The UFMylation Machinery Is a Protein-Level Co-Expression Module Uncoupled from the Transcriptome: Evidence from High-Grade Serous Ovarian Cancer

Background: UFMylation is an evolutionarily conserved ubiquitin-like modification catalyzed by an enzymatic cascade comprising UBA5 (E1), UFC1 (E2), and UFL1 (E3). UFL1 acquires catalytic activity only upon assembly with DDRGK1 and CDK5RAP3 into a ternary complex, while UFSP2 mediates de-UFMylation. In the existing literature, the prevailing practice is to use the mRNA expression of these seven genes as a proxy for UFMylation activity and to associate such transcript-level scores with clinical outcomes. The reliability of this approach rests on the premise that transcript abundance reflects the true state of the pathway at the protein level—a premise that has not been validated. Methods: Using ovarian cancer datasets, we employed the mean of all pairwise Pearson correlations within a gene set (mean r) as a metric, compared it against expression-abundance-matched random gene sets to derive z-scores, and tested whether the seven UFMylation genes exist as a coordinately varying module across transcriptomic data and two mass spectrometry platforms in three independent ovarian cancer populations (five measurement cohorts). Results: The UFMylation machinery formed a coordinated module at the protein level, with z-scores of +5.14, +5.42, +7.94, and +3.24 across all four proteomic cohorts. In contrast, the same gene set did not hold at the transcript level (z = +0.07, indistinguishable from random gene sets). Protein-level coordination was precisely concentrated in the UFL1–DDRGK1–CDK5RAP3 ternary complex (pairwise correlations +0.57 to +0.89), whereas the enzymatic pair UBA5-UFC1 was weak and directionally unstable (r = −0.06 to +0.28), a pattern most consistent with coordination arising from assembly of the ternary complex. Clinically, the module score showed only a suggestive association with platinum resistance (FFPE cohort Δ = +0.249, p = 0.015; p = 0.375 after adjustment for center and neoadjuvant chemotherapy; direction reversed upon removal of a single center). In an exploratory analysis, intratumoral eosinophil infiltration was the only immune association passing all robustness checks (ρ = +0.38, q = 1.4 × 10−5), but this association could not be validated orthogonally and is reported as hypothesis-generating. At the single-cell level, the widely reported “tumor vs. normal” score difference was predominantly attributable to two artifacts—cell composition differences (101.7%; 95% CI 69–144%) and sequencing depth differences (approximately 94%). Conclusions: The UFMylation machinery is a protein-level module whose state cannot be reflected by the transcriptome; transcript-based UFMylation scores cannot serve as proxies for this pathway. This finding reconciles contradictory conclusions regarding UFMylation and cancer in the existing literature and provides a criterion for when gene-set scores are interpretable.