Molecular module of CmMYB15-like-Cm4CL2 regulating lignin biosynthesis of chrysanthemum (Chrysanthemum morifolium) in response to aphid (Macrosiphoniella sanborni) feeding.

Lignin is a major component of plant cell walls and a conserved basic defense mechanism in higher plants deposited in response to aphid infection. However, the molecular mechanisms of lignin biosynthesis in response to aphid infection and the effect of lignin on aphid feeding behavior remain unclear. We report that 4-Coumarate:coenzyme A ligase 2 (Cm4CL2), a gene encoding a key enzyme in the lignin biosynthesis pathway, is induced by aphid feeding, resulting in lignin deposition and reduced aphid attack. Upstream regulator analysis showed that the expression of Cm4CL2 in response to aphid feeding was directly upregulated by CmMYB15-like, an SG2-type R2R3-MYB transcription factor. CmMYB15-like binds directly to the AC cis-element in the promoter region of Cm4CL2. Genetic validation demonstrated that CmMYB15-like was induced by aphid infection and contributed to lignin deposition and cell wall thickening, which consequently enhanced aphid resistance in a Cm4CL2-dependent manner. This study is the first to show that the CmMYB15-like-Cm4CL2 module regulates lignin biosynthesis in response to aphid feeding.

Biochemical properties and crystal structure of formate-tetrahydrofolate ligase from Methylobacterium extorquens CM4.

Methylobacterium extorquens is a methylotroph model organism that has the ability to assimilate formate using the tetrahydrofolate (THF) pathway. The formate-tetrahydrofolate ligase from M. extorquens (MeFtfL) is an enzyme involved in the THF pathway that catalyzes the conversion of formate, THF, and ATP into formyltetrahydrofolate and ADP. To investigate the biochemical properties of MeFtfL, we evaluated the metal usage and enzyme kinetics of the enzyme. MeFtfL uses the Mg ion for catalytic activity, but also has activity for Mn and Ca ions. The enzyme kinetics analysis revealed that Km value of farmate was much higher than THF and ATP, which shows that the ligation activity of MeFtfL is highly dependent on formation concentration. We also determined the crystal structure of MeFtfL at 2.8 Å resolution. MeFtfL functions as a tetramer, and each monomer consists of three domains. The structural superposition of MeFtfL with FtfL from Moorella thermoacetica allowed us to predict the substrate binding site of the enzyme.

TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein.

A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan blue staining and MTT analyses showed that, similar to a TRAIL positive control, Jurkat cell proliferation was significantly inhibited. Flow cytometry analyses using Annexin V-fluorescein revealed that Jurkat cells treated with the TRAIL-CM4 fusion protein exhibited increased apoptosis. Laser confocal microscopy showed that APB-CM4 and the fusion protein TRAIL-CM4 can bind to Jurkat cell membranes and initiate their destruction. ABP-CM4 enhances the antitumor activity of TRAIL by targeting and damaging the tumor cell membrane. In antibacterial experiments, agar well diffusion and bacterial growth inhibition curve assays revealed concentration-dependent TRAIL-CM4 antibacterial activity against Escherichia coli K12D31. The expressed TRAIL-CM4 fusion protein exhibited enhanced antitumor and antibacterial activities. Fusion protein expression allowed the two different proteins to function in combination.

Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

Novel peptide myristoly-CM4 induces selective cytotoxicity in leukemia K562/MDR and Jurkat cells by necrosis and/or apoptosis pathway.

Purpose: There is an urgent need for the development of novel, effective, and less toxic drugs to treat leukemia. Antimicrobial peptides (AMPs) have received much more attention as alternative chemotherapeutic agents. This study aimed to examined the cytotoxicity of a novel AMP myristoly-CM4 against chronic myeloid leukemia cells (K562/MDR) and acute lymphocytic leukemia cells (Jurkat), and further investigated its selectivity to clarify the cytotoxic mechanism. Materials and methods: In this study, the cytotoxicity and selectivity of myristoly-CM4 against K562/MDR and Jurkat cells were assessed in vitro, and the anticancer mechanism responsible for its cytotoxicity and selectivity was further investigated. Results: Myristoly-CM4 was cytotoxic to these leukemia cell lines (IC50 2-4 µM) and was less cytotoxic to normal cells (HEK-293, L02 cells, peripheral blood mononuclear cells, and erythrocytes). Myristoyl-CM4 had stronger affinity to K562/MDR and Jurkat cells than to normal cells, while the contents of phosphatidylserine and sialic acids on the cell surfaces of K562/MDR and Jurkat cells were significantly higher than that of HEK293 cells. The myristoyl group effectively mediated the internalization of myristoyl-CM4 to leukemia cells. After internalization, myristoyl-CM4 could target mitochondria and affected mitochondrial function, including disruption of Δψm, increasing the accumulation of ROS, increasing the Bax/Bcl-2 ratio, activating caspase 9 and 3, and PARP to induce mitochondria-dependent apoptosis in both K562/MDR and Jurkat cells. Myristoyl-CM4 also induced K562/MDR cell necrosis by directive membrane disruption, and significantly decreased the level of P-glycoprotein in K562/MDR cells. Conclusion: These results suggested that myristoyl-CM4 showed selective cytotoxicity to leukemia K562/MDR and Jurkat cells by apoptosis and/or necrosis pathway. Myristoyl-CM4, thus, appears to be a promising candidate for leukemia treatment, including multidrug-resistant leukemia.

N-myristoylation of Antimicrobial Peptide CM4 Enhances Its Anticancer Activity by Interacting With Cell Membrane and Targeting Mitochondria in Breast Cancer Cells.

Development of antimicrobial peptides (AMPs) as highly effective and selective anticancer agents would represent great progress in cancer treatment. Here we show that myristoyl-CM4, a new synthetic analog generated by N-myristoylation of AMPs CM4, had anticancer activity against MCF-7, MDA-MB-231, MX-1 breast cancer cells (IC50 of 3-6 µM) and MDA-MB-231 xenograft tumors. The improved activity was attributed to the effect of myristoyl on the cell membrane. Flow cytometry and confocal laser scanning microscopy results showed that N-myristoylation significantly increased the membrane affinity toward breast cancer cells and also effectively mediated cellular entry. Despite increasing cytotoxicity against HEK293 and NIH3T3 cells and erythrocytes associated with its anticancer activity, myristoyl-CM4 maintained a certain selectivity toward breast cancer cells. Accordingly, the membrane affinity toward breast cancer cells was two to threefold higher than that of normal cells. Glycosylation analysis showed that sialic acid-containing oligosaccharides (including O-mucin and gangliosides) were important targets for myristoyl-CM4 binding to breast cancer cells. After internalization, co-localization analysis revealed that myristoyl-CM4 targeted mitochondria and induced mitochondrial dysfunction, including alterations in mitochondrial transmembrane potential, reactive oxygen species (ROS) generation and cytochrome c release. Activation of caspase 9, caspase 3 and cleavage of PARP were observed in MX-1, MCF-7, and MDA-MB-231 cells after myristoyl-CM4 treatment. The current work indicates that increasing hydrophobicity by myristoylation to modulate peptide-membrane interactions and then target mitochondria is a good strategy to develop AMPs as anticancer agents in the future.

Identification of a cyclodextrin inclusion complex of antimicrobial peptide CM4 and its antimicrobial activity.

Antibacterial peptide CM4 (ABP-CM4) is a natural product isolated from the silkworm Bombyx mori. It is a small cationic peptide with broad-spectrum activities against harmful microorganisms and may be used as a novel food preservative. However, ABP-CM4 lacks tertiary structure in water-like solutions, which makes it more susceptible to proteases and labile when exposed to air. In this study, ß-cyclodextrin (ß-CD) was chosen to form an inclusion complex with ABP-CM4, which enhanced the physical and chemical properties of ABP-CM4 but did not decrease its antibacterial activity. The storage stability and susceptibility to proteinases of ABP-CM4 were apparently improved under the protection of ß-CD. This technology could also be widely applied to other AMPs as an antimicrobial system to be used in the food industry.

THE ANTIBACTERIAL PEPTIDE CM4 INHIBITS REGENERATION OF SACCHAROMYCES CEREVISIAE PROTOPLAST BY DAMAGING ON THE PLASMA MEMBRANES / 微生物学通报

The antibacterial peptide CM4 having potent antifungal activity on inhibitiong the cell wall regeneration of Saccharomyces cerevisiae protoplasts.When the peptide increased,the ratio of the regenerated colonies drop obviously.To study the antifungal mechanism of the antibacterial peptide,fluorescence\|labeled peptide mixted with the protoplast of yeast,then confocal laser scanning microscopy were performed.The results indicated that the peptides interactted with the protoplast membrane and damaged the structure of the membrane,then the permeation of protoplast changed.Finally the protoplasts with the peptide failed to regenerate the cell walls leading to killing the cell.