Development of a Nitric Oxide-Responsive Labeling Reagent for Proteome Analysis of Live Cells

Abstract

Nitric oxide (NO) is a pleiotropic signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems. To understand the biological functions of NO in detail, comprehensive characterization of proteins found in high-NO concentration environments is crucial. Herein, we describe the design of NO-responsive protein labeling reagents based on N-alkoxyacyl-o-phenylenediamine as an optimal reactive scaffold. The designed molecules can label proteins in murine macrophage cells in response to endogenously produced NO. The combination of NO-responsive protein labeling and liquid chromatography–tandem mass spectrometry technology allowed the characterization of the proteome under NO-generated conditions. Moreover, we demonstrated that our reagent was able to selectively mark and be used to fluorescently visualize NO-producing cells in a mixed cell culture system.

Chemical Tools for Endogenous Protein Labeling and Profiling

“…Use of AIZin has successfully identified proteins localized in Zn 2+ -rich vesicles generated in glioma cells stimulated with nitric oxide, by which these vesicles were able to be characterized as ER-and Golgi-related vesicles. The conditional proteomics approach recently has been extended to nitric oxide (NO)-responsive protein labeling reagents that are activated only in the presence of a high concentration of NO (Nishikawa et al, 2019). In this case, N-alkoxyacyl-o-phenylenediamine that can be converted to an electrophilic acyl benzotriazole in the presence of NO was harnessed as a novel NO-responsive reactive moiety (Figure 7F).…”

Mitochondrial mechanisms by which gasotransmitters (H2S, NO and CO) protect cardiovascular system against hypoxia

“…Formation of NO and RNS in cells is controlled by hormones, neurotransmitters, cytokines, and growth factors. With regard to the latter, NO and its derivatives act as secondary paracrine factors that transmit a signal from NO-producing cells to neighboring cells [28]. Intracellular NO and RNS receptors, which include Src proteintyrosine kinases, Ras family proteins, cytochrome oxidase, and soluble guanylate cyclase (sGC), are mainly proteins containing heme, active SH-and iron-sulfur groups.…”

“…They are localized both on the surface of the plasma membrane and in the internal compartments of cells. Most of the NO receptors are key components of intracellular signaling systems that regulate transcription factors AP-1, HIF-1, NF-kappaB, FoxO and the expression of their subordinate genes [28,29,74]. A feature that distinguishes NO from other high molecular weight signaling molecules is that the change in the redox potential of the cells switches the redoxdependent NO receptor and modifies the action of NO.…”

Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper

“…This approach is inspired by ligand-directed acyl imidazole (LDAI) reagents for proteomics and activity-based protein profiling (ABPP). [66][67][68][69][70][71][72] One of the advantages of utilizing this LDAI strategy for sensing and imaging purposes is to overcome a major challenge that small-molecule fluorescent probes can diffuse away from their target upon binding. CD probes address this problem by leveraging the direct coordination of Lewis acidic copper ions, resulting in covalent bond formation between fluorescent reporters and proximal proteins in the cell to preserve spatial information of localized copper hotspots by minimizing diffusion of the dye away from the site of the ABS reaction.…”