Lighting Up and Identifying Metal-Binding Proteins in Cells

Abstract

Metal ions, either essential or therapeutic, play critical roles in life processes or in the treatment of diseases. Proteins and enzymes are involved in metal homeostasis and the action of metallodrugs. Imaging and identifying these metal-binding proteins will facilitate the elucidation of metal-mediated life processes. The emerging research field of metallomics and metalloproteomics has significantly advanced our understanding of metal homeostasis and the roles that metals play in biology and medicine. Fluorescence-based metalloproteomics offers the possibility of not only visualization but also identification of metal-binding proteins in living cells and tissues. Herein, we summarize different strategies of labeling and tracking of metal-binding proteins with the aid of fluorescent probes. We highlight several examples as showcases of how this fluorescence-based metalloproteomics approach could be utilized in metallobiology and chemical biology. In conclusion, we also discuss the advantages and limitations of fluorescence-based metalloproteomics approaches and point out future directions of metalloproteomics including development of more sensitive and selective fluorescence probes, integration with other omics approaches, as well as application of emerging advanced super-resolution imaging techniques that utilize fluorescent molecules or proteins. We aim to attract more scientists to engage in this exciting field.

Figure 1

(a) Diagram illustrates the binding of As(III) to thiols in a protein. (b) Identification of the captured arsenic-binding proteins by PAzPAO. Adapted with permission from ref (39). Copyright 2016 Wiley-VCH. (c) Chemical structure of organoarsenic probe As-AC. (d) GO enrichment analysis of 37 As-binding proteins detected by As-AC probe. (e) Immunolocalization of endogenous Hsp60 (green) with p53 (red) and survivin (red), showing the disruption of the protein–protein interactions upon ATO treatment in cell. (d, e) were reproduced with permission from ref (40). Available under Creative Commons Attribution 4.0 International License.

Figure 2

(a) Chemical structures of NTA-FITC-Ni²⁺, Bis-NTA-Fluo, and ^FEW646 tris-NTA. (b) Confocal image of ^FEW646 tris-NTA labeling a plasma membrane protein. Adapted with permission from ref (55). Copyright 2016 American Chemical Society. (c) Schematic diagram of photoaffinity labeling of protein by metal-NTA based fluorescence probes. (d) Chemical structures of Ni-NTA-based photoaffinity labeling probes with NTA conjugated with different fluorophores. (e) Cartoon representation of transferrin with the Fe³⁺-TRACER binding site at the N-lobe (Light blue). The binding site is enlarged with Fe³⁺-TRACER interacting residues shown in sticks. Reproduced with permission from ref (58). Copyright 2018 the Royal Society of Chemistry. (f) Confocal imaging of P. aeruginosa cells treated with Ga³⁺-NTA-AC (also known as Ga(III)–TRACER). (g) SDS-PAGE separation of P. aeruginosa cell lysate showing Ga³⁺-NTA-AC labeled proteins. (h) Volcano plot showing the fold change and significance of protein intensities detected in the competitive Ga-IMAC experiment. Nodes in green color represent proteins with fold change >2 and p-value (of fold change) < 0.01. (f,g,h) were reproduced with permission from ref (60). Available under Creative Commons Attribution 4.0 International License.

Figure 3

(a) Chemical structure of chromium probe Cr³⁺-NTA-AC. (b) Colocalization confocal images of Cr³⁺-NTA-AC in mitochondria. (c) Dose-dependent inhibition of ATP synthase activity by Cr³⁺ treatment in HepG2 cells under hyperglycaemia condition. ATP5B inhibitor Octyl-α-ketoglutarate (O-KG) is used as a control. n = 3; mean ± SEM (d) Cr³⁺ activates AMPK and ACC in db/db mice. n = 6; mean ± SEM (e) The proposed scheme shows that Cr³⁺ ameliorates hyperglycemia stress by inhibiting ATP synthase and subsequent activation of AMPK in diabetic mice. All images were reproduced with permission from ref (26). Available under Creative Commons Attribution 4.0 International License.

Figure 4

(a) Schematic diagram illustrating conditional protein labeling. (b) Chemical structures of Alzin-1 and Alzin-2. (c) Chemical structures of CD694 and CD433. (d) Chemical structure of Cu(I) ligand (Blue) with cross-linking moiety quinone methide (Orange) and fluorophores FL and AcFL. (e) Chemical structure of IG1- FM.

Figure 5

(a) Schematic diagram illustrating ligand-directed protein labeling. (b) Chemical structure of NAP8 that comprised fluorescent tag (red), war head (yellow) and affinity moiety (blue). (c) Immunofluorescence analysis of the presence of MMP14 on mammary spheroids. (d) Confocal images of active MMP-14 which are shown with white arrow. Noted that the 260C is a mutant form of MMP-14 without interfering with its activity. (e) Chemical structures of probe 1 and 2. (c,d) were adapted with permission from ref (67). Copyright 2018 American Chemical Society.

Figure 6

(a) Chemical structure of probe 3. (b) Wild-field and STORM imaging comparisons of mitochondrial membranes labeled with probe 3. Adapted with permission from ref (72). Copyright 2020 Elsevier B.V. (c) Structure of Halo tagged probe Rh-Gly-Halo and its bidding with HaloTag-protein. (d) PALM image of H₂B-Halo fusion proteins labeled with Rh-Gly-Halo. Panels (c,d) were adapted with permission from ref (74). Copyright 2019 American Chemical Society.