A HECT Domain E3 Enzyme Assembles Novel Polyubiquitin Chains

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Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2⅐Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetraubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.

Section: Fig 3 Assembly and Disassembly Of Lys-63 Chains (Coomassiesupporting

confidence: 67%

In Vitro Assembly and Recognition of Lys-63 Polyubiquitin Chains

Hofmann¹,

Pickart²

2001

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“…YDJ-1, the yeast ortholog of Hsp40, was expressed in bacteria and purified by sequential chromatography on DE52 and hydroxylapatite as described previously (22). GST-tagged UbcH5a (E2, ubiquitin carrier protein) was bacterially expressed and purified by GSHSepharose affinity chromatography as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

Clapp

Peng

Morishima

et al. 2010

It is established that suicide inactivation of neuronal nitricoxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N G -nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.Nitric-oxide synthases (NOS) are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to nitric oxide and citrulline by a process that requires NADPH and molecular oxygen (1). There are three major mammalian isoforms as follows: neuronal NOS (nNOS), 2 endothelial NOS, and inducible NOS. NOS is bidomain in structure with an oxygenase domain, which contains the binding site for the heme, L-arginine, and tetrahydrobiopterin, and a reductase domain, which contains the binding sites for FMN, FAD, and NADPH (2). NOS is a highly regulated enzyme requiring homodimerization and bound calmodulin for efficient electron transfer from the flavins to the heme moiety to enable synthesis of NO. Another mechanism of regulation is the ubiquitination and proteasomal degradation of NOS (3). Of particular pharmacological interest is the finding that certain drugs cause the suicide inactivation, covalent alteration, ubiquitination, and proteasomal degradation of nNOS (3-8). This phenomenon is not unique to nNOS as it is well documented that the suicide inactivation of other P450 cytochromes leads to covalent alteration, enhanced ubiquitination, and proteasomal turnover of the enzymes (9). The C terminus of Hsc70-interacting protein (CHIP) has been shown to be an E3 ligase that ubiquitinates cytochromes P450 3A4 and 2E1 as well as nNOS (10 -12). The...

“…However, this shortened UBE3C remains able to extend Ub chains in vitro (33). We wanted to build upon this work and asked whether the N-terminal 132 residues are necessary for UBE3C function in the more physiologically relevant context of mammalian cells.…”

Section: Identification and Validation Of Ube3c As Important For Ddmentioning

confidence: 99%

“…These ligases form a covalent thioester bond with Ub using a conserved cysteine residue in the HECT domain before transferring Ub to substrates. Prior studies reported that UBE3C associates with the proteasome and assembles Lys-29-and Lys-48-linked polyUb chains (32)(33)(34). The yeast ortholog, Hul5p, ubiquitinates substrates at the proteasome to mediate protein turnover (15,16).…”

mentioning

confidence: 99%

The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates

Chu

Kovary

Guillaume

et al. 2013

Background: An RNAi screen identified UBE3C as a key player in degradation of a model unfolded protein.Results: UBE3C knockdown results in incomplete degradation of relatively stable substrates. Conclusion: UBE3C enhances proteasome processivity to prevent the accumulation of potentially harmful protein fragments. Significance: This advances our understanding of proteasome processivity and the consequences of defects therein.