An Azidoribose Probe to Track Ketoamine Adducts in Histone Ribose Glycation

Abstract

Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose (5-AR), as well as the synthesis of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe's utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.

Figure 1.

5-AR glycates histones in vitro. (A) Schematic depicting incubation of histones with probes followed by click labeling with DBCO Cy5. (B) Incubation of recombinant histones with PBS, 5 mM of d-ribose, 1-AR, or 5-AR for 8 h followed by click-labeling and in-blot fluorescence analysis. (C) Histones glycated with 5 mM of 5-AR, labeled with DBCO Cy5, analyzed side-by-side by in-blot fluorescence and Coomassie Brilliant Blue (CBB) for loading control.

Figure 2.

Ribose permeates cells and nuclei to glycate histones. (A) Glycation of isolated 293T nuclei with PBS or 5 mM of d-ribose, 1-AR, or 5-AR for 8 h, followed by DBCO Cy5 labeling and in-blot fluorescence visualization and western blot analysis. (B) Glycation of live 293T cells with PBS or 5 mM of d-ribose, 1-AR, or 5-AR, followed by hypotonic isolation of nuclei, DBCO Cy5 click labeling, in-blot fluorescence and western blot analysis. (C) Quantification of RiboLys on chromatin isolations from 293T cells incubated with 5-AR, 1-AR, or increasing concentrations of d-ribose. (D) Quantification of Az-RiboLys on chromatin isolations from 293T cells incubated with 5-AR or 1-AR. The MRM transitions used for quantification are shown. (E) Glycation of live 293T cells with increasing amounts of d-ribose followed by high-salt histone extraction, and western blot post-translational modification (PTM) analysis with the indicated antibodies.

Figure 3.

Azidoribose probes can be used to selectively enrich ribose glycation adducts from 293T cells for further analyses. (A) Schematic depicting treatment of isolated nuclei with 5-AR and 1-AR, DBCO Cy5 click labeling, enrichment, digestion, and label-free quantitative proteomics (LFQ) analysis. (B) In-blot fluorescence and western blot showing background labeling and selective enrichment of glycated proteins from AR-treated isolated nuclei. (C) Volcano plot depicting differences in iBAQ values from 5-AR- over 1-AR-enriched sample groups against negative logarithmic (base 10) p-values of the t-test performed from multiple replicates. All identified proteins represented in gray, validated proteins BMI-1 and SMARCC1 labeled, and select protein groups highlighted in separate colors. (D) Western blot of representative output fractions validating the labeling and enrichment of two chromatin remodeling proteomic hits. (E) Binding protein (BP) gene ontology analysis of gene list from set of protein target dataset (p < 0.05 and unique peptides >2).

Figure 4.

FN3K deglycates 5-AR-glycated cytosolic proteins in live cells. (A) Cytosolic and nuclear fractions of FN3K-transfected and wild-type 293T cells after 5-AR glycation and DBCO Cy5 labeling. (B) Model depicting cytosolic but not nuclear ribose deglycation activity of FN3K.

Scheme 1.

Synthesis of (A) 5-Azidoribose (5-AR) and (B) 1-Azidoribose (1-AR) Probes to Track Ribose Glycation