The Non-Proteolytic Role of the Ubiquitin-Associated Motif and Ubiquitin Cascade
Bibliographic record
Abstract
The heterotrimeric AMP-dependent protein kinase (AMPK) family is activated upon low energy states, conferring a switch towards energy-conserving metabolic pathways through immediate kinase actions on enzyme targets, and delayed alterations in gene expression through its nuclear re-localization. This family has known roles in stress resistance, lifespan, and nutrient adaptation, making it a relevant target for improving human health. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) AMPK, the SNF1 kinase, is readily activated by low glucose growth conditions making it a simple model to study the activation and activity of this enzyme class. These 3-subunit enzymes contain a highly conserved ubiquitin-associated (UBA) motif in most catalytic subunits whose function we have confirmed to be inhibitory. We demonstrated that UBA motif mutations significantly enhanced SNF1 kinase activation and biological activity including oxidative stress resistance and lifespan. Significantly, the enhanced UBA-dependent longevity and oxidative stress response is dependent on the Forkhead 1 (Fkh1) and Fkh2 stress response transcription factors, which in turn influence Snf1 catalytic subunit expression. The catalytic subunits are poly-ubiquitinated in yeast and humans with negative regulatory impacts on enzyme activity, yet the ubiquitin cascade enzymes involved are unknown. S. cerevisiae has 13 ubiquitin conjugating enzymes (E2) and a simplified repertoire of ubiquitin ligases (E3) that were screened for their impact on SNF1 kinase activity. We discovered that a discrete cluster of stress-responsive E2s (Ubc1, Ubc4, and Ubc5) and their associated E3 enzyme, Rsp5, affected discrete aspects of SNF1 kinase regulation, none through proteolytic degradation. The deletion of Ubc1 lowered catalytic subunit abundance, due to a synchronized decrease in Fkh1/2 proteins resulting in impaired SNF1 gene transcription. Furthermore, the Fkh1/2 cognate transcription factor, Hcm1, failed to enter the nucleus in the absence of Ubc1. This implies that Ubc1 acts indirectly through transcriptional effects to modulate SNF1 kinase activity. We also determined that yeast strains disrupted for Ubc4/5 and Rsp5 functions were markedly impaired for nuclear import of the SNF1 kinase despite its full phosphorylation and activity, all without effecting protein stability. Together, this study supports a positive role for ubiquitin enzyme cascade in SNF1 kinase activation at multiple steps and is not limited to proteolytic degradation.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.006 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".