New chemical probes for chemoproteomic profiling and redox biology investigation

Abstract

Emerging evidence presented the significance of cellular redox signaling in regulating protein expression and activity, hence modulating cancer proliferation and metastasis. The signaling was mediated by redox-active species that induce reversible redox modifications on amino acid residues, such as cysteine Oxidative post-translational modification (OxiPTM). This led to dynamic regulation of cellular events, including survival, proliferation, and metastasis. Yet, the underlying molecular mechanism of many redox signaling pathways remained undetermined. The transient and short-lived characteristics of redox species and the corresponding redox modification challenged the capture of the signaling process and recognition of the associated protein targets. The thesis presented the synthesis and application of two chemical probes incorporated with the chemoproteomic approach to study cellular redox signaling from two perspectives: (1) the cysteine OxiPTM upon ROS stimulation and (2) the superoxide-induced proximity modification of proteins associated with superoxide biology. In the first section, an acrylamide-based cysteine-reactive probe, NAIA-5, was developed with escalated reactivity due to pi-electrons delocalization from indole aromaticity. NAIA-5 demonstrated superior sensitivity, selectivity, stability, and low cytotoxicity over current cysteine-reactive probes. Unique functional proteins and cysteines identified by NAIA-5 in chemoproteomic analysis expanded the pool of profiled proteins and cysteines in the proteome. Applying NAIA-5 to a live cell cysteine trapping workflow, NAI/NAIA-5, enabled the identification of cysteines that underwent OxiPTM upon H2O2 stimulation. Unreported oxidation-sensitive cysteines in redox-associated protein classes, including kinases, reductases, and proteins related to gene processing and immunity. This highlights the advantage of live cell cysteine trapping available with NAI/NAIA-5 in preserving cysteine OxiPTM for redox signaling investigation. In the second section, superoxide-responsive probes, QMP-SOs, were synthesized, which carried a sensitive trigger for superoxide. This enables the release of highly electrophilic quinone methide for proximity protein labeling around superoxide hotspots in biological samples. QMP-SOs were coupled to tandem mass tag (TMT) to establish QMP-SO-TMT for identifying superoxide-associated proteins in the superoxide hotspots generated through chemical stimulations and in the hypoxic cancer model. In particular, QMP-SO-TMT identified DJ-1 as the redox effector in superoxide-signaling, which modulated cell pathways among proliferation, autophagy, and apoptosis in liver cancer cells under menadione treatment. QMP- SO guided enrichment assay has also been developed to study proteins associated with superoxide signaling in liver cancer cells stimulated with hepatocyte growth factor (HGF). In summary, two new chemical probes and chemoproteomic platforms have been developed, enabling redox-associated protein profiling and facilitating redox biology research.