| 2012 |
Tepsin was identified as the first accessory protein of the AP-4 coat complex, co-purifying with AP-4-coated vesicles from HeLa cells as determined by multivariate SILAC-based quantitative mass spectrometry and siRNA knockdown profiling. |
SILAC-based quantitative mass spectrometry, siRNA knockdown, principal component analysis of coated vesicle fractions |
The Journal of cell biology |
High |
22472443
|
| 2014 |
Recruitment of tepsin to the membrane is abolished when AP-4 complex assembly is disrupted by loss-of-function mutations in AP4S1, demonstrating that tepsin membrane recruitment depends on intact AP-4 complex. |
Patient fibroblast cell line analysis; immunofluorescence/Western blot showing loss of AP-4 subunits and tepsin membrane recruitment upon AP4S1 frameshift/nonsense mutations |
Human molecular genetics |
Medium |
25552650
|
| 2015 |
Tepsin contains two phylogenetically conserved peptide motifs in its unstructured C-terminus—[GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN—that interact with the C-terminal ear/appendage domains of the β4 and ε subunits of AP-4, respectively. Both interactions are required for efficient association of tepsin with AP-4 and for tepsin recruitment to the TGN. Bivalent interaction increases avidity and may cross-link AP-4 heterotetramers to contribute to coat assembly. |
Protein interaction assays (GST pulldown, yeast two-hybrid, phage display), structure-based mutagenesis, cellular localization assays |
The Journal of biological chemistry |
High |
26542808
|
| 2016 |
The tepsin C-terminal LFxG[M/L]x[L/V] motif binds directly and specifically to the AP-4 β4 appendage domain. NMR chemical shift mapping defined the binding site on the β4 appendage surface. Point mutations in either the tepsin motif or the cognate β4 surface abolish in vitro binding and greatly reduce (but do not completely abolish) tepsin–AP-4 interaction in cells, suggesting additional interaction sites exist. |
NMR chemical shift mapping, in vitro binding assays, point mutagenesis, co-immunoprecipitation in cells |
Traffic (Copenhagen, Denmark) |
High |
26756312
|
| 2017 |
X-ray crystal structures of the tepsin ENTH and VHS/ENTH-like domains revealed that: (1) the tepsin ENTH domain lacks helix0, helix8, and a lipid-binding pocket present in epsin1/2/3, explaining why tepsin requires AP-4 for membrane recruitment rather than binding lipids directly; (2) the tepsin VHS domain lacks helix8 and does not mediate known VHS functions such as recognition of dileucine-based cargo motifs or ubiquitin binding. |
X-ray crystallography, biochemical/biophysical binding assays, phylogenetic and comparative genomic analysis |
Traffic (Copenhagen, Denmark) |
High |
28691777
|
| 2024 |
Tepsin directly binds LC3B (preferentially over other mammalian ATG8 family members) via a canonical LC3-Interacting Region (LIR) motif, with micromolar affinity at the LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN and ATG9A distribution at the cell periphery. Tepsin depletion increases autophagosome volume and number without affecting autophagic flux. Reintroduction of wild-type tepsin partially rescues ATG9A trafficking defects, while tepsin with a mutated LIR motif or missing N-terminus fails to fully rescue ATG9A distribution. |
In silico LIR motif prediction, recombinant protein biochemistry (pulldown, calorimetry), structural modeling, siRNA knockdown, fluorescence microscopy (mRFP-GFP-LC3B reporter), rescue experiments with LIR-mutant tepsin |
Molecular biology of the cell |
High |
38381558
|
| 2022 |
Loss of tepsin in CRISPR-edited zebrafish embryos causes abnormal head morphology and neural necrosis, and alters expression levels and patterns of autophagy genes atg9a and map1lc3b, linking tepsin function to AP-4-dependent ATG9A trafficking and autophagy in a developmental context. |
CRISPR-ExoCas9 knockout in zebrafish, morphological analysis at 24 hpf, gene expression analysis |
Advances in biological regulation |
Medium |
36642642
|
| 2025 |
Computational modeling (AlphaFold Multimer) combined with bio-layer interferometry (BLI) and biochemical experiments identified three additional LC3B-binding motifs beyond the canonical LIR in tepsin's disordered regions, all engaging the LC3B LIR docking site. All four motifs must be mutated to abrogate LC3B binding in vitro. Stoichiometry data indicate one tepsin molecule likely binds two LC3B molecules simultaneously, suggesting multivalent LC3B engagement that could dynamically modulate binding strength in response to LC3B membrane concentrations. |
AlphaFold Multimer structural modeling, bio-layer interferometry (BLI), biochemical binding assays, mutagenesis, thermodynamic/kinetic analysis |
Advances in biological regulation |
Medium |
41198464
|