Chemical and Genetic Strategies for Manipulating Polyubiquitin Chain Structure

Volk, Sara; Wang, Min; Pickart, Cecile M.

doi:10.1016/s0076-6879(05)99001-0

Cited by 31 publications

(25 citation statements)

References 64 publications

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“…Monomeric Ub mutants, E2 conjugating enzymes, and human E1 were obtained recombinantly as described (Nakasone et al, 2013; Volk et al, 2005). Enzymatically synthesized K48-, and K63-linked Ub chains were assembled by combining a proximally blocked Ub mutant (Ub-His 6 ) in combination with pLeu8 or pLeu73 modified Ub (Castaneda et al, 2013; Nakasone et al, 2013).…”

Section: Star Methodsmentioning

confidence: 99%

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Chojnacki

Mansour

Hameed

et al. 2017

Cell Chemical Biology

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SUMMARY Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and the polyUb chains to a range of cellular protein targets. Best documented Ub signaling pathway is the one that delivers polyUb-proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb-conjugates. Our work demonstrates the value of UbPT and we expect that its future uses will help define and investigate the ubiquitin interactome.

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Section: Star Methodsmentioning

confidence: 99%

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Chojnacki

Mansour

Hameed

et al. 2017

Cell Chemical Biology

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“…Preparation of Dimeric Ub, Ub 4 , Ub 6ϩ , and Ubch5b-Ub n Conjugates-Monomeric Ub mutants, E2 conjugating enzymes, and human E1 were obtained recombinantly as described (8,48 -Ub 6ϩ chains were created using reaction conditions identical to the dimers with 30 mg of wild type Ub as the only monomer. Following the Superdex 75 column, fractions containing the desired chain lengths were detected by SDS-PAGE and pooled.…”

Section: C118amentioning

confidence: 99%

“…Subsequently, any of eight positions on the first Ub (an ⑀-amine at Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , Lys 48 , or Lys 63 or the N terminus at Met 1 ) can be covalently attached to additional Ub molecules (5-7). The resulting Ub-Ub linkage is typically directed by an E2 conjugating enzyme; for example, E2-25K (Ube2K) forms chains through Lys 48 , whereas Ubc13 (Ube2N) paired with Uev1a (Ube2v1) forms chains through Lys 63 (8). Each linkage results in a unique three-dimensional conformation of the polyUb chain (supplemental Fig.…”

mentioning

confidence: 99%

Disassembly of Lys11 and Mixed Linkage Polyubiquitin Conjugates Provides Insights into Function of Proteasomal Deubiquitinases Rpn11 and Ubp6

Mansour

Nakasone

Delbrück

et al. 2015

Journal of Biological Chemistry

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“…The stabilization of proteins that are targeted for proteasome degradation by proteasome inhibitors is a common observation (i.e. very little accumulation of polyubiquitinated proteins takes place in vivo) (40,41), even in the presence of proteasome inhibitors. This is attributable to the rapid in vivo depolymerization of ubiquitin chains by deubiquitinating enzymes (some of which are associated with the 26 S proteosomes) to promote a dynamic process of deubiquitination coupled to protein degradation (42,43).…”

Section: P12 Depletion Is Due To An Accelerated Rate Of Proteolysis Thatmentioning

confidence: 99%

“…and as yet no cell-permeable inhibitors of deubiquitinating enzymes are available to offset this problem (40).…”

Section: D Ts20tgmentioning

confidence: 99%

A Novel DNA Damage Response

Zhang

Zhou

Trusa

et al. 2007

Journal of Biological Chemistry

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Mammalian DNA polymerase (Pol) ␦ is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol ␦ in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol ␦ by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol ␦ function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR ؊/؊ cells but not in ATM ؊/؊ cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol ␦ in both DNA replication and DNA repair.Three eukaryotic DNA polymerases, ␣, ␦, and ⑀, are involved in chromosomal DNA replication (1, 2). Pol 2 ␦ (3) and Pol ⑀ possess proofreading 3Ј to 5Ј exonuclease activities, which allows them to replicate DNA with high fidelity (1, 2). Mammalian Pol ␦ consists of a tightly associated dimer of the 125-kDa catalytic subunit and p50 (4, 5) that is associated with the p68 (6 -8) and p12 (9) subunits. The current model for DNA synthesis at the replication fork is that Pol ␣/primase synthesizes RNA primers plus short stretches of DNA. These are then elongated to Okazaki fragments of ϳ200 nucleotides by Pol ␦ on the lagging strand of the replication fork (1, 2). Leading strand synthesis requires highly processive synthesis and was originally thought to require Pol ␦, since the latter was able to perform the replication of the SV40 genome in an in vitro system (10). The role of Pol ⑀ has been extensively studied in yeast, where it is essential for viability in both Saccharomyces cerevisiae and Schizosaccharomyces pombe (1, 2). However, it is the C-terminal checkpoint domain and not the N-terminal catalytic domain that is essential (i.e. Pol ⑀ activity per se is dispensable, and it has been suggested that Pol ␦ activity may be able to replace Pol ⑀ activity) (11,12). In mammalian cells, it has been found that although Pol ␦ alone can replicate SV40 DNA replication, both Pol ␦ and Pol ⑀ are involved in cellular DNA replication (13). Localization and cross-linking studies suggest that Pol ␦ and Pol ⑀ may function independently of each other and that their involvement in ch...

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Chemical and Genetic Strategies for Manipulating Polyubiquitin Chain Structure

Cited by 31 publications

References 64 publications

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Disassembly of Lys11 and Mixed Linkage Polyubiquitin Conjugates Provides Insights into Function of Proteasomal Deubiquitinases Rpn11 and Ubp6

A Novel DNA Damage Response

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