Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored.
The transcription factor NF-E2-related nuclear factor 2 (Nrf2) regulates expression of genes that protect cells from oxidative damage. Here, we characterized nitric oxide (
Fluorescent nanosensor probes have
suffered from limited molecular
recognition and a dearth of strategies for spatial-temporal operation
in cell culture. In this work, we spatially imaged the dynamics of
nitric oxide (NO) signaling, important in numerous pathologies and
physiological functions, using intracellular near-infrared fluorescent
single-walled carbon nanotubes. The observed spatial-temporal NO signaling
gradients clarify and refine the existing paradigm of NO signaling
based on averaged local concentrations. This work enables the study
of transient intracellular phenomena associated with signaling and
therapeutics.
Nitrosothiols
(RSNOs) have been proposed as important intermediates
in nitric oxide (NO•) metabolism, storage, and transport
as well as mediators in numerous NO-signaling pathways. RSNO levels
are finely regulated, and dysregulation is associated with the etiology
of several pathologies. Current methods for RSNO quantification depend
on indirect assays that limit their overall specificity and reliability.
Recent developments of phosphine-based chemical probes constitute
a promising approach for the direct detection of RSNOs. We report
here results from a detailed mechanistic and kinetic study for trapping
RSNOs by three distinct phosphine probes, including structural identification
of novel intermediates and stability studies under physiological conditions.
We further show that a triarylphosphine-thiophenyl ester can be used
in the absolute quantification of endogenous GSNO in several cancer
cell lines, while retaining the elements of the SNO functional group,
using an LC–MS-based assay. Finally, we demonstrate that a
common product ion (m/z = 309.0),
derived from phosphine–RSNO adducts, can be used for the detection
of other low-molecular weight nitrosothiols (LMW-RSNOs) in biological
samples. Collectively, these findings establish a platform for the
phosphine ligation-based, specific and direct detection of RSNOs in
biological samples, a powerful tool for expanding the knowledge of
the biology and chemistry of NO•-mediated phenomena.
Hypoxia-inducible factor-1 alpha (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation and proteasomal degradation. During hypoxia, degradation decreases and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α, and thus NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous-NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.
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