A mitochondrial-targeted activity-based sensing probe for ratiometric imaging of formaldehyde reveals key regulators of the mitochondrial one-carbon pool.

Formaldehyde (FA) is both a highly reactive environmental genotoxin and an endogenously produced metabolite that functions as a signaling molecule and one-carbon (1C) store to regulate 1C metabolism and epigenetics in the cell. Owing to its signal-stress duality, cells have evolved multiple clearance mechanisms to maintain FA homeostasis, acting to avoid the established genotoxicity of FA while also redirecting FA-derived carbon units into the biosynthesis of essential nucleobases and amino acids. The highly compartmentalized nature of FA exposure, production, and regulation motivates the development of chemical tools that enable monitoring of transient FA fluxes with subcellular resolution. Here we report a mitochondrial-targeted, activity-based sensing probe for ratiometric FA detection, MitoRFAP-2, and apply this reagent to monitor endogenous mitochondrial sources and sinks of this 1C unit. We establish the utility of subcellular localization by showing that MitoRFAP-2 is sensitive enough to detect changes in mitochondrial FA pools with genetic and pharmacological modulation of enzymes involved in 1C and amino acid metabolism, including the pervasive, less active genetic mutant aldehyde dehydrogenase 2*2 (ALDH2*2), where previous, non-targeted versions of FA sensors are not. Finally, we used MitoRFAP-2 to comparatively profile basal levels of FA across a panel of breast cancer cell lines, finding that FA-dependent fluorescence correlates with expression levels of enzymes involved in 1C metabolism. By showcasing the ability of MitoRFAP-2 to identify new information on mitochondrial FA homeostasis, this work provides a starting point for the design of a broader range of chemical probes for detecting physiologically important aldehydes with subcellular resolution and a useful reagent for further studies of 1C biology.

One Carbon to Rule Them All: Formaldehyde is a One-Carbon Signal Connecting One-Carbon Metabolism and Epigenetic Methylation.

Formaldehyde is commonly thought of as an environmental toxin or laboratory fixation reagent, but there is a growing appreciation for its broader physiological contributions as a naturally generated one-carbon metabolite across all kingdoms of life. In this In Focus article, we summarize emerging advances in the field that show how formaldehyde plays diverse roles as a one-carbon signal in DNA damage, one-carbon metabolism, and epigenetic regulation.

A Transfer Hydrogenation Approach to Activity-Based Sensing of Formate in Living Cells.

Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

Metalloallostery and Transition Metal Signaling: Bioinorganic Copper Chemistry Beyond Active Sites.

Transition metal chemistry is essential to life, where metal binding to DNA, RNA, and proteins underpins all facets of the central dogma of biology. In this context, metals in proteins are typically studied as static active site cofactors. However, the emergence of transition metal signaling, where mobile metal pools can transiently bind to biological targets beyond active sites, is expanding this conventional view of bioinorganic chemistry. This Minireview focuses on the concept of metalloallostery, using copper as a canonical example of how metals can regulate protein function by binding to remote allosteric sites (e.g., exosites). We summarize advances in and prospects for the field, including imaging dynamic transition metal signaling pools, allosteric inhibition or activation of protein targets by metal binding, and metal-dependent signaling pathways that underlie nutrient vulnerabilities in diseases spanning obesity, fatty liver disease, cancer, and neurodegeneration.

T-REX on-demand redox targeting in live cells.

This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t1/2 <1-2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4-24 h, depending on the nature of the pathway and the type of readouts used.