4 research outputs found
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
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
Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein <i>S</i>‑Guanylation
8-Nitroguanosine 3′,5′-cyclic
monophosphate (8-nitro-cGMP)
is a nitrated derivative of guanosine 3′,5′-cyclic monophosphate
(cGMP) formed endogenously under conditions associated with production
of both reactive oxygen species and nitric oxide. It acts as an electrophilic
second messenger in the regulation of cellular signaling by inducing
a post-translational modification of redox-sensitive protein thiols
via covalent adduction of cGMP moieties to protein thiols (protein <i>S</i>-guanylation). Here, we demonstrate that 8-nitro-cGMP potentially <i>S</i>-guanylates thiol groups of cGMP-dependent protein kinase
(PKG), the enzyme that serves as one of the major receptor proteins
for intracellular cGMP and controls a variety of cellular responses. <i>S</i>-Guanylation of PKG was found to occur in a site specific
manner; Cys42 and Cys195 were the susceptible residues among 11 Cys
residues. Importantly, <i>S</i>-guanylation at Cys195, which
is located in the high-affinity cGMP binding domain of PKG, causes
persistent enzyme activation as determined by <i>in vitro</i> kinase assay as well as by an organ bath assay. <i>In vivo</i>, <i>S</i>-guanylation of PKG was demonstrated to occur
in mice without any specific treatment and was significantly enhanced
by lipopolysaccharide administration. These findings warrant further
investigation in terms of the physiological and pathophysiological
roles of <i>S</i>-guanylation-dependent persistent PKG activation
Exposure to Electrophiles Impairs Reactive Persulfide-Dependent Redox Signaling in Neuronal Cells
Electrophiles
such as methylmercury (MeHg) affect cellular functions
by covalent modification with endogenous thiols. Reactive persulfide
species were recently reported to mediate antioxidant responses and
redox signaling because of their strong nucleophilicity. In this study,
we used MeHg as an environmental electrophile and found that exposure
of cells to the exogenous electrophile elevated intracellular concentrations
of the endogenous electrophilic molecule 8-nitroguanosine 3′,5′-cyclic
monophosphate (8-nitro-cGMP), accompanied by depletion of reactive
persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP.
Exposure to MeHg also induced <i>S</i>-guanylation and activation
of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced
activation of redox signaling and the consequent cell damage were
attenuated by pretreatment with a reactive persulfide species donor.
In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity
impair the redox signaling regulatory mechanism, particularly of intracellular
reactive persulfide species and therefore lead to cellular pathogenesis.
Our results suggest that reactive persulfide species may be potential
therapeutic targets for attenuating cell injury by electrophiles