The Low Density Lipoprotein Receptor-related Protein (LRP) Is a Novel β-Secretase (BACE1) Substrate

In the paper

“Multiple intracellular adaptor and scaffolding proteins bind the LRP 100 amino acid cytoplasmic tail (16,17);”

What the sources say

• Partly supported

  The sections “Interactions with intracellular adapter and scaffold proteins” and “Structural organization of LRP” state that multiple cytoplasmic adaptor and scaffold proteins interact with the LRP tail. Our copy does not specify that the tail is 100 amino acids long, so that numerical detail is unsupported. (LLM, conf=0.85)

  cites [16] Herz, 2001, J. Clin. Investig. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [17] Li, 2001, Mol. Neurobiol. source

In the paper

“Furthermore, and as with APP, ␥-secretase cleavage of LRP leads to release of the LRP intracellular domain (LRP-ICD), which can translocate to the nucleus and interact with Tip60 (27,28).”

What the sources say

• Partly supported

  The Discussion says LRP cleavage releases its intracellular domain, which translocates to the nucleus; Fig. 5 reports close interaction with Tip60 there. The Discussion describes γ-secretase as the probable cause of cleavage, so the citing sentence states that mechanism more definitively than the source does. Page numbers are unavailable in our copy. (LLM, conf=0.85)

  cites [27] Kinoshita, 2003, J. Biol. Chem. sourceExperimental — accuracy not yet measured
• Partly supported

  The Abstract reports release of the LRP intracellular domain by a γ-secretase-like activity, and the paper says FE65 can promote its nuclear translocation under certain conditions. It does not establish an LRP-ICD interaction with Tip60: a later passage (indexed as Abstract; page unavailable) raises the possibility that FE65 binding to LRP and recruitment of Tip60 are mutually exclusive, and its reporter assay did not show Tip60-dependent activity. (LLM, conf=0.90)

  cites [28] May, 2002, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“proteolysis of LRP by matrix metalloproteases was recently reported (25);”

What the sources say

• Supported

  The Results report that MT1-MMP proteolyzed LRP and that MT2-MMP, MT3-MMP, and MT4-MMP also degraded it in vitro. The Discussion likewise identifies these matrix metalloproteinases as capable of cleaving LRP. (LLM, conf=0.94)

  cites [25] Rozanov, 2004, J. Biol. Chem. source

In the paper

“In addition, an interaction between the C-terminal domain of APP and LRP, mediated by the cytoplasmic adaptor protein Fe65, impacts APP internalization (18 -23).”

What the sources say

• ?Not checked — no full text of the sourcecites [18] Kounnas, 1995, Cell source
• Partly supported

  The discussion (page unavailable in our copy) says Fe65 binds the cytoplasmic portions of APP and LRP and may link them. It also proposes that APP–LRP interaction modulates APP trafficking through the endocytic pathway. It does not establish that Fe65 mediates an interaction that impacts APP internalization; that mechanism remains a possibility rather than a demonstrated finding. (LLM, conf=0.85)

  cites [19] Ulery, 2000, J. Biol. Chem. sourceExperimental — accuracy not yet measured
• Partly supported

  In “Abnormalities in APP processing are isoform independent” (page not given), the paper reports reduced APP internalization without LRP and its restoration by an LRP C-terminal construct. The Discussion (page not given) proposes an adaptor linking APP and LRP but says whether adaptors coordinate their internalization awaits further study. It mentions Fe65 in connection with transcription, not as an established mediator of APP internalization. (LLM, conf=0.87)

  cites [20] Pietrzik, 2002, EMBO J. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [21] Rebeck, 2001, Brain Res. Mol. Brain Res. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [22] Kinoshita, 2001, J. Neurosci. source
• Partly supported

  The Results show that distinct FE65 domains bind the cytoplasmic tails of LRP and APP, and the Discussion says FE65 could in principle link them. The paper proposes that this might affect APP trafficking or receptor endocytosis, but does not demonstrate an effect on APP internalization; its conclusion says that physiological relevance for endocytosis remains to be tested. (LLM, conf=0.90)

  cites [23] Trommsdorff, 1998, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“its four extracellular binding domains mediate endocytosis of a wide array of ligands, including several of potential importance for Alzheimer disease pathophysiology: APP, apolipoprotein E and ␣ 2 -macroglobulin (16 -18).”

What the sources say

• Supported

  The Structural organization of LRP section describes four clusters of extracellular ligand-binding repeats and their role in ligand internalization. The Potential roles in Alzheimer disease section states that LRP serves as a receptor for APP, apoE, and α2-macroglobulin; Table 1 lists all three among ligands binding its extracellular domain. No page numbers are available in our copy. (LLM, conf=0.91)

  cites [16] Herz, 2001, J. Clin. Investig. source
• ?Not checked — no full text of the sourcecites [17] Li, 2001, Mol. Neurobiol. source
• ?Not checked — no full text of the sourcecites [18] Kounnas, 1995, Cell source

In the paper

“BACE and APP are internalized from the cell surface to early endosomes and cycle between the cell membrane and endosomes (10,11,15).”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Kinoshita, 2003, J. Cell Sci. source
• ?Not checked — no full text of the sourcecites [11] Huse, 2000, J. Biol. Chem. source
• Partly supported

  The introduction says BACE moves from the cell surface to early endosomes and can recycle to the surface; the trafficking results also show BACE entering early endosomes. The paper reports APP colocalization with BACE, but the passages do not establish that APP follows the same internalization and recycling route. (LLM, conf=0.87)

  cites [15] Walter, 2001, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“A family of GGA adaptor proteins and the phospholipid scramblase 1 (PLSCR1) have been shown to directly interact with the BACE tail (42,45).”

What the sources say

• Supported

  The Introduction and Discussion report that the C-terminal region of memapsin 2 (BACE) binds the VHS domains of GGA1 and GGA2. This supports the GGA interaction attributed to this paper; the sentence has a second citation for the separate PLSCR1 claim. (LLM, conf=0.92)

  cites [42] He, 2002, FEBS Lett. source
• Supported

  The Results report that PLSCR1 interacts with the BACE tail in yeast two-hybrid and in vitro binding assays. The Discussion states that GGA adaptor proteins have also been shown to directly interact with the BACE tail. (LLM, conf=0.92)

  cites [45] Kametaka, 2003, J. Biol. Chem. source

In the paper

“Co-transfection with BACE led to a substantial increase in luciferase activity suggesting that LRP undergoes BACE cleavage, leading ultimately to release of the cytoplasmic domain and translocation to the nucleus (Fig. 9).”

What the sources say

• Partly supported

  The paper reports release of LRP’s cytoplasmic domain and, in the Abstract, says FE65 can stimulate its translocation into the nucleus under certain conditions. It does not establish BACE cleavage as the initiating step; the Discussion instead identifies furin and metalloproteinase cleavage and implicates a γ-secretase-like activity in tail release. The citing sentence’s BACE co-transfection result is its own finding, not a result attributed to this source. (LLM, conf=0.88)

  cites [28] May, 2002, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“Besides APP, the few BACE substrates that have been identi-fied include the APP homologues APLP1 and -2 (5), P-selectin glycoprotein ligand-1 (PSGL-1), and a membrane-bound sialyltransferase (6).”

What the sources say

• Supported

  The citation to this paper is attached specifically to APLP1 and APLP2, not to PSGL-1 or the sialyltransferase. The Discussion states that BACE 1 cleaves both APLP1 and APLP2 and summarizes the experimental evidence. (LLM, conf=0.94)

  cites [5] Li, 2004, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [6] Kitazume, 2001, Proc. Natl. Acad. Sci. U. S. A. source

In the paper

“Although LRP-ICD has been shown to translocate to the nucleus and interact with Fe65 and Tip60 (28,47), whether or not it has a transcriptional role under physiological conditions remains unknown.”

What the sources say

• Partly supported

  The Abstract says that FE65 can stimulate translocation of the LRP tail fragment into the nucleus under certain conditions, and the Discussion says LRP binds FE65. But the Discussion (Fig. 6C) suggests that FE65 binding to the LRP tail and binding to Tip60 are mutually exclusive; it does not show LRP-ICD interacting with Tip60. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites [28] May, 2002, J. Biol. Chem. sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Results and Discussion describe APP-CT, not LRP-ICD, translocating to the nucleus and interacting with Fe65 and Tip60; no page numbers are available in our copy. Searches for LRP-ICD, its expanded name, and LRP found no passage addressing whether LRP-ICD does so. (LLM, conf=0.77)

  cites [47] Kinoshita, 2002, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“However, both LRP (48,49) and other members of the LDL receptor-related family (50) have been implicated as having signaling roles in neurons, and it seems likely that the cleavage of LRP we observe could modulate such signaling.”

What the sources say

• Supported

  The citation to this paper is attached to LRP’s signaling role, not the separate claim about other family members or the authors’ suggestion about cleavage. The paper’s Discussion reports that stimulation through LRP elicits a calcium response in neurons and concludes that LRP also serves as a signaling receptor. (LLM, conf=0.94)

  cites [48] Bacskai, 2000, Proc. Natl. Acad. Sci. U. S. A. source
• ?Not checked — no full text of the sourcecites [49] Boucher, 2002, J. Biol. Chem. source
• Supported

  The Abstract identifies VLDLR and ApoER2 as LDL receptor family members with signaling functions in the Reelin pathway. The Discussion likewise describes their role in neuronal migration signaling. This supports the portion of the sentence attributed to citation 50; the separate claims about LRP and its cleavage are not attributed to this source. (LLM, conf=0.93)

  cites [50] Trommsdorff, 1999, Cell source

In the paper

“Because LRP is a known ␥-substrate, we hypothesized that it might also be cleaved by ␤-secretase.”

What the sources say

• Supported

  The Abstract reports that LRP’s cytoplasmic domain is released by a γ-secretase-like activity. The Discussion reports that γ-secretase inhibitors strongly inhibited LRP tail cleavage, supporting the description of LRP as a γ-secretase substrate. (LLM, conf=0.90)

  cites [28] May, 2002, J. Biol. Chem. source

In the paper

“such an interaction may help explain the observations that directing APP to rafts enhances ␤-cleavage and A␤ generation (13).”

What the sources say

• Partly supported

  The Results section on antibody-induced cross-linking (page not provided) reports increased Aβ secretion when APP and BACE1 were copatched in rafts. The Discussion says this clustering can promote β-cleavage, but also states that partitioning APP and BACE1 into rafts alone is insufficient: cleavage normally depends on endocytosis. The claim omits that distinction and implies that directing APP to rafts by itself produces both effects. (LLM, conf=0.85)

  cites [13] Ehehalt, 2003, J. Cell Biol. sourceExperimental — accuracy not yet measured

In the paper

“LC, which contains the ␤-chain of LRP, and LRP165, a construct that contains only the 100 amino acid intracellular domain, the transmembrane domain and a very small extension beyond the membrane, both interacted strongly with BACE (Table I).”

In the paper

“FITC bound to BACE-V5 in the absence of acceptor has a uniform lifetime and a single lifetime peak (Fig. 4A);”

In the paper

“The presence of BACE further increased and co-transfection with catalytically inactive BACE mutant did not increase this band intensity (Fig. 8).”

In the paper

“The doublet bands of ϳ60 and 75 kDa were detected only in lane 1 and in the control lysate lane (Fig. 3B), suggesting that BACE is present in the mLRP1 immunoprecipitates.”

In the paper

“Co-localization and close proximity in both H4 and N2a cells was shown by confocal microscopy and FRET-based proximity assays, suggesting co-localization primarily in endosomes and at the cell surface.”

In the paper

“Although there was striking co-localization, no lifetime change was observed.”

In the paper

“in the presence of mLRP1-Cy3 acceptor, FITC has two distinct lifetimes and is faster, representing “FRETting” molecules (Fig. 4B).”

In the paper

“In the absence of an acceptor fluorophore, the lifetime of FITC conjugated to IgG (hereafter referred to simply as FITC) is ϳ2300 ps, GFP ϳ2200 ps, and Alexa 488 is ϳ1900 ps.”

In the paper

“However, under the conditions utilized we did not detect any FRET between BACE and Fe65 or between BACE and mDab1 (data not shown).”

In the paper

“The interaction was demonstrated by co-immunoprecipitation of BACE and LRP from overexpressing cells and from endogenous BACE and LRP in human brain samples and primary neurons.”

In the paper

“The lifetime of FITC attached to the C terminus of BACE-V5 alone (2334 Ϯ 72 ps) was significantly shortened when co-expressed mLRP1-Myc was C-terminally labeled by Cy3 (2153 Ϯ 55 ps, p Ͻ 0.0001), indicating FRET between the two fluorophores (Table I).”

In the paper

“We found that BACE activity at endogenous levels leads to an increase of secreted LRP in the medium as well as LRP-CTF, analogous to APP processing.”

In the paper

“The low density lipoprotein receptor-related protein, LRP, is a type I integral membrane protein with a 515-kDa extracellular ␣-chain non-convalently bound to the 85 kDa membranespanning ␤-chain.”

In the paper

“No bleedthrough or mis-excitation of Cy3 was observed under these conditions.”

In the paper

“BACE is a transmembrane protease with ␤-secretase activity that cleaves the amyloid precursor protein (APP).”

• Also stated in: § Abstract · plain

  “BACE 1 (␤ site of APP-cleaving enzyme) is a type I membrane-associated aspartyl protease that cleaves APP (1)(2)(3)(4).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites (1)(2)(3)(4)

In the paper

“Specific knockdown of Ͼ70% of overexpressing BACE was observed starting 24 h after transfection by Western blot and immunostaining (data not shown).”

In the paper

“In contrast to ␥-secretase, where at least 15 substrates have been described (44), BACE has so far appeared to be relatively specific.”

What the sources say

• ?Not checked — no full text of the sourcecites [44] De Strooper, 2003, Neuron source

In the paper

“Measurable amounts of APP and BACE are present on the plasma membrane (10 -12) and in lipid rafts (12)(13)(14).”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Kinoshita, 2003, J. Cell Sci. source
• ?Not checked — no full text of the sourcecites [11] Huse, 2000, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [12] Riddell, 2001, Curr. Biol. source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites (12)(13)(14)

In the paper

“MT1-MMP also cleaves APP (26)”

What the sources say

• ?Not checked — no full text of the sourcecites [26] Higashi, 2003, Biochemistry source

In the paper

“indicating that the observed effect has physiologic relevance and is not restricted to cells overexpressing BACE (Fig. 7B).”

In the paper

“The 60-kDa band represents endogenous BACE, whereas the 75-kDa band represents transfected BACE containing a V5-His tag.”

In the paper

“Post-translational processing of BACE involves N-glycosylation, removal of its prodomain by a furin-like protease, and further complex glycosylation (7)(8)(9).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites (7)(8)(9)

In the paper

“It is also found on the cell surface and cycles between the cell membrane and endosomes.”

In the paper

“Examination of the FLIM images of transfected cells suggests that LRP and BACE interact (red pseudocolor) at the cell surface and in endosomal compartments.”

In the paper

“Ectodomain shedding of LRP has been described (24)”

What the sources say

• ?Not checked — no full text of the sourcecites [24] Quinn, 1999, Exp. Cell Res. source

In the paper

“Authenticity of the PCRgenerated constructs was confirmed by sequencing.”

In the paper

“Thus we now show that LRP is a substrate for both BACE and ␥-secretase, identified as APP-processing enzymes.”

In the paper

“After glycosylation, BACE co-traffics with APP and is rapidly transported to the Golgi apparatus and distal secretory pathway (9).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [9] Creemers, 2001, J. Biol. Chem. source

In the paper

“Control precipitate from samples lacking anti-BACE Ab (lane 2) showed only a weak immunoreactive band, and samples lacking cell extract (lane 3) did not contain this band.”

In the paper

“Sialyltransferase and PSGL-1, in addition to APP and its homologues APLP1 and 2 are the only other reported substrates of BACE (6,43).”

What the sources say

• ?Not checked — no full text of the sourcecites [6] Kitazume, 2001, Proc. Natl. Acad. Sci. U. S. A. source
• ?Not checked — no full text of the sourcecites [43] Lichtenthaler, 2003, J. Biol. Chem. source

In the paper

“This is in good accordance with a recent article showing that ApoE, a LRP ligand, is co-localized with APP and BACE in lipid rafts (46).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [46] Kawarabayashi, 2004, J. Neurosci. source

In the paper

“The LRP C-terminal domain also mediates an interaction with APP in similar cell compartments (22).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [22] Kinoshita, 2001, J. Neurosci. source

In the paper

“Analogous results were obtained using three different fluorophores, multiple different antibody pairs, endogenous or transfected LRP and BACE, and two different cell types.”