Structure of E3 ligase E6AP with a proteasome-binding site provided by substrate receptor hRpn10

Buel, Gwen R.; Chen, Xiang; Chari, Raj; O'Neill, Michael C.; Ebelle, Danielle L.; Jenkins, Conor; Sridharan, Vinidhra; Tarasov, Sergey G.; Tarasova, Nadya I.; Andrésson, Þorkell; Walters, Kylie J.

doi:10.1038/s41467-020-15073-7

Cited by 33 publications

(54 citation statements)

References 126 publications

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“…Evolutionary analysis of the mUBE3A-Iso2/hUBE3A-Iso3-like extensions revealed that the variant lacking the proline at position 9 is only present in human, Apes and Old World monkeys ( Catarrhini ), and must therefore have arisen 25–40 Mya. Although the exact mechanism by which the N-terminal extension mediates differential localization remains to be established, our previous work in mice suggested that the cytosolic mUBE3A-Iso2, following initial targeting to the nucleus through interaction of the UBE3A AZUL domain with PSMD4 ( 9 , 22 ), is actively transported out of the nucleus. Since there is no nuclear export signal in either of the UBE3A extensions, our results suggest that loss of Proline 9 interferes with nuclear export by affecting protein folding and/or interaction with other proteins, ultimately resulting in nuclear retention of UBE3A.…”

Section: Discussionmentioning

confidence: 99%

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms

Zampeta

Sonzogni

Niggl

et al. 2020

Human Molecular Genetics

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The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This mutation is shared with apes and Old-World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a single nucleotide in this non-coding exon, the exon became in frame with the remainder of the UBE3A protein. RNA-seq analysis of human brain samples showed that the human UBE3A isoforms arise by alternative splicing. Consistent with the predominant nuclear enrichment of UBE3A in human neurons, the two nuclear localized isoforms, hUBE3A-Iso1 and -Iso3, are the most abundantly expressed isoforms of UBE3A, while hUBE3A-Iso2 maintains a small pool of cytosolic UBE3A. Our findings provide new insight into UBE3A localization and evolution, and may have important implications for gene therapy approaches in Angelman Syndrome.

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Section: Discussionmentioning

confidence: 99%

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms

Zampeta

Sonzogni

Niggl

et al. 2020

Human Molecular Genetics

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“…Disruption of the RPN10 VWA domain or loss of RPN10 destabilizes the lid-base interaction (Glickman et al 1998;Fu et al 2001;Tomko and Hochstrasser 2011). RPN10 also provides primary binding sites to recruit extrinsic ubiquitin receptors like DSK2/Ubiquilin (Matiuhin et al 2008;Zhang et al 2009a;Chen et al 2019) and certain E3 ligases like E6AP/UBE3A (Buel et al 2020). Binding of the E6AP AZUL (Amino-terminal Zincbinding domain of Ubiquitin E3a Ligase) domain induces refolding of an unstructured C-terminal segment in RPN10 into a helical bundle (Buel et al 2020).…”

Section: Rpn10mentioning

confidence: 99%

“…RPN10 also provides primary binding sites to recruit extrinsic ubiquitin receptors like DSK2/Ubiquilin (Matiuhin et al 2008;Zhang et al 2009a;Chen et al 2019) and certain E3 ligases like E6AP/UBE3A (Buel et al 2020). Binding of the E6AP AZUL (Amino-terminal Zincbinding domain of Ubiquitin E3a Ligase) domain induces refolding of an unstructured C-terminal segment in RPN10 into a helical bundle (Buel et al 2020). The yeast Rpn10 is monoubiquitylated in vivo, at Lys71, Lys84 and Lys99 in the VWA domain, which regulates its interactions with substrates by inhibiting the UIM (Isasa et al 2010).…”

Section: Rpn10mentioning

confidence: 99%

Structure, Dynamics and Function of the 26S Proteasome

Mao

2020

Subcellular Biochemistry

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The 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.

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“…This article is protected by copyright. All rights reserved domain destabilizes lid-base association in both human [116] and yeast [117] proteasomes. The location of the VWA domain is nearby to where ubiquitin binds on hRpn11, however, the hRpn10 portion beyond the VWA, including the UIMs, is not visible in cryoEM density maps of the proteasome [11,12,18,58,118,119].…”

Section: Accepted Articlementioning

confidence: 99%

“…As new functional roles continue to be discovered for the shuttle factors, multiple E3 ligases have been reported to associate with the proteasome as well [31,79,116,131,132]. It is possible that these ligases are recruited to ubiquitinate proteasome components [126,132,133].…”

Section: Ubiquitination Machinery At the Proteasomementioning

confidence: 99%

Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies

et al. 2020

Self Cite

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The 26S proteasome is responsible for regulated proteolysis in eukaryotic cells. Its substrates are diverse in structure, function, sequence length, and amino acid composition, and are targeted to the proteasome by post-translational modification with ubiquitin. Ubiquitination occurs through a complex enzymatic cascade and can also signal for other cellular events, unrelated to proteasome-catalyzed degradation. Like other post-translational protein modifications, ubiquitination is reversible, with ubiquitin chain hydrolysis catalyzed by the action of deubiquitinating enzymes (DUBs), ~90 of which exist in humans and allow for temporal events as well as dynamic ubiquitin-chain remodeling. DUBs have been known for decades to be an integral part of the proteasome, as deubiquitination is coupled to substrate unfolding and translocation into the internal degradation chamber. Moreover, the proteasome also binds several ubiquitinating enzymes as well as shuttle factors that recruit ubiquitinated substrates. The role of this intricate machinery and how ubiquitinated substrates interact with proteasomes remains an area of active investigation. Here, we review what has been learned about the mechanisms used by the proteasome to bind ubiquitinated substrates, substrate shuttle factors, ubiquitination machinery, and DUBs. We also discuss many open questions that require further study or the development of innovative approaches to be answered. Finally, we address the promise of expanded therapeutic targeting that could benefit from such new discoveries.

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Structure of E3 ligase E6AP with a proteasome-binding site provided by substrate receptor hRpn10

Cited by 33 publications

References 126 publications

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms

Structure, Dynamics and Function of the 26S Proteasome

Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies

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