DHA and Its Metabolites Have a Protective Role against Methylmercury-Induced Neurotoxicity in Mouse Primary Neuron and SH-SY5Y Cells.

The consumption of fish now involves a risk of methylmercury (MeHg) exposure but also provides the benefit of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) such as docosahexaenoic acid (DHA). Some epidemiological studies have suggested that the intake of DHA can alleviate the neurotoxicity of MeHg, but the underlying mechanism is not known. Herein, we observed that pretreatment with 0.1-1 µM DHA suppressed MeHg-induced cytotoxicity in human neuroblastoma (SH-SY5Y) cells and mouse primary neuronal cells. These effects of DHA were canceled in the presence of the retinoid X receptor (RXR) antagonist UVI3003. An RXR agonist, bexarotene, suppressed the cytotoxicity of MeHg. DHA also suppressed the MeHg-induced production of reactive oxygen species (ROS) via an induction of antioxidant genes (catalase and SOD1). Pretreatment with DHA did not change the incorporation of MeHg. We showed previously that in the brain, the intake of DHA increased the level of 19,20-DHDP, which is the metabolite produced by cytochrome P450 and soluble epoxide hydrolase from DHA. In the present study, we observed that 19,20-DHDP also suppressed neurotoxicity from MeHg. These results indicate that DHA and its metabolites have a protective role in MeHg-induced neurotoxicity.

Photoinduced in†Vivo Magnetic Resonance Imaging (MRI) with Rapid CO Release from an MnCO-Protein Needle Composite.

Construction of an artificial protein needle (PN), which includes the membrane puncturing needle domain of bacteriophage T4 conjugated to Mn carbonyl (MnCO) complexes, is reported. The responsiveness to visible light of the MnCO complex makes it useful as a photoinduced in vivo magnetic resonance imaging contrast reagent (MRI CR), because the PN carrier has the potential to deliver the MnCO complex into mouse tumors with retention of coordination structure within the in vivo environment. Moreover, the composite has higher relaxivity and longer circulation as an MRI CR than the corresponding MnCO complex. These results demonstrate construction of a responsive in vivo MRI CR by using an artificial metalloprotein.

A Photoactive Carbon-Monoxide-Releasing Protein Cage for Dose-Regulated Delivery in Living Cells.

Protein cages can serve as bioinorganic molecular templates for functionalizing metal compounds to regulate cellular signaling. We succeeded in developing a photoactive CO-releasing system by constructing a composite of ferritin (Fr) containing manganese-carbonyl complexes. When Arg52 adjacent to Cys48 of Fr is replaced with Cys, the Fr mutant stabilizes the retention of 48 Mn-carbonyl moieties, which can release the CO ligands under light irradiation, although wild-type Fr retains very few Mn moieties. The amount of released CO is regulated by the extent of irradiation. This could reveal an optimized dose for cooperatively activating the nuclear factor κB (NF-κB) in mammalian cells and the tumor necrosis factor α (TNF-α). These results suggest that construction of a CO-releasing protein cage will advance of research in CO biology.

A metal carbonyl-protein needle composite designed for intracellular CO delivery to modulate NF-κB activity.

Carbon monoxide (CO) has been recognized as a messenger for signal transduction in living cells and tissues. For intracellular CO delivery, several metal carbonyl complexes have been used as CO-releasing molecules (CO-RMs). To improve the properties of CO-RMs, such as the stability and the CO release rate, ligands and carriers of the metal complexes have been exploited. Here we report the development of an efficient intracellular CO delivery system using a protein scaffold. We used a protein needle reconstructed from gene product 5 of bacteriophage T4, which has high cellular permeability and stability. When ruthenium carbonyl complexes are conjugated to the needle using a His-tag triad at the C-terminus, the resulting composite has a significantly higher cellular uptake efficiency of Ru carbonyl and a 12-fold prolonged CO release rate relative to Ru(CO)3Cl(glycinate), a widely used CO-RM. We demonstrate that CO delivered by the composite activates the transcriptional factor nuclear factor-kappaB (NF-κB), which in turn leads to significant induction of expression of its target genes, HO1, NQO1, and IL6, through generation of reactive oxygen species (ROS). The signaling pathway is distinct from that of tumor necrosis factor (TNF)-α-induced activation of NF-κB. The protein needle-based CO-RM can be exploited to elucidate the biological functions of CO and used in the development of protein-based organometallic tools for modulation of cellular signaling.

Design of biomaterials for intracellular delivery of carbon monoxide.

Carbon monoxide (CO) is recognized as one of the most important gas signaling molecules involved in governing various therapeutic responses. Intracellular generation of CO is spatiotemporally controlled by catalytic reactions of heme oxygenases (HOs). Thus, the ability to control intracellular CO delivery with modulation of the CO-release rate in specific amounts and locations is expected to improve our fundamental understanding of the functions of CO and the development of clinical applications. For this purpose, CO-releasing molecules (CORMs) have been developed and investigated in vitro and in vivo. Most CORMs are based on transition metal carbonyl complexes. Recently, various biomaterials consisting of metal carbonyls with biomacromolecular scaffolds have been reported to improve the properties of bare metal carbonyls. In this mini-review, current progress in CO delivery, recent strategies for the development of CORMs, and future directions in this field are discussed.

Use of the confined spaces of apo-ferritin and virus capsids as nanoreactors for catalytic reactions.

Self-assembled protein cages providing nanosized internal spaces which are capable of encapsulating metal ions/complexes, enzymes/proteins have great potential for use as catalytic nanoreactors in efforts to mimic confined cellular environments for synthetic applications. Despite many uses in biomineralization, drug delivery, bio-imaging and so on, applications in catalysis are relatively rare. Because of their restricted size, protein cages are excellent candidates for use as vessels to exert control over reaction kinetics and product selectivity. Virus capsids with larger internal spaces can encapsulate multiple enzymes and can mimic natural enzymatic reactions. The apo-ferritin cage is known to accommodate various metal ions/complexes and suitable for organic transformation reactions in an aqueous medium. This review highlights the importance, prospects and recent significant research on catalytic reactions using the apo-ferritin cage and virus capsids.

Preparation of a cross-linked porous protein crystal containing Ru carbonyl complexes as a CO-releasing extracellular scaffold.

Protein crystals generally are stable solid protein assemblies. Certain protein crystals are suitable for use as nanovessels for immobilizing metal complexes. Here we report the preparation of ruthenium carbonyl-incorporated cross-linked hen egg white lysozyme crystals (Ru·CL-HEWL). Ru·CL-HEWL retains a Ru carbonyl moiety that can release CO, although a composite of Ru carbonyl-HEWL dissolved in buffer solution (Ru·HEWL) does not release CO. We found that treatment of cells with Ru·CL-HEWL significantly increased nuclear factor kappa B (NF-κB) activity as a cellular response to CO. These results demonstrate that Ru·CL-HEWL has potential for use as an artificial extracellular scaffold suitable for transport and release of a gas molecule.

Intracellular CO release from composite of ferritin and ruthenium carbonyl complexes.

Protein cages have been utilized as templates in the development of biomaterials. Here we report protein engineering of the ferritin (Fr) cage for encapsulating carbon monoxide releasing molecules (CORMs) and release of CO gas which serves as a cell signaling molecule. The protein cages enable us to increase the half-life for CO release, providing a release rate that is 18-fold slower than the rate of a typical CORM, Ru(CO)3Cl(glycinate) (CORM-3). Moreover, the uptake ratio of the composite is about 4-fold greater than that of CORM-3. We found that these effects enhance the activation of nuclear factor κB 10-fold higher than CORM-3. The protein cage of Fr thus provides the basis for new CORMs that can be used for in vitro cell research.