Protein tyrosine sulfation: from molecular mechanisms and pathophysiology to analytical advances and therapeutic translation.

Protein tyrosine sulfation (PTS), catalyzed by the Golgi-resident tyrosylprotein sulfotransferases 1 and 2 (TPST1 and TPST2) using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfuryl donor, regulates extracellular protein-protein interactions but remains analytically challenging because sulfotyrosine is labile and nearly isobaric with phosphotyrosine. This review summarizes the molecular and structural basis of TPST-mediated sulfation, its roles in infection, cancer, immunity, and cardiovascular disease, and recent advances in sulfoproteomics, including open-search mass spectrometry and single-molecule nanopore sensing. We critically evaluate non-canonical cytoplasmic and nuclear sulfation claims and emphasize the need for topology-aware, orthogonal validation. We also propose the PAPS pool hypothesis as a testable framework for examining how PAPS synthesis, Golgi transport, and pathway-specific consumption may shape protein and glycan sulfation, together with a four-step validation framework for non-canonical assignments and a compartmentalized kinase-crosstalk model for selected topological discrepancies. Finally, we assess emerging therapeutic strategies and their key limitations, including selectivity, Golgi access, pharmacokinetics, toxicity, and effects on other PAPS-dependent pathways. Overall, this review provides an evidence-based framework for defining the boundaries of PTS and guiding future analytical and therapeutic studies.