A human ubiquitin conjugating enzyme, L-UBC, maps in the Alzheimer's disease locus on Chromosome 14q24.3

Robinson, Philip A.; Leek, J.P.; Thompson, John; Carr, Ian; Bailey, Andrew M.; Moynihan, Terry P.; Coletta, P. Louise; Lench, Nicholas; Markham, Alexander F.

doi:10.1007/bf00354295

Cited by 17 publications

(11 citation statements)

References 53 publications

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“…UbcH7 was initially identified as the E2 enzyme that interacts with the E3 ligase, E6AP (26)(27)(28). UbcH7 can form a complex with BRCA1 in vitro, but failed to support the BRCA1-mediated Ub ligase activity (29).…”

Section: Resultsmentioning

confidence: 99%

UbcH7 regulates 53BP1 stability and DSB repair

Han¹,

Zhang²,

Chung³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the errorprone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.DNA damage response | UbcH7 | 53BP1 | protein degradation | DSB repair P rompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2-6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7,8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9-11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12)(13)(14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12,16,17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12-14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 sta...

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Section: Resultsmentioning

confidence: 99%

UbcH7 regulates 53BP1 stability and DSB repair

Han¹,

Zhang²,

Chung³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The "bait" plasmid was constructed by cloning the open reading frame of UbcH7 (13,20,21) (CLONTECH). Clones were selected on minimal synthetic dropout medium in the absence of tryptophan.…”

Section: Methodsmentioning

confidence: 99%

“…Interaction of HHARI with Human E2s in Vitro-Four human E2s were assessed for their capacity to form a stable association with HHARI: (i) UbcH1 (24), which does not interact with E6-AP, (ii) UbcH5 (25), (iii) UbcH7 (13,20,21), and (iv) UbcH8 (14), all of which support E6-AP-ubiquitin thioester formation. Plasmids bearing upstream bacteriophage T7 promoters encoding these E2s have been described previously (6,13,25).…”

Section: Methodsmentioning

confidence: 99%

“…Identification of UbcH7 Domains Regulating the Interaction with HHARI-Specific regions of the UbcH7 cDNA (15,20,21) were amplified by PCR using Pfu DNA polymerase and cloned into the BamHI site of pET32a as in-frame thioredoxin (TXD) C-terminal fusion proteins (Novagen). Binding assays were performed as described above using ϳ0.5 g each of TXD fusion protein and GST-HHARI (118 -557).…”

Section: Interaction Of Gst-hhari (118 -557) With Endogenous Ubch7 Inmentioning

confidence: 99%

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The Ubiquitin-conjugating Enzymes UbcH7 and UbcH8 Interact with RING Finger/IBR Motif-containing Domains of HHARI and H7-AP1

Moynihan

Ardley

Nuber

et al. 1999

Journal of Biological Chemistry

110

104

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Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast twohybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.The primary role of the protein ubiquitinylation pathway is the targeting of intracellular substrate proteins for degradation (1). In this process, ubiquitin is first activated in an ATPdependent step forming a thioester bond with an ubiquitinactivating enzyme (E1). Ubiquitin is then transferred to an ubiquitin-conjugating enzyme (E2), 1 retaining the high energy thioester bond. Thereafter, the E2, alone or in conjunction with an ubiquitin-protein ligase (E3), catalyzes the final attachment of ubiquitin to the target protein (2). Ubiquitin itself can then serve as a ubiquitinylation substrate, resulting in the generation of polyubiquitinylated proteins possibly with the aid of an E4 (3). Finally, ubiquitinylated proteins are recognized and degraded by the 26 S proteasome. It has been proposed that the selection of an individual protein for proteasomal degradation via the ubiquitin pathway requires a unique combination of an E2 and E3. In S. cerevisiae, some 13 E2s or E2-related proteins have been identified, and many more are present in higher eukaryotes (2). E2s are characterized by a conserved catalytic domain of approximately 150 amino acid residues. Despite their functional redundancy, individual E2s appear to be involved in different cellular processes and, therefore, in the ubiquitinylation of different substrate proteins. The distinct substrate specificity of E2s is at least in part explained by the observation that different E2s interact with different E3s.Four classes of E3 have been identified to date; in contrast to E2s, they exhibit no overt sequence homology. These are yeast UBR1 and its mammalian homologues (4), mammalian E6-AP

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“…The RING finger domain is a Cys/His-rich, zinc-chelating domain that promotes both protein-protein and protein-DNA interactions and is defined as Cys 1 -X 2 -Cys 2 -X 9 -39 -Cys 3 -X 1-3 -His 4 -X 8 , where X can be any amino acid (15). Proteins containing RING finger motifs are further subgrouped depending on whether a Cys or His residue is found at Cys/His 5 within the motif, so that they are defined as either RING-HC (Cys 5 ) or a RING-H2 (His 5 ) type.…”

mentioning

confidence: 99%

Features of the Parkin/Ariadne-like Ubiquitin Ligase, HHARI, That Regulate Its Interaction with the Ubiquitin-conjugating Enzyme, UbcH7

Ardley

Tan

Rose

et al. 2001

Journal of Biological Chemistry

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We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186 -254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.

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A human ubiquitin conjugating enzyme, L-UBC, maps in the Alzheimer's disease locus on Chromosome 14q24.3

Cited by 17 publications

References 53 publications

UbcH7 regulates 53BP1 stability and DSB repair

UbcH7 regulates 53BP1 stability and DSB repair

The Ubiquitin-conjugating Enzymes UbcH7 and UbcH8 Interact with RING Finger/IBR Motif-containing Domains of HHARI and H7-AP1

Features of the Parkin/Ariadne-like Ubiquitin Ligase, HHARI, That Regulate Its Interaction with the Ubiquitin-conjugating Enzyme, UbcH7

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