Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys^2nd and Cys^6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains.     

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 μL L^?1, 10 mg L^?1, and 80 μmol L^?1.     

Discovery of novel phenoxazinone derivatives as DKK1/LRP6 interaction inhibitors: Synthesis,biological evaluation and structure–activity relationships

Amino derivatives of NCI8642 were synthesized and evaluated as inhibitors of DKK1/LRP6 interactions. The new inhibitors were able to activate the Wnt signaling pathway as indicated by the increased levels of β-catenin, and decrease the DKK1-induced Tau phosphorylation at serine 396.     

Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Nature of Novel 2D van der Waals Heterostructures for Superior Potassium Ion Batteries

Novel 2D van der Waals heterostructures with innovative bimetallic oxychloride (Bi‐ and Sb‐based oxychloride) nanosheets that are well dispersed on reduced graphene oxide nanosheets, are established through element engineering for superior potassium ion battery (PIBs) anodes. This material displays an exceptional electrochemical performance, obtaining a discharge capacity as high as 360 mAh g^?1 at 100 mA g^?1 after running 1000 cycles for over 9 months with a capacity preservation percentage of 88.5% and achieving a discharge capacity as high as 319 mAh g^?1 at 1000 mA g^?1, in addition to the low charge/discharge plateaus for anodes and promising full cell performance. More significantly, the nature of such 2D van der Waals heterostructures, including the element engineering for morphology control, the function of each component of heterostructures, the mechanism of potassium ion storage, and the process of K⁺ intercalation accompanied with the lattice distortion and chemical bond breakages, is explored in depth. This study is critical for not only paving the way for the practical application of PIBs but also shedding light on fundamentals of potassium ion storage in 2D van der Waals heterostructures.     

The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin’s biological interactions and avenues for controlling ghrelin signaling

Ghrelin is a small peptide hormone that requires a unique post-translational modification, serine octanoylation, to bind and activate the GHS-R1a receptor. Initially demonstrated to stimulate hunger and appetite, ghrelin-dependent signaling is implicated in a variety of neurological and physiological processes influencing diseases such as diabetes, obesity, and Prader-Willi syndrome. In addition to its cognate receptor, recent studies have revealed ghrelin interacts with a range of binding partners within the bloodstream. Defining the scope of ghrelin’s interactions within the body, understanding how these interactions work in concert to modulate ghrelin signaling, and developing molecular tools for controlling ghrelin signaling are essential for exploiting ghrelin for therapeutic effect. In this review, we discuss recent findings regarding the biological effects of ghrelin signaling, outline binding partners that control ghrelin trafficking and stability in circulation, and summarize the current landscape of inhibitors targeting ghrelin octanoylation.     

Selective inhibition of Tumor necrosis factor receptor-1 (TNFR1) for the treatment of autoimmune diseases

Anti-TNF biologics have achieved great success in the treatment of autoimmune diseases and have been the most selling biologics on market. However, the anti-TNF biologics have shown some disadvantages such as poor efficacy to some patients and high risk of infection and malignancies during clinical application. Current anti-TNF biologics are antibodies or antibody fragments that bind to TNF-α and subsequently block both TNF-TNFR1 and TNF-TNFR2 signaling. Transgenic animal studies indicate that TNFR1 signaling is responsible for chronic inflammation and cell apoptosis whereas TNFR2 signaling regulates tissue regeneration and inflammation. Recent studies propose to selectively inhibit TNFR1 to enhance efficacy and avoid side effects. In this review, we introduce the biology of TNF-TNFR1 and TNF-TNFR2 signaling, the advantages of selective inhibition of TNF-TNFR1 signaling and research updates on the development of selective inhibitors for TNF-TNFR1 signaling. Antibodies, small molecules and aptamers that selectively inhibit TNFR1 have showed therapeutic potential and less side effects in preclinical studies. Development of selective inhibitors for TNFR1 is a good strategy to enhance the efficacy and reduce the side effects of anti-TNF inhibitors and will be a trend for next-generation of anti-TNF inhibitors.