Schueppert, A. V. | bioRxiv Science

bioRxiv2026

Mapping the mammalian dark metabolome by in vivo isotope tracing

Despite decades of biochemical study, a comprehensive map of the mammalian metabolome remains elusive. Mass spectrometry-based metabolomics detects thousands of small molecule-associated signals in mammalian tissues, but it is currently unclear how many of these reflect products of endogenous metabolism. Here, we leverage systematic in vivo isotope tracing to infer the biosynthetic origins of unidentified metabolites. We administered 26 different isotopically labelled nutrients to mice, measured circulating and tissue metabolite labelling by mass spectrometry, and developed a statistical framework to infer the number of carbon atoms incorporated from each of these precursors into more than 4,000 putative metabolites. We show this information can be harnessed for biosynthesis-aware structure elucidation using a multimodal AI model that co-embeds isotopic labelling patterns with chemical structures. This approach revealed several previously unrecognized families of mammalian metabolites, including cysteine-derived alkylthiazolidines, dithioacetal mercapturic acid derivatives, short-chain N-acyltaurines, acylglycyltaurines, and N-oxidized taurines. It further uncovered a family of mevalonate-derived isoprenoid metabolites that includes 2,3-dihydrofarnesoic acid, which is markedly depleted in both mouse and human aging. Age-related depletion of these isoprenoids is driven by impaired coenzyme A synthesis. Our work establishes the biosynthetic precursors for thousands of unidentified metabolites and reveals multiple previously unrecognized branches of mammalian metabolism.

biochemistry↗

bioRxiv2024

LAT encodes T cell activation pathway balance

Immune cells transduce environmental stimuli into responses essential for host health via complex signaling cascades. T cells, in particular, leverage their unique T cell receptors (TCRs) to detect specific Human Leukocyte Antigen (HLA)-presented peptides. TCR activation is then relayed via linker for activation of T cells (LAT), a TCR-proximal disordered adapter protein, which organizes protein partners and mediates the propagation of signals down diverse pathways including NFAT and AP-1. Here, we studied how balanced downstream pathway activation is encoded in the amino acid sequence of LAT. To comprehensively profile the sequence-function relationship of LAT, we developed a pooled, single-cell, high-content screening approach in which a large series of mutants in the LAT protein were analyzed to characterize their effects on T cell activation. Measuring epigenetic, transcriptomic, and cell surface protein dynamics of single cells harboring distinct LAT mutants, we found functional regions spanning over 40% of the LAT amino acid sequence. Conserved sequence motifs for protein interactions along with charge distribution are critical sequence features, and contribute to interpretation of human genetic variation in LAT. While mutant defect severity spans from moderate to complete loss of function, nearly all defective mutants, irrespective of their position in LAT, confer balanced defects across all downstream pathways. To understand the molecular basis for this observation, we performed proximal protein labeling which demonstrated that disruption of LAT interaction with a single partner protein indirectly disrupts other partner interactions, likely through the dual roles of these proteins as effectors of downstream pathways and bridging factors between LAT molecules. Overall, we report widely distributed functional regions throughout a disordered adapter and a precise physical organization of LAT and interacting molecules which constrains signaling outputs. More broadly, we describe an approach for interrogating sequence-function relationships for proteins with complex activities across regulatory layers of the cell.

genomics↗