Cryo-EM captures FAT10 activation and transfer from UBA6 to E2 UBE2Z, revealing FAT10 monopolises UBA6. Efficient transfer requires E2 engagement of both FAT10 UBL domains, particularly UBL2, and co-o...

Here, Arkinson et al. reconstitute NUB1-mediated FAT10 degradation by the human 26S proteasome and use biochemistry, cryo-EM and hydrogen–deuterium exchange to show that NUB1 acts as an ATP-independen...

Cryo-EM captures FAT10 activation and transfer from UBA6 to E2 UBE2Z, revealing FAT10 monopolises UBA6. Efficient transfer requires E2 engagement of both FAT10 UBL domains, particularly UBL2, and co-o...

Cryo-EM captures FAT10 activation and transfer from UBA6 to E2 UBE2Z, revealing FAT10 monopolises UBA6. Efficient transfer requires E2 engagement of both FAT10 UBL domains, particularly UBL2, and co-ordination of InsP₆ in the UBA6 catalytic domain.

Specificity in ubiquitin (Ub) and Ub-like protein signaling is essential. Here, the authors use cryo-EM to show how UBA6 selectively engages its cognate E2 for dual Ub and FAT10 transfer, revealing a role for an InsP₆-binding site and illuminating molecular rules governing pathway specificity.

Purpose The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directs substrates to the 26 S proteasome, but its role in autophagic protein degradation remains unclear. Methods FAT10 expression was analyzed by qRT-PCR and western blotting in colon cancer (CC) tissues and cells. Bioinformatics revealed FAT10-associated biological processes in CC. In vitro and in vivo assays examined CC cell invasion and metastasis. Autophagy was assessed by western blotting, mRFP-GFP-LC3 reporter (tfLC3), and electron microscopy. In vitro ubiquitination assays measured ubiquitination of zona occludens 1 (ZO-1) and autophagy-related gene 3 (ATG3). liquid chromatography‒tandem mass spectrometry (LC-MS/MS) identified FAT10-interacting proteins. Co-IP and GST pull-down confirmed FAT10–ATG3 binding. Results FAT10 was upregulated in CC tissues and cells. Bioinformatic analyses revealed that FAT10 expression was correlated with extracellular matrix binding and cell migration in CC. FAT10

Nature Structural & Molecular Biology, Published online: 11 April 2025; doi:10.1038/s41594-025-01527-3Here, Arkinson et al. reconstitute NUB1-mediated FAT10 degradation by the human 26S proteasome and use biochemistry, cryo-EM and hydrogen–deuterium exchange to show that NUB1 acts as an ATP-independent chaperone to trap partially folded FAT10 for proteasome delivery.

Nature Structural & Molecular Biology, Published online: 11 April 2025; doi:10.1038/s41594-025-01527-3Here, Arkinson et al. reconstitute NUB1-mediated FAT10 degradation by the human 26S proteasome and use biochemistry, cryo-EM and hydrogen–deuterium exchange to show that NUB1 acts as an ATP-independent chaperone to trap partially folded FAT10 for proteasome delivery.

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the

Under inflammatory conditions, the ubiquitin-like modifier FAT10 serves as a tag for protein degradation by the 26S proteasome. FAT10 is degraded along with its substrates and this process is independent of the segregase VCP/p97, which, in the regular ubiquitin pathway of degradation, is required if a substrate lacks a disordered initiation region. FAT10 itself is loosely folded and its tendency to aggregate has complicated investigations of its structure, interaction, and function. Recently hydrogen-deuterium exchange in combination with mass spectrometry has suggested that, in preparation of degradation by the proteasome, the adapter protein NUB1 traps FAT10 in a mostly unfolded state by capturing a {beta}-strand. {beta}-strand capture was subsequently confirmed by magic-angle spinning (MAS) NMR spectroscopy of a stabilized variant of the N-domain of FAT10 in complex with NUB1L, the longer splice variant of NUB1. MAS NMR, in addition, revealed that the N-domain of FAT10 and NUB1L form a fuzzy complex and that the N-terminus of FAT10 is positioned for initiation of degradation by specific non-covalent interaction with NUB1L. Here, we report the investigation of the wild-type N-domain of FAT10 by MAS NMR. Co-sedimentation with NUB1L yields high-quality spectra, which enable sequential assignment of resonances. Through the lens of MAS NMR, the complexes of the wild-type and stabilized N-domain of FAT10 with NUB1L are identical. The N-terminus of FAT10 again shows up prominently in the spectra, even though the residue is this time an Ala, not a Gly. Our experience suggests that co-sedimentation in combination with MAS NMR is generally helpful in the exploration of conditional folds of intrinsically disordered proteins.