Structural basis for UFM1 transfer from UBA5 to UFC1

Kumar, Manoj; Padala, P.; Fahoum, Jamal; Hassouna, Fouad; Tsaban, Tomer; Zoltsman, Guy; Banerjee, Sayanika; Cohen-Kfir, Einav; Dessau, Moshe; Rosenzweig, Rina; Isupov, Michail N.; Schueler‐Furman, Ora; Wiener, Reuven

doi:10.1038/s41467-021-25994-6

Cited by 28 publications

(51 citation statements)

References 63 publications

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“…UFC1 differs from prototypical E2s since it lacks the catalytic HPN motif, has an exposed catalytic Cys with a lower pKa, lacks the C-terminal α -helix observed in canonical E2s and has an additional α -helix at its N-terminus ( α 0) 25,40,59 . Further, it is unclear if the canonical “backside” interaction can occur in UFC1 28,60 .…”

Section: Discussionmentioning

confidence: 99%

“…The interaction of UFC1 with UBA5 and how UFM1 is transferred from UBA5 to the catalytic Cys is reasonably well understood 23,24,[40][41][42] . We therefore focussed on subsequent events and first analysed the intrinsic reactivity and stability of UFC1~UFM1 (~ denotes the thioester bond between UFM1 G83 and UFC1 C116 ) in comparison to the well characterized ubiquitin E2s, UBE2D3 and UBE2L3 43 .…”

Section: Ufc1 Has Intrinsic Cysteine Reactivitymentioning

confidence: 99%

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Non canonical scaffold-type ligase complex mediates protein UFMylation

Peter

Magnussen

DaRosa

et al. 2022

Preprint

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Protein UFMylation is emerging as a posttranslational modification essential for endoplasmic reticulum and cellular homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported E3 ligase UFL1 is inactive on its own and identify UFBP1 to bind UFL1 to form an active E3 ligase complex. While UFC1 is an intrinsically Cys-reactive E2, we do not identify any catalytic cysteines on UFL1/UFBP1, suggesting a scaffold-type E3 ligase mechanism. Interestingly, the E3 ligase complex consists of winged-helix (WH) domain repeats that activates UFC1 for aminolysis. We identify the adaptor protein CDK5RAP3 to bind to and regulate E3 ligase activity potentially by preventing off-target UFMylation. In summary, our work identifies the minimal requirements for UFMylation and reveals regulatory principles of this atypical E3 ligase complex.

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Section: Discussionmentioning

confidence: 99%

Section: Ufc1 Has Intrinsic Cysteine Reactivitymentioning

confidence: 99%

Non canonical scaffold-type ligase complex mediates protein UFMylation

Peter

Magnussen

DaRosa

et al. 2022

Preprint

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“…For instance, the SUMO-specific E2 Ubc9 (UBE2I) is auto-SUMOylated, a modification that serves to attract proteins with SUMO interacting motifs (SIMs) (Stewart et al, 2016). Of note, a UFM1 interacting motif has been described and may serve a similar purpose here (Padala et al, 2017;Kumar et al, 2021). Alternatively, auto-UFMylation may interfere with E2 catalytic activity.…”

Section: A)mentioning

confidence: 99%

Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation

Millrine

Cummings

Matthews

et al. 2022

Preprint

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An essential first step in the posttranslational modification of proteins with UFM1, UFMylation, is the proteolytic cleavage of pro-UFM1 to expose a C-terminal glycine. Of the two UFM1-specific proteases (UFSPs) identified in humans, only UFSP2 is reported to be active since the annotated sequence of UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs in cells lacking UFSP2 suggesting the presence of another active protease. We hereby identify a long isoform of UFSP1 to be this protease. Cells lacking both UFSPs show complete loss of UFMylation resulting from an absence of mature UFM1. While UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26, UFSP1 acts earlier in the pathway to mature UFM1 and cleave a potential auto-inhibitory modification on UFC1, thereby controlling activation of UFMylation. In summary, our studies reveal important distinctions in substrate specificity and localization-dependent functions for the two proteases in regulating UFMylation.

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“…UFM1 is then activated by UBA5, an E1 activating enzyme. UBA5 transfers UFM1 to UFC1, the E2 conjugating enzyme, through a trans-binding mechanism [18, 19]. Finally, UFM1 is transferred to the substrate by UFL1, which, in complex with the ER membrane protein DDRGK1, form an E3 ligase complex to covalently modify lysine residues on substrates [20, 21].…”

Section: Introductionmentioning

confidence: 99%

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

Picchianti

Hernández

Zhan

et al. 2022

Preprint

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UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.

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Structural basis for UFM1 transfer from UBA5 to UFC1

Cited by 28 publications

References 63 publications

Non canonical scaffold-type ligase complex mediates protein UFMylation

Non canonical scaffold-type ligase complex mediates protein UFMylation

Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

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