Exploring metabolic pathways and regulation through functional chemoproteomic and metabolomic platforms

Abstract

Genome sequencing efforts have revealed a strikingly large number of uncharacterized genes, including poorly or uncharacterized metabolic enzymes, metabolites, and metabolic networks that operate in normal physiology, and those enzymes and pathways that may be rewired under pathological conditions. Although deciphering the functions of the uncharacterized metabolic genome is a challenging prospect, it also presents an opportunity for identifying novel metabolic nodes that may be important in disease therapy. In this review, we will discuss the chemoproteomic and metabolomic platforms used in identifying, characterizing, and targeting nodal metabolic pathways important in physiology and disease, describing an integrated workflow for functional mapping of metabolic enzymes.

Figure 1

Activity-based protein profiling (ABPP). A) Examples of activity-based probes. B) gel-based ABPP and ABPP-MudPIT platforms for fluorescent and mass-spectrometry-based analysis of enzyme activities. “Rh” denotes rhodamine and “B” denotes biotin.

Figure 2

Competitive ABPP platforms. A) ABPP-SILAC; B) fluopol-ABPP; c) isotope-ABPP

Figure 3

Targeted and untargeted metabolomic profiling platforms. Targeted metabolomic approaches oftentimes are performed by multiple reaction monitoring-based LC-MS/MS methods. Untargeted metabolomic approaches are performed through collected all mass spectra and using bioinformatics tools (e.g. XCMS) to identify, integrate, and compare ions between comparison groups.

Figure 4

Examples of metabolic pathways elucidated by metabolomic profiling. A) ABHD12 was characterized as a LPS hydrolase. ABHD12 deficiency leads to an accumulation in LPS, activates toll-like receptor 2, induces neuroinflammation, and causes a neurodegenerative disease known as PHARC. B) MAGL was shown to play critical roles of controlling endocannabinoid and eicosanoid signaling in the brain and fatty acid and fatty acid-derived oncogenic signaling lipids in cancer. MAGL blockade in brain leads to elevations in 2-AG endocannabinoid signaling and anxiolytic and anti-nociceptive effects, while also lowering the primary arachidonic acid precursor pool for pro-inflammatory eicosanoid production, leading to reduced inflammation, and neuroprotection against neurodegenerative diseases. In cancer, MAGL blockade leads to reduced fatty acids and fatty acid derived signaling lipids such as prostaglandins and lysophosphatidic acid, which impairs cancer pathogenicity. C) In cancer, AGPS was shown to not only control ether lipid synthesis, but also fatty acid metabolism that balances structural lipids with signaling lipids that fuel cancer. AGPS knockdown in cancer cells leads to reductions in the tumor-promoting lipid lysophosphatidic acid-ether, leading to a diversion of arachidonic acid away from oncogenic prostaglandins and towards structural lipids, leading to a net impairement in cancer aggressiveness.

Figure 5

Examples of post-translational modifications regulated by specific metabolites. a) isoTOP-ABPP was used to map distinct hyper-reactive cysteines that were particularly sensitive to modification by the endogenously produced reactive electrophile 4-hydro2-nonenal. B) A novel post-translational regulation by a glycolytic product 1,3-bisphosphoglycerate that modifies lysines on glycolytic enzymes and inhibits their activity.

Figure 6

Metabolic control of epigenetic features. A) Mutant IDH1 R132H generates 2-HG, inhibiting histone demethylases to fuel cancer progression through multiple mechanisms. 2) ACL activity controls acetyl-CoA levels to confer nutrient-responsive histone acetylation and gene expression, altering glucose metabolism and cellular programming of macromolecular synthesis and energy production.C) Increased expression of NNMT in cancer decreases histone methylation, increasing expression of tumor promoting genes. In white adipose tissue, inactivation of NNMT increases polyamine synthesis and histone methylation, elevating gene expression and activity of enzymes critical for high energy expenditure.