Ubiquitin regulates GGA3-mediated degradation of BACE1

Alzheimer's Disease Research Laboratory, Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2010; 285(31):24108-19. DOI: 10.1074/jbc.M109.092742
Source: PubMed


BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of beta-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1-3 (Golgi-localized gamma-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and beta-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.

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    • "GGA3 controls post-endocytic trafficking of transmembrane proteins through multiple mechanisms. GGA3 was found to interact with ubiquitin and to be involved in the endosomal sorting of ubiquitinated receptors, such as EGFR and BACE1, to lysosomes (Kang et al., 2010; Puertollano and Bonifacino, 2004). It was also involved in the endosomal sorting of Met to the recycling pathway through an interaction with Crk (Parachoniak et al., 2011). "
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    ABSTRACT: Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood. Here, we demonstrate that GGA3 (Golgi-localized γ adaptin-ear-containing ADP ribosylation factor-binding protein 3) interacts directly with the TrkA cytoplasmic tail through an internal DXXLL motif and mediates the functional recycling of TrkA to the plasma membrane. We found that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation and reduces cell survival. We also showed that GGA3's effect on TrkA recycling was dependent on the activation of Arf6. This work identifies GGA3 as a key player in a novel DXXLL-mediated endosomal sorting machinery that targets TrkA to the plasma membrane, where it prolongs the activation of Akt signaling and survival responses.
    Molecular Biology of the Cell 10/2015; Mol Biol Cell. 2015 Oct 7. pii: mbc.E15-02-0087. [Epub ahead of print]. DOI:10.1091/mbc.E15-02-0087 · 4.47 Impact Factor
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    • "While the mechanism of BACE1 elevation in AD and APP Tg brains is not fully understood, recent data indicate that BACE1 levels are upregulated during stresses associated with AD risk such as energy deprivation [22], [27], hypoxia and stroke [28]–[30], oxidative stress [31] and traumatic brain injury [32], [33]. Several molecular pathways have been proposed to mediate the BACE1 elevation, such as caspase-3-induced cleavage of the trafficking adaptor protein GGA3 leading to impaired lysosomal degradation of BACE1 [34], [35], Cdk5 phosphorylation of transcription factor Stat3 [36], altered levels of microRNAs [37]–[41], increased transcription factor HIF1α activity [29], and elevated BACE1 mRNA translation via phosphorylation of the translation initiation factor eIF2α [22]. "
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    ABSTRACT: β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) initiates the production of β-amyloid (Aβ), the major constituent of amyloid plaques in Alzheimer's disease (AD). BACE1 is elevated ∼2-3 fold in AD brain and is concentrated in dystrophic neurites near plaques, suggesting BACE1 elevation is Aβ-dependent. Previously, we showed that phosphorylation of the translation initiation factor eIF2α de-represses translation of BACE1 mRNA following stress such as energy deprivation. We hypothesized that stress induced by Aβ might increase BACE1 levels by the same translational mechanism involving eIF2α phosphorylation. To test this hypothesis, we used three different genetic strategies to determine the effects of reducing eIF2α phosphorylation on Aβ-dependent BACE1 elevation in vitro and in vivo: 1) a two-vector adeno-associated virus (AAV) system to express constitutively active GADD34, the regulatory subunit of PP1c eIF2α phosphatase; 2) a non-phosphorylatable eIF2α S51A knockin mutation; 3) a BACE1-YFP transgene lacking the BACE1 mRNA 5' untranslated region (UTR) required for eIF2α translational regulation. The first two strategies were used in primary neurons and 5XFAD transgenic mice, while the third strategy was employed only in 5XFAD mice. Despite very effective reduction of eIF2α phosphorylation in both primary neurons and 5XFAD brains, or elimination of eIF2α-mediated regulation of BACE1-YFP mRNA translation in 5XFAD brains, Aβ-dependent BACE1 elevation was not decreased. Additionally, robust inhibition of eIF2α phosphorylation did not block Aβ-dependent APP elevation in primary neurons, nor did it reduce amyloid pathology in 5XFAD mice. We conclude that amyloid-associated BACE1 elevation is not caused by translational de-repression via eIF2α phosphorylation, but instead appears to involve a post-translational mechanism. These definitive genetic results exclude a role for eIF2α phosphorylation in Aβ-dependent BACE1 and APP elevation. We suggest a vicious pathogenic cycle wherein Aβ42 toxicity induces peri-plaque BACE1 and APP accumulation in dystrophic neurites leading to exacerbated Aβ production and plaque progression.
    PLoS ONE 07/2014; 9(7):e101643. DOI:10.1371/journal.pone.0101643 · 3.23 Impact Factor
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    • "GGAs (Golgi-localized Gammaear containing ARF-binding) are monomeric adapter proteins that are ubiquitously expressed and involved in trafficking of proteins from the trans-Golgi network to the endosome/lysosome system. GGA3 specifically, binds to and transports ubiquitinated BACE1 to lysosomes to mediate its degradation (Kang et al., 2010; Tesco et al., 2007). Elevated BACE1 levels in AD are due in part to the stabilization of BACE1 protein caused by decreased levels of GGA3 due to its cleavage by caspase-3 (Tesco et al., 2007). "
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    ABSTRACT: Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69- and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in release of endogenous Aβ1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. Expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aβ. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aβ production, with this potentiating the decline in function of cholinergic neurons.
    Neurobiology of Disease 05/2014; 69. DOI:10.1016/j.nbd.2014.05.008 · 5.08 Impact Factor
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