LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

Stieglitz, Benjamin; Morris-Davies, Aylin C.; Koliopoulos, Marios G.; Christodoulou, Evangelos; Rittinger, Katrin

doi:10.1038/embor.2012.105

Cited by 206 publications

(321 citation statements)

References 21 publications

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“…The highly homologous E2 enzyme UBE2D3 (also known as UbcH5C), which can assemble linear ubiquitin chains in vitro [6,8,9], failed to interact with any LUBAC components by co-IP (Supplementary information, Figure S1B). Furthermore, UBE2D3 did not trigger NF-κB reporter activity ( Figure 1A).…”

Section: Dear Editormentioning

confidence: 99%

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The ubiquitin conjugating enzyme UBE2L3 regulates TNFα-induced linear ubiquitination

Wang

et al. 2013

Cell Res

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Section: Dear Editormentioning

confidence: 99%

“…LUBAC is a 600 kDa multimeric complex [6,8,9]. However, the stoichiometry and additional components associated with the LUBAC protein complex remain unknown.…”

Section: Dear Editormentioning

confidence: 99%

The ubiquitin conjugating enzyme UBE2L3 regulates TNFα-induced linear ubiquitination

Wang

et al. 2013

Cell Res

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“…Cloning, expression and purification of His‐Ube1, UBE2D1, UBE2D3, UBE2N, UBE2V1, His 6 ‐M1C‐ubiquitin and mutants have been described before (Carvalho et al , 2012; Stieglitz et al , 2012). TRIM25 constructs RING (1–82), RB1 (1–152), RB1B2 (1–202), RBCC (1–433) and mutants present in this study were cloned into a modified pET‐52b with a SUMO‐tag to produce cleavable His 6 ‐SUMO fusion proteins.…”

Section: Methodsmentioning

confidence: 99%

“…Media were supplemented with 100 μM ZnCl 2 , and cells were induced at 0.6 OD 600 with 150 μM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) and incubated at 16°C for 16 h. Protein was purified by affinity chromatography, followed by ion‐exchange chromatography (after removal of the His‐SUMO or GST‐tag by HRV‐3C) and size‐exclusion chromatography (SEC). His 6 ‐M1C‐ubiquitin was labelled with Atto 647N maleimide (Sigma) as described for Cy5 labelling (Stieglitz et al , 2012). All plasmids were verified by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

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TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.

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“…Traditional RING E3 ligases activate direct transfer of ubiquitin from E2~Ub to substrate; in contrast, HECT domain E3 ligases first transfer ubiquitin to a catalytic cysteine before passing it to substrate [26]. It was recently found, however, that HHARI and HOIP, both members of the RBR E3 ligase family, appear to function like HECT domain E3 ligases [27][28][29]. An intermediate cysteine~ubiquitin transfer step can be detected with both the HHARI and HOIP RBR domains in an in vitro assay.…”

Section: Introductionmentioning

confidence: 99%

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Zheng

Hunter

2013

Cell Res

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Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine~ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin's in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteinemediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.

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LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

Cited by 206 publications

References 21 publications

The ubiquitin conjugating enzyme UBE2L3 regulates TNFα-induced linear ubiquitination

The ubiquitin conjugating enzyme UBE2L3 regulates TNFα-induced linear ubiquitination

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

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