bound together to form a tetramer at a small crossing angle. This tetramer structure allows two RING domains to exist on each side to form an active homodimer in supporting ubiquitin transfer from E2 to its nearby substrate recruited by the C-terminal domains on the same side. These findings suggest that the coiled-coil domain-mediated tetramer is a feasible scaffold for facilitating the recruitment and transfer of ubiquitin to accomplish E3 ligase activity.

Structural studies of the coiled-coil domain of TRIM75 reveal a tetramer architecture facilitating its E3 ligase complex.

Protein ubiquitination plays a vital role in controlling the degradation of intracellular proteins and in regulating cell signaling pathways. Functionally, E3 ubiquitin ligases control the transfer of ubiquitin to the target substrates. As a major family of ubiquitin E3 ligases, the structural assembly of RING E3 ligases required to exert their ubiquitin E3 ligase activity remains poorly defined. Here, we solved the crystal structure of the coiled-coil domain of TRIM75, a member of the RING E3 ligase family, which showed that two disulfide bonds stabilize two antiparallel dimers at a small crossing angle. This tetrameric conformation confers two close RING domains on the same side to form a dimer. Furthermore, this architecture allows the RING dimer to present ubiquitin to a substrate on the same side. Overall, this structure reveals a disulfide bond-mediated unique tetramer architecture and provides a tetrameric structural model through which E3 ligases exert their function.

Microfluidic triple-gradient generator for efficient screening of chemical space.

Generation of a combinatorial gradient for multiple chemicals is essential for studies of biochemical stimuli, chemoattraction, protein crystallization and others. While currently available platforms require complex design/settings to obtain a double-gradient chemical matrix, we herein report for the first time a simple triple-gradient matrix (TGM) device for efficient screening of chemical space. The TGM device is composed of two glass slides and works following the concept of SlipChip. The device utilizes XYZ space to distribute three chemicals and establishes a chemical gradient matrix within 5 min. The established matrix contains 24 or 104 screening conditions depending on the device used, which covers a concentration range of [0.117-1, 0.117-1 and 0.686-1] and [0.0830-1, 0.0830-1, 0.686-1] respectively for the three chemicals. With the triple gradients built simultaneously, this TGM device provides order-of-magnitude improvement in screening efficiency over existing single- or double-gradient generators. As a proof of concept, we applied the device to screen the crystallization conditions for two model proteins of lysozyme and trypsin and confirmed the crystal structures using X-ray diffraction. Furthermore, we successfully obtained the crystallization condition of adhesin competence repressor, a protein that senses the alterations in intracellular zinc concentrations. We expect the TGM system to be widely used as an analytical platform for material synthesis and chemical screening beyond for protein crystallization.

Identification of the E3 Ligase TRIM29 as a Critical Checkpoint Regulator of NK Cell Functions.

NK cells play an important role in immune surveillance and protective immunity, mainly through rapid cytokine release and cytolytic activities. But how such responses are negatively regulated remains poorly defined. In this study, we demonstrated that the E3 ubiquitin ligase TRIM29 is a crucial regulator of NK cell functions. We found that TRIM29 was not expressed in resting NK cells, but was readily upregulated following activation, especially after IL-12 plus IL-18 stimulation. The levels of TRIM29 expression were inversely correlated with IFN-γ production by NK cells, suggesting that TRIM29 inhibits NK cell functions. Indeed, deficiency of TRIM29, specifically in NK cells, resulted in an enhanced IFN-γ production and consequently protected mice from murine CMV infection. Mechanistically, we showed that once induced in NK cells, TRIM29 ubiquitinates and degrades the TGF-ß-activated kinase 1 binding protein 2 (TAB2), a key adaptor protein in IFN-γ production by NK cells. These results identify TRIM29 as a negative regulator of NK cell functions and may have important clinical implications.

Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation.

Th9 cells are prominently featured in allergic lung inflammation, but the mechanism that regulates IL-9 induction in T helper cells remains poorly defined. Here we demonstrate that formation of super-enhancers (SEs) is critical in robust induction of IL-9 and that assembly of the Il9 SEs in Th cells requires OX40-triggered chromatin acetylation. Mechanistically, we found that OX40 costimulation induces RelB expression, which recruits the histone acetyltransferase p300 to the Il9 locus to catalyze H3K27 acetylation. This allows binding of the SE factor Brd4 to organize assembly of the SE complex, which in turn drives robust IL-9 expression and Th9 cell induction. Thus, Th9 cells are strongly induced upon OX40 stimulation, and disruption of SEs abolished Th9 cell induction in vitro and inhibited Th9 cell-mediated allergic airway inflammation in vivo. Together, our data suggest that formation of SEs is essential in IL-9 expression and Th9 cell induction. These findings may have important clinical implications.

TNF superfamily receptor OX40 triggers invariant NKT cell pyroptosis and liver injury.

Tissue-resident immune cells play a key role in local and systemic immune responses. The liver, in particular, hosts a large number of invariant natural killer T (iNKT) cells, which are involved in diverse immune responses. However, the mechanisms that regulate survival and homeostasis of liver iNKT cells are poorly defined. Here we have found that liver iNKT cells constitutively express the costimulatory TNF superfamily receptor OX40 and that OX40 stimulation results in massive pyroptotic death of iNKT cells, characterized by the release of potent proinflammatory cytokines that induce liver injury. This OX40/NKT pyroptosis pathway also plays a key role in concanavalin A-induced murine hepatitis. Mechanistically, we demonstrated that liver iNKT cells express high levels of caspase 1 and that OX40 stimulation activates caspase 1 via TNF receptor-associated factor 6-mediated recruitment of the paracaspase MALT1. We also found that activation of caspase 1 in iNKT cells results in processing of pro-IL-1ß to mature IL-1ß as well as cleavage of the pyroptotic protein gasdermin D, which generates a membrane pore-forming fragment to produce pyroptotic cell death. Thus, our study has identified OX40 as a death receptor for iNKT cells and uncovered a molecular mechanism of pyroptotic cell death. These findings may have important clinical implications in the development of OX40-directed therapies.

A screening cascade to identify ERß ligands.

The establishment of effective high throughput screening cascades to identify nuclear receptor (NR) ligands that will trigger defined, therapeutically useful sets of NR activities is of considerable importance. Repositioning of existing approved drugs with known side effect profiles can provide advantages because de novo drug design suffers from high developmental failure rates and undesirable side effects which have dramatically increased costs. Ligands that target estrogen receptor ß (ERß) could be useful in a variety of diseases ranging from cancer to neurological to cardiovascular disorders. In this context, it is important to minimize cross-reactivity with ERα, which has been shown to trigger increased rates of several types of cancer. Because of high sequence similarities between the ligand binding domains of ERα and ERß, preferentially targeting one subtype can prove challenging. Here, we describe a sequential ligand screening approach comprised of complementary in-house assays to identify small molecules that are selective for ERß. Methods include differential scanning fluorimetry, fluorescence polarization and a GAL4 transactivation assay. We used this strategy to screen several commercially-available chemical libraries, identifying thirty ERß binders that were examined for their selectivity for ERß versus ERα, and tested the effects of selected ligands in a prostate cancer cell proliferation assay. We suggest that this approach could be used to rapidly identify candidates for drug repurposing.

Structural and functional insight into TAF1-TAF7, a subcomplex of transcription factor II D.

Transcription factor II D (TFIID) is a multiprotein complex that nucleates formation of the basal transcription machinery. TATA binding protein-associated factors 1 and 7 (TAF1 and TAF7), two subunits of TFIID, are integral to the regulation of eukaryotic transcription initiation and play key roles in preinitiation complex (PIC) assembly. Current models suggest that TAF7 acts as a dissociable inhibitor of TAF1 histone acetyltransferase activity and that this event ensures appropriate assembly of the RNA polymerase II-mediated PIC before transcriptional initiation. Here, we report the 3D structure of a complex of yeast TAF1 with TAF7 at 2.9 Å resolution. The structure displays novel architecture and is characterized by a large predominantly hydrophobic heterodimer interface and extensive cofolding of TAF subunits. There are no obvious similarities between TAF1 and known histone acetyltransferases. Instead, the surface of the TAF1-TAF7 complex contains two prominent conserved surface pockets, one of which binds selectively to an inhibitory trimethylated histone H3 mark on Lys27 in a manner that is also regulated by phosphorylation at the neighboring H3 serine. Our findings could point toward novel roles for the TAF1-TAF7 complex in regulation of PIC assembly via reading epigenetic histone marks.

Structure of the retinoid X receptor α-liver X receptor ß (RXRα-LXRß) heterodimer on DNA.

Nuclear receptors (NRs) are conditional transcription factors with common multidomain organization that bind diverse DNA elements. How DNA sequences influence NR conformation is poorly understood. Here we report the crystal structure of the human retinoid X receptor α-liver X receptor ß (RXRα-LXRß) heterodimer on its cognate element, an AGGTCA direct repeat spaced by 4 nt. The complex has an extended X-shaped arrangement, with DNA- and ligand-binding domains crossed, in contrast to the parallel domain arrangement of other NRs that bind an AGGTCA direct repeat spaced by 1 nt. The LXRß core binds DNA via canonical contacts and auxiliary DNA contacts that enhance affinity for the response element. Comparisons of RXRα-LXRßs in the crystal asymmetric unit and with previous NR structures reveal flexibility in NR organization and suggest a role for RXRα in adaptation of heterodimeric complexes to DNA.

GPS2 is required for cholesterol efflux by triggering histone demethylation, LXR recruitment, and coregulator assembly at the ABCG1 locus.

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom.

A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data set was collected to 2.35 A resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 43.2, c = 147.9 A. There are two molecules in the crystallographic asymmetric unit.

Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel.

Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms. Some members of these CRISPs recently have been found to block L-type Ca(2+) channels or cyclic nucleotide-gated ion (CNG) channels. Here, natrin purified from Naja atra venom, a member of the CRISP family, can induce a further contractile response in the endothelium-denuded thoracic aorta of mouse which has been contracted by a high-K(+) solution. Further experiments show it can block the high-conductance calcium-activated potassium (BK(Ca)) channel in a concentration-dependent manner with an IC(50) of 34.4 nM and a Hill coefficient of 1.02, which suggests that only a single natrin molecule is required to bind an ion channel to block BK(Ca) current. The crystal structure of natrin displaying two domains in tandem shows its cysteine-rich domain (CRD) has relatively independent flexibility, especially for the C-terminal long loop (loop I) of CRD to participate in the interface of two domains. On the basis of previous studies of CNG channel and L-Ca(2+) channel blockers, and the sequence and structural comparison of natrin and stecrisp, the deviation of the vital loop I of CRD is suggested to contribute to different effects of some CRISPs in protein-protein interaction.

The atomic resolution crystal structure of atratoxin determined by single wavelength anomalous diffraction phasing.

By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.

Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain alpha-neurotoxin from Naja atra venom.

Atratoxin-b, a short-chain alpha-neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD(50) of about 0.18 mg kg(-1). Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other alpha-neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R(free) of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 x 0.1 x 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 A resolution using X-rays of wavelength 1.1516 A generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 49.28, c = 44.80 A, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 A(3) Da(-1).

Purification, N-terminal sequencing, crystallization and preliminary X-ray diffraction analysis of atratoxin, a new short-chain alpha-neurotoxin from the venom of Naja naja atra.

Atratoxin, a new alpha-neurotoxin purified to homogeneity by a series of liquid chromatographies from the venom of Naja naja atra (mainland Chinese cobra), is a small single-polypeptide alkaline protein with a pI of about 9.5 and molecular weight of 6952 Da estimated by mass spectrometry. Although the sequencing of the N-terminal 15 residues (LECHNQQTTQQPEGG) shows that this neurotoxic protein contains most of the residues, especially at the conserved positions, of the consensus sequence of short-chain alpha-neurotoxins, the natural mutations in the N-terminal Loop-1 presented by the sequence alignment may have structural or functional implications for the interactions between alpha-neurotoxins and related receptors. Single crystals of atratoxin have been grown from drops containing the necessary Cu(2+) ions by the conventional hanging-drop vapour-diffusion method. The crystals diffract X-rays to 1.6 A resolution and belong to space group C222(1), with unit-cell parameters a = 47.36, b = 47.83, c = 91.31 A, corresponding to a volume-to-mass ratio of 1.85 A(3) Da(-1) and two molecules in each asymmetric unit.