X11-family proteins, including X11, X11-like (X11L) and X11-like 2 (X11L2), bind to the cytoplasmic domain of amyloid β-protein precursor (APP) and regulate APP metabolism. Both X11 and X11L are expressed specifically in brain, while X11L2 is expressed ubiquitously. X11L is predominantly expressed in excitatory neurons, in contrast to X11, which is strongly expressed in inhibitory neurons. In vivo gene-knockout studies targeting X11, X11L, or both, and studies of X11 or X11L transgenic mice have reported that X11-family proteins suppress the amyloidogenic processing of endogenous mouse APP and ectopic human APP with one exception: knockout of X11, X11L or X11L2 has been found to suppress amyloidogenic metabolism in transgenic mice overexpressing the human Swedish mutant APP (APPswe) and the mutant human PS1, which lacks exon 9 (PS1dE9). Therefore, the data on X11-family protein function in transgenic human APP metabolism in vivo are inconsistent.
Results
To confirm the interaction of X11L with human APP ectopically expressed in mouse brain, we examined the amyloidogenic metabolism of human APP in two lines of human APP transgenic mice generated to also lack X11L. In agreement with previous reports from our lab and others, we found that the amyloidogenic metabolism of human APP increased in the absence of X11L.
Conclusion
X11L appears to aid in the suppression of amyloidogenic processing of human APP in brain in vivo, as has been demonstrated by previous studies using several human APP transgenic lines with various genetic backgrounds. X11L appears to regulate human APP in a manner similar to that seen in endogenous mouse APP metabolism.
Retrograde transport of several transmembrane proteins from endosomes to the trans-Golgi network (TGN) occurs via Rab 5-containing endosomes, mediated by clathrin and the recently characterized retromer complex. This complex and one of its putative sorting receptor components, SorLA, were reported to be associated to late onset Alzheimer's disease (AD). The pathogenesis of this neurodegenerative disorder is still elusive, although accumulation of amyloidogenic Abeta is a hallmark. This peptide is generated from the sucessive β- and γ- secretase proteolysis of the Alzheimer's amyloid precursor protein (APP), events which are associated with endocytic pathway compartments. Therefore, APP targeting and time of residence in endosomes would be predicted to modulate Abeta levels. However, the formation of an APP- and retromer-containing protein complex with potential functions in retrieval of APP from the endosome to the TGN had, to date, not been demonstrated directly. Further, the motif(s) in APP that regulate its sorting to the TGN have not been characterized.
Results
Through the use of APP-GFP constructs, we show that APP containing endocytic vesicles targeted for the TGN, are also immunoreactive for clathrin-, Rab 5- and VPS35. Further, they frequently generate protruding tubules near the TGN, supporting an association with a retromer-mediated pathway. Importantly, we show for the first time, that mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. The phosphomimetic APP S655E displays decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life.
Conclusions
We reported for the first time the importance of APP phosphorylation on S655 in regulating its retromer-mediated sorting to the TGN or lysosomes. Significantly, the data are consistent with known interactions involving the retromer, SorLA and APP. Further, these findings add to our understanding of APP targeting and potentially contribute to our knowledge of sporadic AD pathogenesis representing putative new targets for AD therapeutic strategies. 相似文献
The amyloid precursor protein (APP) is implied both in cell growth and
differentiation and in neurodegenerative processes in Alzheimer disease.
Regulated proteolysis of APP generates biologically active fragments such as
the neuroprotective secreted ectodomain sAPPα and the neurotoxic
β-amyloid peptide. Furthermore, it has been suggested that the intact
transmembrane APP plays a signaling role, which might be important for both
normal synaptic plasticity and neuronal dysfunction in dementia. To understand
APP signaling, we tracked single molecules of APP using quantum dots and
quantitated APP homodimerization using fluorescence lifetime imaging
microscopy for the detection of Förster resonance energy transfer in
living neuroblastoma cells. Using selective labeling with synthetic
fluorophores, we show that the dimerization of APP is considerably higher at
the plasma membrane than in intracellular membranes. Heparan sulfate
significantly contributes to the almost complete dimerization of APP at the
plasma membrane. Importantly, this technique for the first time structurally
defines the initiation of APP signaling by binding of a relevant physiological
extracellular ligand; our results indicate APP as receptor for neuroprotective
sAPPα, as sAPPα binding disrupts APP dimers, and this disruption
of APP dimers by sAPPα is necessary for the protection of neuroblastoma
cells against starvation-induced cell death. Only cells expressing reversibly
dimerized wild-type, but not covalently dimerized mutant APP are protected by
sAPPα. These findings suggest a potentially beneficial effect of
increasing sAPPα production or disrupting APP dimers for neuronal
survival.The amyloid precursor protein
(APP)4 is known both
for its important role in the development and plasticity of the nervous system
(1–6)
and for its involvement in Alzheimer disease (AD)
(7,
8). Despite intensive research
efforts, the initial events that lead to the prevalent sporadic, i.e.
non-familial, forms of AD are still unclear. Furthermore, although a higher
gene dose of APP (9) or the
presence of pathological APP mutations is sufficient to induce familial AD
(for review, see Ref. 10), the
exact pathological mechanism that is triggered by APP is still under
debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are
thought to contribute to synaptic dysfunction and neurotoxicity
(11,
12). On the other hand, the
α-secretase-derived extracellular fragment of APP (sAPPα), which
is present at lower levels in AD patients than in controls
(13), has been shown to be
beneficial for memory function, to possess neuroprotective properties, and to
counteract the effects of Aβ
(14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration
in AD
(19–24);
however, the signal transduction pathway for transmembrane APP remains
unknown, although several potential regulatory proteins, glycosaminoglycans,
and metal ions are known to bind with high affinity to APP and sAPPα
(25,
26). The most common form of
signal transduction for single-pass transmembrane proteins is the
ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the
dimerization of transmembrane APP has been implied several times in the past.
Several studies have investigated the effects of presumed dimer-breaking
perturbations on biological read-outs, such as the production of Aβ
(27,
28), but without directly
measuring the APP aggregation state, or have investigated the aggregation
state of APP subdomains, often reconstituted in cell-free systems
(27–32).
Dimerization interfaces in both the extracellular and the transmembrane domain
have been suggested.In the studies investigating the aggregation state of full-length APP, most
of the employed methods, such as chemical cross-linking and
co-immunoprecipitation, do not lend themselves readily to a rigorous
quantitative analysis of the abundance of potentially instable dimers
(31,
33), whereas in other cases
the use of chimeras may have influenced the dimerization potential or
precluded the search for a natural stimulus
(23,
34). The only previously
reported direct observation of APP dimerization by Förster resonance
energy transfer (FRET) microscopy uses an assay in which the FRET efficiency
varies with the level of overexpression
(35). Therefore, a
concentration-dependent FRET component due to nonspecific stochastic
encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the
selective detection of APP dimers on the surface of live cells, where they
would encounter ligands, they could not differentiate between subpopulations
of APP. This may be one reason why no natural ligand of APP has ever been
shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective
sAPPα
(36–39).
The ligand-dependent dimerization of sAPPα in solution
(40) and its origination from
transmembrane APP suggest that APP might serve as receptor for sAPPα,
but this binding has never been experimentally shown. 相似文献
Processing of amyloid precursor protein (APP) into amyloid‐β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface‐bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co‐immunoprecipitation and co‐localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post‐endocytic pathway. We introduce functional Venus‐tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP‐positive transport vesicles contain SorCS1. Co‐expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP‐positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.
The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites. 相似文献
Accumulation of the amyloid β (Aβ) peptide derived from the
proteolytic processing of amyloid precursor protein (APP) is the defining
pathological hallmark of Alzheimer disease. We previously demonstrated that
the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP)
robustly promoted Aβ generation independent of FE65 and specifically
interacted with Ran-binding protein 9 (RanBP9). In this study we found that
RanBP9 strongly increased BACE1 cleavage of APP and Aβ generation. This
pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the
Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell
surface APP and accelerated APP internalization, consistent with enhanced
β-secretase processing in the endocytic pathway. The N-terminal half of
RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with
APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP
interactions with LRP and BACE1 and increased lipid raft association of APP.
Importantly, knockdown of endogenous RanBP9 significantly reduced Aβ
generation in Chinese hamster ovary cells and in primary neurons,
demonstrating its physiological role in BACE1 cleavage of APP. These findings
not only implicate RanBP9 as a novel and potent regulator of APP processing
but also as a potential therapeutic target for Alzheimer disease.The major defining pathological hallmark of Alzheimer disease
(AD)2 is the
accumulation of amyloid β protein (Aβ), a neurotoxic peptide derived
from β- and γ-secretase cleavages of the amyloid precursor protein
(APP). The vast majority of APP is constitutively cleaved in the middle of the
Aβ sequence by α-secretase (ADAM10/TACE/ADAM17) in the
non-amyloidogenic pathway, thereby abrogating the generation of an intact
Aβ peptide. Alternatively, a small proportion of APP is cleaved in the
amyloidogenic pathway, leading to the secretion of Aβ peptides
(37–42 amino acids) via two proteolytic enzymes, β- and
γ-secretase, known as BACE1 and presenilin, respectively
(1).The proteolytic processing of APP to generate Aβ requires the
trafficking of APP such that APP and BACE1 are brought together in close
proximity for β-secretase cleavage to occur. We and others have shown
that the low density lipoprotein receptor-related protein (LRP), a
multifunctional endocytosis receptor
(2), binds to APP and alters
its trafficking to promote Aβ generation. The loss of LRP substantially
reduces Aβ release, a phenotype that is reversed when full-length
(LRP-FL) or truncated LRP is transfected in LRP-deficient cells
(3,
4). Specifically, LRP-CT
lacking the extracellular ligand binding regions but containing the
transmembrane domain and the cytoplasmic tail is capable of rescuing
amyloidogenic processing of APP and Aβ release in LRP deficient cells
(3). Moreover, the LRP soluble
tail (LRP-ST) lacking the transmembrane domain and only containing the
cytoplasmic tail of LRP is sufficient to enhance Aβ secretion
(5). This activity of LRP-ST is
achieved by promoting APP/BACE1 interaction
(6), although the precise
mechanism is unknown. Although we had hypothesized that one or more
NPXY domains in LRP-ST might underlie the pro-amyloidogenic
processing of APP, we recently found that the 37 C-terminal residues of LRP
(LRP-C37) lacking the NPXY motif was sufficient to robustly promote
Aβ production independent of FE65
(7). Because LRP-C37 likely
acts by recruiting other proteins, we used the LRP-C37 region as bait in a
yeast two-hybrid screen, resulting in the identification of 4 new LRP-binding
proteins (7). Among these, we
focused on Ran-binding protein 9 (RanBP9) in this study, which we found to
play a critical role in the trafficking and processing of APP. RanBP9, also
known as RanBPM, acts as a multi-modular scaffolding protein, bridging
interactions between the cytoplasmic domains of a variety of membrane
receptors and intracellular signaling targets. These include Axl and Sky
(8), MET receptor
protein-tyrosine kinase (9),
and β2-integrin LFA-1
(10). Similarly, RanBP9
interacts with Plexin-A receptors to strongly inhibit axonal outgrowth
(11) and functions to regulate
cell morphology and adhesion
(12,
13). Here we show that RanBP9
robustly promotes BACE1 processing of APP and Aβ generation. 相似文献
Summary 1. The amyloid precursor protein (APP) is widely distributed among eukaryotic cells, however, its precise role in cellular functioning is not fully clarified. APP is glycoprotein membrane constituent and it may facilitate membrane associated functions.2. The aim of the present study was to examine the possibility that APP may play a role in mediating cellular trophic responses. The methods made use of an antisense oligonucleotide that was prepared to the 5 terminus of APP and shown specifically to reduce the level of APP isoforms.3. In sequential mixing experiments it was observed that the APP antisense oligonucleotide did not significantly modify the trophic response of PC12 cells pretreated with nerve growth factor (NGF). However, pretreatment of cells with the antisense oligonucleotide diminished NGF-induced increases in cellular size and neurite length.4. These observations suggest that APP may play a role in modulating the trophic response. The combined use of APP antisense oligonucleotides and neurotrophic agents may find clinical utility in the treatment of Alzheimertype dementia since it is known that NGF normally causes increases in APP levels. 相似文献
Actinobacillus pleuropneumoniae (APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits.
Results
Proteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge.
Conclusions
We identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates. 相似文献
Seasonal change in the abundance of autotrophic picoplankton (APP) was investigated once or twice a week in relation to some environmental variables in a hypereutrophic pond, from July 1999 to June 2000. Cell density of APP ranged between 0.3×105 and 10.1×105 cells ml–1, overlapping the lower range of APP abundances given in the literature for hypereutrophic systems. The pattern of seasonal change in concentration of dissolved inorganic phosphorus (0.3–20.3 mol P l–1) was similar to that of cell density of APP, suggesting that phosphorus limitation on APP abundance. By contrast, nitrogen limitation seemed unlikely since the pattern of seasonal change in concentration of dissolved inorganic nitrogen was different from that of APP cell density. We could not find any coupled oscillations between APP abundance and heterotrophic nanoflagellates, or between that of APP and ciliates. The dominant ciliate taxa, based on their cell densities, were Cinetochilum margaritaceum, Cyclidium glaucoma, Halteria grandinella, Strobilidium sp. and Urotricha spp. The relative contribution of the <2 m fraction to total chlorophyll concentration was seasonally high (up to 16.2%), indicating seasonal importance of APP abundance as food for heterotrophs. 相似文献
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