Characterization of novel regulatory components in the dynamic protein palmitoylation cycle
Bibliographic record
Abstract
Protein palmitoylation represents the only reversible lipid modification in the cell. As a post-translational modification, it is highly dynamic and plays an important role in protein trafficking and localization. Two families of enzymes mediate dynamic palmitoylation: palmitoyl-acyl transferases (PATs) catalyze palmitate addition, and acyl-protein thioesterases (APTs) catalyze palmitate removal. In mammalian cells, twenty-three PATs have been identified; however, the mechanisms that regulate their enzymatic activity are largely unexplored. Only two APTs, APT1 and APT2, have been identified, but it is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. To determine if APT1 and APT2 are responsible for the depalmitoylation of all cytosolic substrates, in this dissertation, I first examined the roles of APT1 and APT2 in protein depalmitoylation. Using a dual pulse-chase strategy to compare protein and palmitate half-lives, I found that simultaneous knockdown or inhibition of APT1 and APT2 strongly blocked palmitate removal from the N-terminal domain of Huntingtin (N-HTT), but had no effect on the depalmitoylation of post-synaptic density-95 (PSD-95). By activity-based protein profiling (ABPP), I showed that the APT1/2 inhibitor Palmostatin B has additional serine hydrolase targets that may play a role in PSD-95 depalmitoylation. Moreover, Palmostatin B induced PSD-95-GFP re-distribution in COS-7 cells, a phenotype not observed with APT1- and APT2-selective inhibitors. These results demonstrate that serine hydrolases other than APT1 and APT2 mediate the substrate-specific removal of palmitate from cytosolic proteins. I also investigated a possible novel regulator of the PAT HTT-interacting protein 14 (HIP14), Optineurin (OPTN), a cargo adaptor known to interact with HTT to mediate post-Golgi vesicle trafficking. I validated this interaction by co-immunoprecipitation and showed that OPTN is not a palmitoylated substrate. Furthermore, HIP14, OPTN, and HTT formed a trimeric complex. I mapped the binding of OPTN and HTT to the HIP14 ankyrin repeat domain, and identified mutations that selectively destabilized the HIP14/OPTN interaction. I hypothesize that OPTN transports HIP14 to distinct subcellular compartments to regulate its access to substrates. In summary, these results reveal potential novel regulatory components in the dynamic palmitoylation cycle.
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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 | 0.000 | 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 | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".