Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract Metals are essential micronutrients that are required for proper functioning of cells and organisms, and their distribution and speciation (the metallome) is tightly controlled by complex homeostatic machinery. Deviations from metal homeostasis are associated with multiple pathologies, environmental metal contamination, and metal deficiencies, which all have important effects on cellular function. The metallome is defined by two main metal ion pools: the labile metal ion pool in which metals are weakly bound and exchangeable and the tightly-bound metal ion pool in which metals are ligated to biomolecules with high affinity. Metalloproteins that constitute the tightly-bound metal ion pool represent one third of the proteome and within these proteins, metals have diverse structural and catalytic functions. These metalloproteins exist in several metalation states, including apo (metal- free), holo (metal-bound), and mismetalated. These states are controlled by several factors, including metal ion availability and the presence of metal transporters or metallochaperones. The Que lab is interested in studying how the metalation state of metalloproteins in the cellular environment responds to changes in metal ion availability and other biological stimuli. There are several broad questions that drive our research: (1) are there biological or abiological scenarios in which metals in metalloproteins are labile and exchangeable rather than tightly-bound, making these proteins vulnerable to metal ion loss or demetalation? (2) What factors govern how vulnerable a metalloprotein is to demetalation? (3) How does this knowledge impact our understanding of the biological function of these enzymes and their relation to human health and disease? Our strategy to tackle these questions is to develop fluorescent probes that allow us to monitor metalloprotein expression and metalation dynamics in live cells. We specifically target metalloenzymes due to the important chemical reactions they catalyze and the presence of an open coordination site in their active site that can be targeted using metal- binding, inhibitor-inspired fluorogenic molecules. In the previous granting period, we demonstrated our ability to produce fluorescence turn-on probes for the ubiquitously expressed carbonic anhydrase (CA) and antibiotic resistance enzyme New Delhi Metallo--lactamase (NDM). These probes revealed that CA-bound zinc is not labile in cells whereas NDM-bound zinc is labile, with metal loss being observed after NDM-expressing E. coli were treated with metal chelators. In the next five years, our first goal is to improve the properties of our fluorescent probes to increase their sensitivity and enable multiplexed imaging. Our second goal is to target additional metalloenzymes in order to expand our biological scope and elaborate on our probe design principles. Our third goal is to further explore the lability of zinc sites in three clinically relevant metallo--lactamases, including NDM, and the consequences of this lability on antibiotic resistant infections, the nutritional immunity response, and therapeutic strategies. Metalloenzyme probes developed as part of this award also have potential applications in diagnostics and high-throughput drug screening for pathologies associated with these species.
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