18 research outputs found
Controlled Delivery of an Anti-Inflammatory Toxin to Macrophages by Mutagenesis and Nanoparticle Modification
Advances in drug delivery systems (DDSs) have enabled the specific delivery of drugs to target cells. Subtilase cytotoxin (SubAB) produced by certain enterohemorrhagic Escherichia coli strains induces endoplasmic reticulum (ER) stress and suppresses nitric oxide generation in macrophages. We previously reported that modification of SubAB with poly(D,L-lactide-co-glycolic) acid (PLGA) nanoparticles (SubAB-PLGA NPs) increased intracellular uptake of SubAB and had an anti-inflammatory effect on macrophages. However, specific delivery of SubAB to macrophages could not be achieved because its effects on other cell types were not negligible. Therefore, to suppress non-specific SubAB binding, we used low-binding mutant SubABS35A (S35A) in which the 35th serine of the B subunit was mutated to alanine. In a macrophage cell line, PLGA NPs modified with S35A (S35A-PLGA NPs) induced ER stress and had anti-inflammatory effects similar to WT-PLGA NPs. However, in an epithelial cell line, S35A-PLGA NPs induced lower ER stress than WT-PLGA NPs. These results suggest that S35A is selectively delivered to macrophages rather than epithelial cells by modification with PLGA NPs and exerts anti-inflammatory effects. Our findings provide a useful technique for protein delivery to macrophages and encourage medical applications of DDSs for the treatment of inflammatory diseases
Synthesis of Pegylated Manganese Protoporphyrin as a Catalase Mimic and Its Therapeutic Application to Acetaminophen-Induced Acute Liver Failure
Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor ÎşB signaling in macrophages
Identification of Subtilase Cytotoxin (SubAB) Receptors Whose Signaling, in Association with SubAB-Induced BiP Cleavage, Is Responsible for Apoptosis in HeLa Cellsâ–ż
Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), causes the 78-kDa glucose-regulated protein (GRP78/BiP) cleavage, followed by induction of endoplasmic reticulum (ER) stress, leading to caspase-dependent apoptosis via mitochondrial membrane damage by Bax/Bak activation. The purpose of the present study was to identify SubAB receptors responsible for HeLa cell death. Four proteins, NG2, α2β1 integrin (ITG), L1 cell adhesion molecule (L1CAM), and hepatocyte growth factor receptor (Met), were identified to be SubAB-binding proteins by immunoprecipitation and purification, followed by liquid chromatography-tandem mass spectrometry analysis. SubAB-induced Bax conformational change, Bax/Bak complex formation, caspase activation, and cell death were decreased in β1 ITG, NG2, and L1CAM small interfering RNA-transfected cells, but unexpectedly, BiP cleavage was still observed. Pretreatment of cells with a function-blocking β1 ITG antibody (monoclonal antibody [MAb] P5D2) enhanced SubAB-induced caspase activation; MAb P5D2 alone had no effect on caspase activation. Furthermore, we found that SubAB induced focal adhesion kinase fragmentation, which was mediated by a proteasome-dependent pathway, and caspase activation was suppressed in the presence of proteasome inhibitor. Thus, β1 ITG serves as a SubAB-binding protein and may interact with SubAB-signaling pathways, leading to cell death. Our results raise the possibility that although BiP cleavage is necessary for SubAB-induced apoptotic cell death, signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways
Structural Determination of the Nanocomplex of Borate with Styrene–Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug
The
development of effective anticancer drugs is essential for
chemotherapy that specifically targets cancer tissues. We recently
synthesized a multifunctional water-soluble anticancer polymer drug
consisting of styrene–maleic acid copolymer (SMA) conjugated
with glucosamine and boric acid (BA) (SGB complex). It demonstrated
about 10 times higher tumor-selective accumulation compared with accumulation
in normal tissues because of the enhanced permeability and retention
effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial
damage, and thermal neutron irradiation. Gaining insight into the
anticancer effects of this SGB complex requires a determination of
its structure. We therefore investigated the chemical structure of
the SGB complex by means of nuclear magnetic resonance, infrared (IR)
spectroscopy, and liquid chromatography–mass spectrometry.
To establish the chemical structure of the SGB complex, we synthesized
a simple model compoundmaleic acid–glucosamine (MAG)
conjugateby using a maleic anhydride (MA) monomer unit instead
of the SMA polymer. We obtained two MAG–BA complexes (MAGB)
with molecular weights of 325 and 343 after the MAG reaction with
BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable
complex via an amide bond between MA and glucosamine and that BA bound
to glucosamine via a diol bond. As a result of this chemical design,
identified via analysis of MAGB, the SGB complex can release BA and
demonstrate toxicity to cancer cells through inhibition of lactate
secretion in mild hypoxia that mimics the tumor microenvironment.
For clinical application of the SGB complex, we confirmed that this
complex is stable in the presence of serum. These findings confirm
that our design of the SGB complex has various advantages in targeting
solid cancers and exerting therapeutic effects when combined with
neutron irradiation
Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons
Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein S-Guanylation
8‑Nitro-cGMP Enhances SNARE Complex Formation through S‑Guanylation of Cys90 in SNAP25
Nitrated
guanine nucleotide 8-nitroguanosine 3′,5′-cyclic
monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen
species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP
formation and its protein targets in the normal brain have not been
identified. Here, we investigated 8-nitro-cGMP generation and protein
S-guanylation in the rodent brain. Immunohistochemistry indicated
that 8-nitro-cGMP was produced by neurons, such as pyramidal cells
and interneurons. Using liquid chromatography-tandem mass spectrometry,
we determined endogenous 8-nitro-cGMP levels in the brain as 2.92
± 0.10 pmol/mg protein. Based on S-guanylation proteomics, we
identified several S-guanylated neuronal proteins, including SNAP25
which is a core member of the soluble <i>N</i>-ethylmaleimide
sensitive factor attachment protein receptor (SNARE) complex. SNAP25
post-translational modification including palmitoylation, phosphorylation,
and oxidation, are known to regulate neurotransmission. Our results
demonstrate that S-guanylation of SNAP25 enhanced the stability of
the SNARE complex, which was further promoted by Ca<sup>2+</sup>-dependent
activation of neuronal nitric oxide synthase. Using site-directed
mutagenesis, we identified SNAP25 cysteine 90 as the main target of
S-guanylation which enhanced the stability of the SNARE complex. The
present study revealed a novel target of redox signaling via protein
S-guanylation in the nervous system and provided the first substantial
evidence of 8-nitro-cGMP function in the nervous system