This proposal focuses on understanding the role of zinc finger (ZF) proteins in H2S signaling. ZFs are highly abundant proteins that play critical roles in transcriptional and translational regulation. ZFs are cysteine rich and use zinc as a cofactor. The role of zinc in ZFs has long been considered to be solely structural; however, evidence for a new paradigm is emerging in which the zinc ion plays a more reactive role. This evidence includes the finding that hydrogen sulfide (H2S) targets ZF proteins. H2S is an endogenous gasotransmitter that has gained attention in recent years as a biological signaling molecule via cysteine persulfidation. This process is now proposed as a type of protein post-translational modification that plays a critical role in cell signaling. It has been shown that H2S signaling is associated with inflammation, neuromodulation, transcription and metabolism, with aberrant H2S activity linked to cardiovascular, neurological, inflammatory and metabolic diseases. Despite the growing appreciation for the importance of H2S in biology, our understanding of the biological roles of H2S is limited. The macromolecular targets of H2S are not clearly defined, and the mechanism of H2S action has not been determined in most cases. We have obtained exciting proteomics data that identifies numerous ZFs as cellular targets for H2S. We have also reported that a specific ZF, tristetraprolin (TTP), reacts with H2S in a zinc and O2 dependent manner to give persulfidated cysteine intermediates and modulate TTP structure. These structural changes transmit the `signal' from H2S by abrogating the RNA binding ability of TTP. In this proposal we aim to (i) identify the ZFs that are targeted by H2S in cells using a persulfide selective proteomics strategy (ii) determine the reactivity and mechanism of H2S with ZFs by applying a suite of biochemical and biophysical methods (including cryo-ESI-mass spectrometry, fluorescence quenching, EPR, XAS and NMR spectroscopies, and fluorescence anisotropy binding assays) to ZF proteins, ZF peptides and synthetic model complexes and (iii) determine how zinc speciation impacts H2S targeting of ZFs by using a combined LC-ICP-MS, proteomics and antibody/microarray strategy. This `molecules to proteomics' approach will allow us to understand the largely unexplored role of ZF proteins in H2S signaling, and has the potential to transform our understanding of H2S signaling.
The proposed studies will provide fundamental knowledge on how H2S interacts with zinc fingers in cell signaling pathways. H2S and zinc finger proteins both play important roles in inflammation, transcription, metabolism and neuromodulation. Deciphering the interplay between H2S and zinc on ZF protein function will contribute to our understanding of their significance in human health.
