Distinct Functional Domains Contribute to Degradation of the Low Density Lipoprotein Receptor (LDLR) by the E3 Ubiquitin Ligase Inducible Degrader of the LDLR (IDOL)

A mutant ubiquitin K63R completely abolishes UBC13/UEV1a-mediated ubiquitin chain formation independently of the RING domain.

“a mutant ubiquitin in which lysine 63 is replaced by an arginine (K63R) completely abolished UBC13/ UEV1a-mediated ubiquitin chain formation independently of the RING domain (19,20) (Fig. 4A).”

• cites [19] Deng, 2000, Cell sourcePartly supported

  The Results report that ubiquitin with lysine 63 replaced by arginine (R63) abolished ubiquitin’s ability to stimulate IKK activation. They do not show that this mutant completely abolished Ubc13/Uev1A-mediated chain formation independently of the RING domain. Instead, the Results and Discussion say that TRAF6-facilitated chain assembly requires an intact RING domain. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [20] Nakada, 2010, Nature sourceNot checked — no full text of the source

None of the generated RING mutations, including V389A and V420A, resulted in marked changes in degradation of the LDLR or VLDLR.

“None of the mutations we generated, including V389A and V420A that correspond to the detrimental Ile-383 and Phe-418 in c-Cbl (17), resulted in marked changes in degradation of the LDLR (Fig. 2B) or VLDLR (Fig. 2C).”

• cites [17] Zheng, 2000, Cell sourcePartly supported

  The c-Cbl–UbcH7 interface passage (section labeled Abstract; page unavailable) identifies Ile-383 and Phe-418 as residues that contact UbcH7, supporting the cited comparison of RING residues. Our copy does not show that mutations of those two c-Cbl residues are detrimental. The LDLR and VLDLR degradation results are the citing authors’ own findings, not findings attributed to this source. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

IDOL promotes the appearance of ubiquitinated LDLR in cells.

“We have previously demonstrated that IDOL promotes appearance of ubiquitinated LDLR in cells (11).”

• cites [11] Zelcer, 2009, Science sourceSupported

Recent genome-wide association studies identify IDOL as a novel modulator of LDL in humans.

“Recent genome-wide association studies identify IDOL as a novel modulator of LDL in humans, thus implicating IDOL as a potential therapeutic target (35)(36)(37).”

• cites [35] Waterworth, 2010, Arterioscler. Thromb. Vasc. Biol. sourcePartly supported

  The Results section (‘MYLIP–GMPR’) reports a genome-wide association between LDL cholesterol and SNPs at a locus near MYLIP, also called IDOL, and notes a report implicating IDOL in LDL regulation. The associated SNP lies between MYLIP and GMPR, however, so this study does not establish that IDOL itself—rather than the locus—is responsible for the association. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [36] Teslovich, 2010, Nature sourceSupported
• cites [37] Chasman, 2009, PLoS Genet. sourcePartly supported

  The Results (Secondary genome-wide analysis; page unavailable) report a novel lipoprotein-associated locus at 6p22.3 that implicates GMPR or MYLIP, supporting a possible connection to the gene identified as IDOL in the claim. The paper does not establish that MYLIP specifically modulates LDL in humans: it leaves GMPR as an alternative and says the lead SNP was associated with at least one of LDL-C, HDL-C, or triglycerides. It does not assess IDOL as a therapeutic target. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Lrp1b is not degraded by IDOL.

“Notably, a closely related LDLR family member, Lrp1b, is not degraded by IDOL (13).”

• cites [13] Hong, 2010, J. Biol. Chem. sourceSupported

Differential E2 ligase recruitment to IDOL determines the outcome of the ubiquitination reaction.

“However, we find it more plausible that differential E2 ligase recruitment to IDOL determines the outcome of the ubiquitination reaction as most E2 ligases have a preference for forming specific polyubiquitin chain linkages (24,25).”

• cites [24] Ye, 2009, Nat. Rev. Mol. Cell Biol. sourceSupported
• cites [25] Rodrigo-Brenni, 2010, Mol. Cell sourcePartly supported

  The Introduction and Discussion describe linkage preferences for several E2s, including Ubc1 and Cdc34 for K48 and Ubc13 for K63. They do not establish that *most* E2s have such preferences; the Discussion also describes general E2s. The IDOL recruitment proposal is framed as the citing authors’ own explanation, not a finding attributed to this source. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Analysis of the homology model of the IDOL PTB and the DAB1-APOER2 crystal structure identified a structurally conserved α-helix that forms part of a receptor-binding pocket.

“Analysis of the homology model of the IDOL PTB and the DAB1-APOER2 crystal structure (15) identified a structurally conserved ␣-helix that forms part of a receptor-binding pocket.”

• cites [15] Stolt, 2003, Structure sourceSupported

• AssumptionRests on an assumption: The homology model of the IDOL PTB domain accurately represents its structure and the DAB1-APOER2 crystal structure is a valid template for identifying the IDOL-LDLR interaction interface.

The predicted α-helix within the PTB domain of IDOL is highly evolutionarily conserved.

“the sequence of the predicted ␣-helix within the PTB domain of IDOL is highly evolutionarily conserved (Fig. 8C).”

• Also stated in: § RESULTS · plain

  “In fact, it is even conserved in the distant fly homolog, Dnr1, which we have recently shown to also promote LDLR degradation (13).”

• cites [13] Hong, 2010, J. Biol. Chem. sourcePartly supported

  The Discussion (page unavailable) says IDOL itself is highly evolutionarily conserved, with homologs in vertebrates and nonvertebrates. It does not establish that the sequence of a predicted α-helix within a PTB domain is conserved; searches for the helix and PTB domain found no such passage. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

IDOL interacts with the LDLR at or near the plasma membrane.

“As such, the straightforward interpretation of this result is that IDOL interacts with the LDLR at or near the plasma membrane, akin to other adaptor proteins like DAB1 and ARH involved in receptor endocytosis (6,30,31).”

• cites [6] Garcia, 2001, Science sourceNot checked — no full text of the source
• cites [30] Gotthardt, 2000, J. Biol. Chem. sourceSupported
• cites [31] Michaely, 2004, J. Biol. Chem. sourceSupported

Modulation of LDLR family receptor levels by IDOL is a post-transcriptional event.

“Importantly, modulation of the levels of these receptors by IDOL is a post-transcriptional event.”

• cites [11] Zelcer, 2009, Science sourcePartly supported

  The indexed copy (section labeled Abstract; page unavailable) reports that Idol knockdown changed LDLR protein levels without affecting LDLR mRNA, supporting post-transcriptional regulation of LDLR. It does not support extending that effect to LDLR family receptors generally: Lrp4 and SorLA were unaffected, and ApoER2 was only marginally affected. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [13] Hong, 2010, J. Biol. Chem. sourceSupported

Several residues within this helix contribute to APOER2 binding to DAB1.

“Several residues within this helix contribute to APOER2 binding to DAB1”

• cites [15] Stolt, 2003, Structure sourceSupported

Unlike the LDLR and Pcsk9, IDOL is not regulated by SREBP.

“Unlike the LDLR and Pcsk9, IDOL is not regulated by SREBP.”

• cites [11] Zelcer, 2009, Science sourcePartly supported

  The paper says LXR-induced Idol expression is not secondary to SREBP-1c and describes the LXR–Idol pathway as independent of SREBPs (copy section labeled Abstract; no page supplied). That supports SREBP-independent induction, but does not establish that SREBP never regulates IDOL or the claimed contrast with both LDLR and PCSK9. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [12] Zelcer, 2006, J. Clin. Invest. sourceCould not verify

  The passages on LXR signaling in intermediary metabolism and energy balance and on LXRs as potential drug targets discuss SREBP-1c regulation, but not whether SREBP regulates IDOL. Searches for IDOL and for LDLR, Pcsk9, and SREBP together found no passage addressing the claimed contrast. Page numbers were not available in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

In addition to the LDLR, VLDLR and APOER2 are subject to IDOL-mediated degradation.

“We recently reported that in addition to the LDLR, two other closely related receptors, the VLDLR and APOER2, are also subject to IDOLmediated degradation (13).”

• cites [13] Hong, 2010, J. Biol. Chem. sourceSupported

Ubiquitination of these receptors by IDOL marks them for subsequent lysosomal degradation.

“Ubiquitination of these receptors by IDOL marks them for subsequent lysosomal degradation.”

• cites [11] Zelcer, 2009, Science sourceSupported
• cites [13] Hong, 2010, J. Biol. Chem. sourceSupported

Expression of wild type IDOL dramatically decreases LDLR abundance in a HEK 293T co-transfection assay.

“In this assay, expression of wild type IDOL dramatically decreases abundance of the LDLR (Fig. 1B).”

The IDOL RING domain promotes Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or LXR activation.

“and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation.”

• Also stated in: § RESULTS · plain

  “we determined that transfection of IDOL together with different ubiquitin mutants promotes Lys-63-specific ubiquitination of the LDLR in cells (Fig. 5A).”

• Also stated in: § RESULTS · plain

  “concomitant to activation of the LXR-IDOL axis, we observed appearance of Lys-63-specific ubiquitinated LDLR (Fig. 5B).”

Mutation of Leu-415, Pro-419, Val-420, and Arg-422 leads to a varied increase in IDOL protein stability and abundance.

“Rather, mutation of Leu-415, Pro-419, Val-420, and Arg-422 led to a varied increase in IDOL protein stability and abundance (Fig. 2, B and C and data not shown).”

Only the full-length IDOL protein is capable of degrading the LDLR.

“We found that only the full IDOL protein was capable of degrading the LDLR.”

The IDOL FERM domain interacts with the LDLR.

“The FERM domain interacts with the LDLR”

• Also stated in: § RESULTS · plain

  “we used an IDOL FERM construct (IDOL residues 1-344) and found that it interacted with two LDLR family members and degradation targets of IDOL, the LDLR and VLDLR (Fig. 6, A and B).”

In living cells, the IDOL FERM domain co-localizes with the LDLR at the plasma membrane.

“in living cells co-localizes with the receptor at the plasma membrane.”

• Also stated in: § RESULTS · plain

  “co-expression of both constructs shifted the localization of the FERM domain toward the plasma membrane where it largely co-localized with the LDLR (Fig. 7, C-H).”

IDOL strongly enhances Lys-63-specific polyubiquitin chain formation in a RING- and ATP-dependent manner.

“We observed that IDOL also strongly enhanced Lys-63-specific polyubiquitin chain formation in a RING-and ATP-dependent manner (Fig. 3C).”

Deleting LDLR residues 817-824 results in a receptor insensitive to IDOL-mediated degradation.

“Deleting these residues in the LDLR resulted in a receptor insensitive to IDOL-mediated degradation (Fig. 10B).”

Independent expression of either the FERM or RING domain does not influence LDLR abundance.

“Independent expression of either the FERM or RING domains did not influence LDLR abundance (Fig. 1B)”

• Also stated in: § DISCUSSION · plain

  “Independently, neither of these functionally defined protein domains is able to enhance LDLR degradation.”

The IDOL RING domain promotes ubiquitination in vitro.

“The RING domain promotes ubiquitination in vitro”

• Also stated in: § RESULTS · plain

  “recombinant IDOL RING promoted polyubiquitin chain formation in an E2-and ATP-dependent manner (Fig. 3B).”

Recombinant IDOL RING promotes polyubiquitin chain formation in an E2- and ATP-dependent manner.

“recombinant IDOL RING promoted polyubiquitin chain formation in an E2-and ATP-dependent manner (Fig. 3B).”

Homology modeling revealed a phosphotyrosine-binding element embedded in the IDOL FERM domain.

“Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain.”

• Also stated in: § RESULTS · hedged

  “Bioinformatic analysis revealed the presence of a putative phosphotyrosine-binding domain (PTB) also seen in the modeled structure.”

• AssumptionRests on an assumption: The homology model of the IDOL FERM domain based on protein 4.1 and DAB1 accurately predicts the IDOL-LDLR interaction interface.

The inability of truncated IDOL proteins to degrade the LDLR is not due to their decreased abundance.

“Importantly, the inability of the truncated proteins to degrade the LDLR is likely not a result of their decreased abundance.”

• Also stated in: § RESULTS · plain

  “Titrating the level of full-length IDOL to a level comparable with that of the truncated forms still resulted in potent degradation of the LDLR (Fig. 1D).”

Mutating LDLR residues Phe-823 and Asp-824 results in an LDLR substantially less sensitive to IDOL-mediated degradation.

“Mutating these residues resulted in an LDLR substantially less sensitive to IDOL-mediated degradation.”

Mutating IDOL residues Tyr-265 and His-272 strongly attenuates LDLR degradation by IDOL.

“Mutating these residues strongly attenuated degradation of the LDLR by IDOL (Fig. 8D)”

RING residues differentially influence ubiquitination of the LDLR or stability of IDOL.

“We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL.”

IDOL-mediated lysosomal degradation of the LDLR requires both the conserved FERM and RING domains of IDOL.

“We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL.”

• Also stated in: § Abstract · hedged

  “Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL.”

• Also stated in: § RESULTS · hedged

  “Collectively, these results indicate that degradation of the LDLR by IDOL is dependent on both protein domains”

In the absence of LDLR, the FERM protein localizes intracellularly and vesicularly.

“This was in stark contrast to the intracellular and vesicular localization of the FERM protein in the absence of the LDLR.”

Mutation of Tyr-265 and His-272 attenuated LDLR degradation by IDOL.

“Their mutation attenuated LDLR degradation by IDOL, most likely by reducing the affinity of IDOL to the LDLR.”

Deleting the predicted α-helix (IDOL residues 265-272) results in an IDOL protein unable to degrade the LDLR.

“deleting this helix (IDOL residues 265-272, underlined in Fig. 8C) results in an IDOL protein that is unable to degrade the LDLR (data not shown).”

Co-expression of LDLR and the FERM domain shifts FERM localization toward the plasma membrane where it largely co-localizes with LDLR.

“co-expression of both constructs shifted the localization of the FERM domain toward the plasma membrane where it largely co-localized with the LDLR (Fig. 7, C-H).”

The cellular localization of Y265A and H272A mutants is indistinguishable from that of WT IDOL.

“The cellular localization of the Y265A and H272A mutants was indistinguishable from that of WT IDOL as assessed by indirect immunofluorescence”

Bioinformatic analysis revealed a putative PTB domain in IDOL also seen in the modeled structure.

“Bioinformatic analysis revealed the presence of a putative phosphotyrosine-binding domain (PTB) also seen in the modeled structure.”

• AssumptionRests on an assumption: The homology model of the IDOL FERM domain based on protein 4.1 and DAB1 accurately predicts the IDOL-LDLR interaction interface.

Treatment of HepG2 cells with GW3965 increases ABCA1 expression by 196 ± 19% (n=5, p<0.01).

“treatment of HepG2 cells with GW3965, a synthetic LXR agonist, increased expression of the canonical LXR target gene ABCA1 (196 Ϯ 19%, n ϭ 5, p Ͻ 0.01)”

RING mutants L415A, V420A, and R422A did not abrogate degradation of the LDLR by IDOL.

“Unexpectedly, a set of RING mutants (L415A, V420A, and R422A) that we generated based on reported E2-E3 co-structures did not abrogate degradation of the LDLR by IDOL.”

Mutation of adjacent Ile-268 and Tyr-269 has no effect on LDLR degradation.

“whereas mutation of the adjacent Ile-268 and Tyr-269 had no effect (Fig. 8D and data not shown).”

Mutating the two residues immediately preceding the NPVY motif is sufficient to strongly attenuate degradation of the LDLR by IDOL.

“Consistent with the DAB1-APOER2 structure, it was sufficient to mutate the two residues immediately preceding this motif to strongly attenuate degradation of the LDLR by IDOL.”

With a mutant ubiquitin containing only a single lysine at position 63, the RING domain potently stimulates formation of Lys-63 polyubiquitin chains.

“When we used a mutant ubiquitin containing only a single lysine at the 63 position, the RING domain could potently stimulate formation of Lys-63 polyubiquitin chains (Fig. 4B).”

Concomitant to activation of the LXR-IDOL axis, Lys-63-specific ubiquitinated LDLR appears.

“concomitant to activation of the LXR-IDOL axis, we observed appearance of Lys-63-specific ubiquitinated LDLR (Fig. 5B).”

The region immediately preceding the NPVY motif is highly conserved in different receptors marked by IDOL for degradation.

“In fact, this region is highly conserved in the different receptors marked by IDOL for degradation (Fig. 10A).”

Treatment of HepG2 cells with GW3965 increases IDOL expression by 702 ± 125% (n=5, p<0.01).

“and of IDOL (702 Ϯ 125%, n ϭ 5, p Ͻ 0.01).”

RING mutants L415A, V420A, and R422A increased IDOL protein stability.

“Rather, these increased IDOL protein stability.”

Activation of LXR in HepG2 cells does not redistribute the FERM-DsRed2 protein toward the plasma membrane.

“In preliminary experiments, we ruled out the possibility that activation of LXR in HepG2 cells is able to redistribute the FERM-DsRed2 protein toward the plasma membrane.”

At higher IDOL/LDLR ratios, Y265A and H272A mutants degrade the LDLR, albeit to a lower extent than WT IDOL.

“at higher IDOL/LDLR ratios, the Y265A and H272A mutants were able to degrade the LDLR (Fig. 9, A and B), albeit to a lower extent than an equivalent level of wild type IDOL.”

Introducing corresponding Lrp1b residues into the LDLR identified Phe-823 and Asp-824 as important specificity determinants for interaction with IDOL.

“we introduced corresponding Lrp1b residues into the LDLR and identified the highly conserved Phe-823 and Asp-824 as important specificity determinants for interaction with IDOL (Fig. 10C).”

The IDOL FERM domain interacts with the target receptors LDLR and VLDLR.

“Accordingly, we demonstrate an interaction between the IDOL FERM domain and the target receptors LDLR and VLDLR.”

Mutation of the equivalent residues in IDOL identified Tyr-265 and His-272 as important determinants of LDLR degradation.

“mutation of the equivalent residues in IDOL identified Tyr-265 and His-272 as important determinants of LDLR degradation.”

The IDOL RING domain stimulates polyubiquitin chain formation in vitro.

“The ability of the IDOL RING domain to stimulate polyubiquitin chain formation in vitro”

The sequence preceding the NPVY endocytosis motif in the LDLR is critical for IDOL-dependent degradation.

“In a reciprocal manner, we could also demonstrate that the sequence preceding the NPVY endocytosis motif in the LDLR is critical for IDOL-dependent degradation.”

Transfection of IDOL together with different ubiquitin mutants promotes Lys-63-specific ubiquitination of the LDLR in cells.

“we determined that transfection of IDOL together with different ubiquitin mutants promotes Lys-63-specific ubiquitination of the LDLR in cells (Fig. 5A).”

The results support the presence of a well defined binding interface between IDOL and the LDLR with residues from both proteins contributing to the interaction network.

“Overall, our results support the presence of a well defined binding interface between IDOL and the LDLR with residues from both proteins contributing to the interaction network.”

The LXR-IDOL-LDLR axis defines a complementary but distinct pathway for sterol-dependent control of cellular cholesterol uptake through the LDLR.

“As such, the LXR-IDOL-LDLR axis defines a complementary but distinct pathway for sterol-dependent control of cellular cholesterol uptake through the LDLR.”

Ubiquitination of the LDLR by IDOL acts as a sorting signal that directs the receptor toward the lysosomal degradation pathway.

“We therefore propose that ubiquitination of the LDLR by IDOL acts as a sorting signal that directs the receptor toward the lysosomal degradation pathway.”

Mutation of Tyr-265 and His-272 reduces the affinity of IDOL to the LDLR.

“most likely by reducing the affinity of IDOL to the LDLR.”

The second functional domain present in IDOL is the N-terminal FERM.

“The second functional domain present in IDOL is the N-terminal FERM.”

The model predicts that residues immediately preceding the NPVY endocytosis motif in the LDLR interact with IDOL.

“Our model predicts that the residues immediately preceding the NPVY endocytosis motif in the LDLR interact with IDOL.”

• AssumptionRests on an assumption: The homology model of the IDOL FERM domain based on protein 4.1 and DAB1 accurately predicts the IDOL-LDLR interaction interface.

Within the endocytic system, Lys-63 ubiquitin linkage is specifically involved in receptor sorting and trafficking.

“There is accumulating evidence that within the endocytic system, Lys-63 ubiquitin linkage is specifically involved in receptor sorting and trafficking (33).”

• cites [33] Mukhopadhyay, 2007, Science sourceNot checked — no full text of the source

These studies provide the first structural-functional characterization of the IDOL-LDLR interaction network.

“These studies provide the first structural-functional characterization of the IDOL-LDLR interaction network.”

IDOL forms a unique interaction interface with the LDLR and its E2 counterpart.

“We propose that IDOL forms a unique interaction interface with the LDLR and its E2 counterpart.”

Tyr-265 and His-272 are predicted from the model to contribute to the IDOL-LDLR interaction.

“Within this helix, Tyr-265 and His-272 are predicted from our model to contribute to the IDOL-LDLR interaction.”

• AssumptionRests on an assumption: The homology model of the IDOL FERM domain based on protein 4.1 and DAB1 accurately predicts the IDOL-LDLR interaction interface.

Ubiquitination of the LDLR by IDOL is not a prerequisite step for receptor endocytosis.

“However, it is important to emphasize that ubiquitination of the LDLR by IDOL is not a prerequisite step for receptor endocytosis.”

The most parsimonious explanation for the reduced activity of the PTB mutants is that mutation of Tyr-265 and His-272 decreased the affinity of IDOL for the LDLR.

“Therefore, the most parsimonious explanation for the reduced activity of the PTB mutants is that mutation of Tyr-265 and His-272 decreased the affinity of IDOL for the LDLR.”

Specific E2-E3 interactions can control the nature of the ubiquitin linkage that is formed.

“Furthermore, it is now clear that specific E2-E3 interactions can control the nature of the ubiquitin linkage that is formed, as was shown for the BRCA1 RING (28).”

• cites [28] Christensen, 2007, Nat. Struct. Mol. Biol. sourceNot checked — no full text of the source

The recycling LDLR pool is the target for IDOL-mediated degradation.

“This localization points toward the recycling LDLR pool as the target for IDOL-mediated degradation.”

IDOL is a bona fide E3 ubiquitin ligase.

“formally establishes IDOL as a bona fide E3 ubiquitin ligase.”