Analysis of modification and proteolytic targeting by the ubiquitin-like modifier FAT10

Aichem, Annette; Boehm, Annika N; Catone, Nicola; Schmidtke, Gunter; Groettrup, Marcus

doi:10.1016/bs.mie.2018.12.040

Cited by 9 publications

(13 citation statements)

References 30 publications

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“…The generation of tagless human FAT10 via a pSUMO vector and ULP1 digestion was performed as described before ( Aichem et al, 2019 ). Human NUB1L and the deletion mutants delta UBA and delta UBL were cloned into the pSUMO vector via BsaI and XhoI with the primers: 5′-BsaI Nub1L 5′-CCAGTGGGTCTCAGGTGGTGCACAAAAGAAATATCTTCAAGC-3′; 3′-XhoI-Nub1L 5′-CCGCTCGAGTTAGTTTTTCTTTGTTGCTGACTTCC-3′; 3′-XhoI-Nub1L-UBA Domains 5′-CCGCTCGAGTTATCCTCCGTTGTGAGCAAGGG-3′; 5′-BsaI-Nub1L-3xUBA 5′-CCAGTGGGTCTCAGGTGGTGATCCATCAAAAGTGGACAATTTGTTGC-3′; 5′-BsaI-Nub1L-dUBL 5′-CCAGTGGGTCTCAGGTGGTTCAGAAAGAAAAGCCCTTATGTTAGC-3′; 3′-XhoI-Nub1L-UBA Domains 5′-CCGCTCGAGTTATCCTCCGTTGTGAGCAAGGG-3′.…”

Section: Methodsmentioning

confidence: 99%

FAT10 and NUB1L cooperate to activate the 26S proteasome

Brockmann,

Catone,

Wünsch

et al. 2023

Life Sci. Alliance

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The interaction of the 19S regulatory particle of the 26S proteasome with ubiquitylated proteins leads to gate opening of the 20S core particle and increases its proteolytic activity by binding of the ubiquitin chain to the inhibitory deubiquitylation enzyme USP14 on the 19S regulatory subunit RPN1. Covalent modification of proteins with the cytokine inducible ubiquitin-like modifier FAT10 is an alternative signal for proteasomal degradation. Here, we report that FAT10 and its interaction partner NUB1L facilitate the gate opening of the 20S proteasome in an ubiquitin- and USP14-independent manner. We also show that FAT10 is capable to activate all peptidolytic activities of the 26S proteasome, however only together with NUB1L, by binding to the UBA domains of NUB1L and thereby interfering with NUB1L dimerization. The binding of FAT10 to NUB1L leads to an increased affinity of NUB1L for the subunit RPN1. In conclusion, the herein described cooperation of FAT10 and NUB1L is a substrate-induced mechanism to activate the 26S proteasome.

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Section: Methodsmentioning

confidence: 99%

FAT10 and NUB1L cooperate to activate the 26S proteasome

Brockmann,

Catone,

Wünsch

et al. 2023

Life Sci. Alliance

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“…For the induction of endogenous FAT10 expression, cells were seeded with a density of 1–2 × 10 6 cells/cell culture dish (10 cm). After 24 h, the expression of endogenous FAT10 was induced by the addition of 300 U/ml IFNγ and 600 U/ml TNFα (both from PeproTech), as described in Aichem et al (2019a) . For transient transfection, cells were seeded as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant 6His-tagged E2 conjugating enzymes were purified as described above. The purification of 6His-tagged USE1 has been described earlier ( Aichem et al, 2010 ; Boehm et al, 2020 ), and purification of FAT10 and its conjugation-incompetent mutant FAT10-AV was described earlier ( Aichem et al, 2019a ). HA-tagged stabilized FAT10 HA-FAT10 C0 (C134L)-GG or its conjugation-incompetent variant HA-FAT10 C0 (C134L)-GC was purified in the same way as described earlier for FAT10 ( Aichem et al, 2019a ).…”

Section: Methodsmentioning

confidence: 99%

Tumor necrosis factor mediates USE1-independent FAT10ylation under inflammatory conditions

Schnell,

Zubrod,

Catone

et al. 2023

Life Sci. Alliance

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The ubiquitin-like modifier FAT10 is up-regulated in many different cell types by IFNγ and TNFα (TNF) and directly targets proteins for proteasomal degradation. FAT10 gets covalently conjugated to its conjugation substrates by the E1 activating enzyme UBA6, the E2 conjugating enzyme USE1, and E3 ligases including Parkin. To date, USE1 was supposed to be the only E2 enzyme for FAT10ylation, and we show here that a knockout of USE1 strongly diminished FAT10 conjugation. Remarkably, under inflammatory conditions in the presence of TNF, FAT10 conjugation appears to be independent of USE1. We report on the identification of additional E2 conjugating enzymes, which were previously not associated with FAT10. We confirm their capacity to be charged with FAT10 onto their active site cysteine, and to rescue FAT10 conjugation in the absence of USE1. This finding strongly widens the field of FAT10 research by pointing to multiple, so far unknown pathways for the conjugation of FAT10, disclosing novel possibilities for pharmacological interventions to regulate FAT10 conjugation under inflammatory conditions and/or viral infections.

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“…Expression of endogenous FAT10 was induced by treating HEK293 cells with a combination of the two pro-inflammatory cytokines tumor necrosis factor-α (TNF, 600 U/mL) and interferon-γ (IFN-γ, 300 U/mL) (both from Peprotech GmbH) [ 67 ], as described previously [ 67 ].…”

Section: Methodsmentioning

confidence: 99%

The ubiquitin-like modifier FAT10 covalently modifies HUWE1 and strengthens the interaction of AMBRA1 and HUWE1

Mueller,

Bialas,

Ryu

et al. 2023

PLoS ONE

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The ubiquitin-like modifier FAT10 is highly upregulated under inflammatory conditions and targets its conjugation substrates to the degradation by the 26S proteasome. This process termed FAT10ylation is mediated by an enzymatic cascade and includes the E1 activating enzyme ubiquitin-like modifier activating enzyme 6 (UBA6), the E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1) and E3 ligases, such as Parkin. In this study, the function of the HECT-type ubiquitin E3 ligase HUWE1 was investigated as a putative E3 ligase and/or conjugation substrate of FAT10. Our data provide strong evidence that HUWE1 is FAT10ylated in a UBA6 and FAT10 diglycine-dependent manner in vitro and in cellulo and that the HUWE1-FAT10 conjugate is targeted to proteasomal degradation. Since the mutation of all relevant cysteine residues within the HUWE1 HECT domain did not abolish FAT10 conjugation, a role of HUWE1 as E3 ligase for FAT10ylation is rather unlikely. Moreover, we have identified the autophagy-related protein AMBRA1 as a new FAT10 interaction partner. We show that the HUWE1-FAT10 conjugate formation is diminished in presence of AMBRA1, while the interaction between AMBRA1 and HUWE1 is strengthened in presence of FAT10. This implies a putative interplay of all three proteins in cellular processes such as mitophagy.

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Analysis of modification and proteolytic targeting by the ubiquitin-like modifier FAT10

Cited by 9 publications

References 30 publications

FAT10 and NUB1L cooperate to activate the 26S proteasome

FAT10 and NUB1L cooperate to activate the 26S proteasome

Tumor necrosis factor mediates USE1-independent FAT10ylation under inflammatory conditions

The ubiquitin-like modifier FAT10 covalently modifies HUWE1 and strengthens the interaction of AMBRA1 and HUWE1

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