IndraLab

Statements

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"OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2~ubiquitin thioester and preventing ubiquitin transfer."
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"During non‐canonical deubiquitination, OTUB1 preferentially binds to ubiquitin‐conjugated E2, which restricts ubiquitin transfer. [ xref ] Moreover, the binding surface of OTUB1/E2 interferes with the interaction between E2 and E3, which further inhibits ubiquitination. [ xref ] "
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"Juang et al. reported that OTUB1 is a lysine 48 linkage-specific deubiquitinating enzyme and elucidated the structural mechanism by which OTUB1 binds E2 ligases to inhibit ubiquitin transfer (7)."
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"OTUB1 also binds to the activated E2‐conjugating enzyme Ub∼UbcH5b, and the binding is enhanced by the presence of Ub [140]."
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"In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes."
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"Our quantitative analysis provides a basis for further exploring the biological roles of OTUB1-E2 complexes in cells."
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"We next mapped the position of OTUB1 mutations identified in our genetic screen onto the structure of the Ub ~ E2 and OTUB1-Ub complex."
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"The biological roles of other E2 complexes with OTUB1 remain to be explored."
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"For example, OTU family member OTUB1 binds to E2 conjugating enzymes to prevent their activity [175]."
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"Together, the inhibitory profile of the OTUB1 mutants was fully consistent with the crystal structure of the Ub ~ E2 and OTUB1-Ub complex and demonstrated (1) that the E2 interaction surface on OTUB1, remote from its catalytic site, was dispensable for its DUB function, and (2) that both Ub interaction surfaces on OTUB1 were required for its isopeptidase activity, as they likely engage K48 linked di-Ub substrates."
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"Our mutagenesis results and the structure of the Ub ~ E2 and OTUB1-Ub complex however, offers two possible avenues for consideration."
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"These observations suggested a potential network of cross-regulation in cells in which OTUB1:E2 complexes have different enzymatic activity depending on whether or not the E2 enzyme is charged with ubiquitin xref ."
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"The contribution of E2 binding to OTUB1 DUB activity in vivo , as well as the relative balance between active and repressed OTUB1 E2 complexes, is unknown."
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"Indeed, other DUBs regulate H2A ubiquitination at DNA damage foci indirectly, as for example OTUB1 inhibits the ubiquitin ligase activity of RNF168 by binding to its E2 conjugating enzyme UBC13 (Nakad[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"These parameters provide a framework for investigating the diverse in vivo roles of OTUB1-E2 complexes."
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"The ratio of charged to uncharged E2s and the concentration of free ubiquitin determine whether the OTU deubiquitinase, ubiquitin aldehyde binding 1-E2 complex can function as a deubiquitinating enzymes or polyubiquitination inhibitor ( xref )."
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"Formation of both active and repressive OTUB1-E2 complexes are governed, in part, by the intrinsic affinity of OTUB1 for each of its E2 partners."
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"In the case of the OTUB1-E2 ~ Ub interaction, we have an unusual example of product inhibition mimicry; the assembly of the Ub ~ E2 and OTUB1-Ub complex completes the formation of a pseudo DUB cleavage product that inhibits a second enzyme, in this case the E2."
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"These data indicated that the OTUB1-E2 interaction disrupted the E3-E2 interaction, thus inhibiting FGFR2 ubiquitination catalyzed by SMURF1."
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"In turn, E2 binding to OTUB1 also controls its DUB enzyme activity [XREF_BIBR]."
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"To further characterize how E2 binding to OTUB1 effects DUB activity, we assayed OTUB1 activity as a function of E2 concentration to determine the half-maximal effective E2 concentration (EC 50 ) for stimulation of OTUB1."
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"In addition, OTUB1-E2 interactions stimulate OTUB1-mediated cleavage of K48-linked Ub chains through conformational changes of this DUB."
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"This approach allowed us to directly measure K48 diubiquitin binding to the OTUB1:E2 complex without dilution of the E2."
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"D88A disrupts the interaction between OTUB1 and E2 and hence the non-canonical function of OTUB1 in most scenarios (Nakada et al., 2010; Sun et al., 2012)."
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"To further characterize how E2 binding to OTUB1 effects DUB activity, we assayed OTUB1 activity as a function of E2 concentration to determine the half-maximal effective E2 concentration (EC 50) for stimulation of OTUB1."
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"Formation of DUB-active OTUB1:E2 complexes therefore depends on the presence of sufficient uncharged E2, which has been observed to vary among cell lines."
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"Moreover, it is possible that responses to a variety of stresses cause transient changes in the E2:E2~Ub ratio, which could shift the balance of OTUB1:E2 complexes from a repressed to active state, or vice versa."
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"Particularly in the case of the UBE2D isoforms, which are present in cells at micromolar concentrations xref , xref , an increase in K48 polyubiquitin could thereby drive formation of OTUB1-E2 complexes that would, in turn, degrade the chains."
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"In the later condition, the D88 residue is responsible for the interaction between OTUB1 and E2-conjugating enzymes, thereby blocking the synthesis of polyubiquitin chains on target proteins."
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"B1.1 was identified as a ubiquitin mutant that binds to the distal ubiquitin binding site of OTUB1, effectively inhibiting its catalytic activity and interfering with OTUB1-E2 complex formation [ xref ]."
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"We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination."
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"The contribution of E2 binding to OTUB1 DUB activity in vivo, as well as the relative balance between active and repressed OTUB1 E2 complexes, is unknown."
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"Higher concentrations of E2 substantially rescued this inhibition suggesting that binding of OTUB1 to E2 is likely to be the rate limiting step in the inhibition of polyubiquitylation of SMAD3 (XREF_FIG)."
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"OTUB1 binds to E2 ~ Ub thioester intermediates and prevent ubiquitin transfer, thereby non catalytically inhibiting accumulation of polyubiquitin."
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"OTUB1 binding to the charged E2 ~ Ub is allosterically regulated by an additional free ubiquitin monomer that binds to the OTUB1 distal ubiquitin binding site, triggering conformational changes that favor binding of the UBC13 ~ Ub donor ubiquitin in the OTUB1 proximal site 13."
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"Thus OTUB1 simultaneously binds to E2 charged Ub and a free Ub, and the arrangement of these two ubiquitins mimics K48 di-Ub."
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"For example, OTU family member OTUB1 binds to E2 conjugating enzymes to prevent their activity [ xref ]."
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"Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13)."
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"A charged E2 ~ Ub conjugate, however, will bind to OTUB1 with the conjugated donor ubiquitin bound in the OTUB1 proximal site, while free ubiquitin binds to the OTUB1 distal site and allosterically stimulates binding of OTUB1 to the E2 ~ Ub conjugate."
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"These observations reveal a novel role for OTUB1 binding to E2 ubiquitin-conjugating enzymes in regulating E2 stability within the cell."
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"Substitution of the active site cysteine with serine (C91S) inactivates OTUB1 (9), whereas a T134R substitution abrogates OTUB1 binding to E2 ubiquitin-conjugating enzymes (18, 28), thereby disrupting the ability of OTUB1 to prevent ubiquitin transfer."
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"OTUB1 D88 binds E2 enzymes and suppresses Ub-conjugating activity (47)."
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"We therefore conclude that OTUB1 regulates UBE2E1 levels through the previously reported ability of OTUB1 to bind to E2 enzymes (18–20), and not as a consequence of OTUB1 DUB activity."
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"When the E2 partners of OTUB1 are not charged with ubiquitin, E2 binding to OTUB1 stimulates its Lys-48–specific deubiquitinating activity ( xref ), although the physiological role of this stimulation remains to be shown."
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"The ability of OTUB1 to bind to E2 enzymes and inhibit ubiquitin transfer in a manner that does not depend on OTUB1 catalytic activity was first discovered in studies of DNA damage signaling, in which OTUB1 inhibits the E2, UBE2N/UBC13 (17)."
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"The K48-specific activity of OTUB1 depends on its catalytic cysteine 91 (C91), aided by C23, which facilitates recognition of the ubiquitin C-terminus through electrostatic interactions with negatively charged residues on the catalytic core. xref In addition to their enzymatic roles, Otubains also inhibit ubiquitination through a non-catalytic mechanism by directly binding to E2 enzymes and blocking ubiquitin transfer to substrates. xref This inhibitory mode is exemplified by OTUB1, which uses aspartate 88 (D88) to interact with E2 enzymes like UBC13 (also known as UBE2N) or UbcH5, preventing the formation of K63-linked ubiquitin chains. xref The OTUB1-E2 binding is allosterically regulated by free ubiquitin binding a second site on OTUB1, including a conformational rearrangement that forms an N-terminus ubiquitin-binding helix, stabilizing OTUB1’s association with E2-ubiquitin conjugates and preventing the donor ubiquitin from being transferred to substrates. xref A classic example is OTUB1 binding to UbcH5, inhibiting MDM2-mediated p53 ubiquitination, leading to apoptosis and p53-dependent suppression of cell proliferation. xref "
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"Binding of OTUB1 and E2 enzymes, including UbcH5 and Ubc13, also regulates OTUB1 enzymatic activity towards K48 ubiquitin chains [XREF_BIBR]."
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"Therefore, the authors suggested an elegant model wherein OTUB1-E2 complexes dynamically regulate the level of polyubiquitin chains in cells by either activating chain hydrolysis or inhibiting E2-medi[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"Recent structural studies of the E2 and Otub1 complex [XREF_BIBR - XREF_BIBR] would likely promote structure based design of such E2 inhibitors."
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"Liu and associates reported that inactivation of the catalytic activity of OTUB1 (C91A) does not decrease SLC7A11 levels, whereas disrupting the interaction between the E2-conjugating enzyme and OTUB1 (D88A) significantly reduces the stability of SLC7A11 xref ."
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"E2 binding to OTUB1 stabilizes an N-terminal disordered helix that interacts with the proximal ubiquitin and enhances the deubiquitinating activity of OTUB1 for K48-linked chains ( xref A–C)."
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"In the latter case, OTUB1 binds ubiquitin-conjugated E2 and blocks it in an inactive state to prevent ubiquitin transfer."
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"This work revealed that OTUB1 directly binds E2 ~ Ub together with a second non covalently bound ubiquitin and shed light on how OTUB1 inhibits a subset of E2s independent of its isopeptidase activity."
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"D88A disrupts the interaction between OTUB1 and E2 and hence the non-canonical function of OTUB1 in most scenarios ( xref ; xref )."
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"Remarkably, depletion of OTUB1 restored a pharmacologically disabled DSB response, indicating that targeting the OTUB1-E2 interaction might be an attractive route for the development of agents that bolster DSB repair."
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"The OTUB1 N-terminal extension also binds to the E2 linked donor ubiquitin in a similar manner to a UIM domain."
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"The canonical activity, dependent on the C91 residue, enables OTUB1 to directly and specifically cleave K48‐linked polyubiquitination from substrates. xref , xref In addition, OTUB1 can interact with E2 ubiquitin‐conjugating enzymes and obstruct ubiquitin transfer to substrates in a non‐canonical way, thereby blocking the synthesis of polyubiquitin chains on target proteins. xref , xref , xref , xref Although the canonical catalytic activity of OTUB1 is specific for K48 polyubiquitination, OTUB1 can also reduce other types of ubiquitination, especially K63 ubiquitination, on substrates through the non‐canonical mechanism. xref , xref , xref The non‐canonical function is dependent on the N‐terminal Ub‐binding domain and the D88 residue. xref , xref , xref We found that the D88A mutant, rather than the C91S mutant, of OTUB1 failed to inhibit the ubiquitination and degradation of CCN6, indicating that OTUB1 blocks the synthesis of ubiquitination on CCN6 in a non‐canonical way."
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"A second role of OTUB1–E2 interactions, which does not require E2 charging, is to stimulate the DUB activity of OTUB1 against Lys48 polyubiquitin."
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"It is proposed that OTUB1–E2 complexes provide a sensing mechanism that allows switching between these activities according to the pool of free ubiquitin and the charged:uncharged E2s ratio [59] ."
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"It is proposed that OTUB1–E2 complexes provide a sensing mechanism that allows switching between these activities according to the pool of free ubiquitin and the charged:uncharged E2s ratio [59] ."
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"B1.1 also obviously restrains OTUB1 activity by binding to the terminal ubiquitin binding site of OTUB1, further cutting off the K48 linked ubiquitin and disturbing the formation of OTUB1-E2 complex [[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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