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Ubiquitin regulates GGA3-mediated degradation of BACE1.

ABSTRACT: 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.

SUBMITTER: Kang EL

PROVIDER: S-EPMC2911305 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Ubiquitin regulates GGA3-mediated degradation of BACE1.

Kang Eugene L EL Cameron Andrew N AN Piazza Fabrizio F Walker Kendall R KR Tesco Giuseppina G

The Journal of biological chemistry 20100518 31

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 ...[more]

PMID: 20484053

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Project description:Seladin-1 is a neuroprotective protein selectively down-regulated in brain regions affected in Alzheimer disease (AD). Seladin-1 protects cells against beta-amyloid (Abeta) peptide 42- and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the beta-secretase (BACE1) function and beta-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that approximately 60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or Abeta secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized gamma-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced beta-amyloidogenic processing of APP and ultimately increased Abeta production. These adverse changes associated with decreased cell viability in seladin-1 siRNA-transfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased beta-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis.

Project description:CCAAT/enhancer-binding protein alpha (C/EBP?) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBP? for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBP?, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBP? protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42?kDa CCAAT-enhancer-binding protein alpha (p42C/EBP?) and mutant isoform 30?kDa CCAAT-enhancer-binding protein alpha (p30C/EBP?), this may explain perturbed p42C/EBP?/p30C/EBP? ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBP? inducible K562- p42C/EBP?-estrogen receptor (ER) cells inhibits ?-estradiol (E2)-induced C/EBP? degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBP? in C/EBP? stably transfected cells (?-estradiol inducible K562 cells stably expressing p42C/EBP?-ER (K562-C/EBP?-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBP?-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBP? protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBP? often experienced in myeloid leukemia.

Dataset Information

Ubiquitin regulates GGA3-mediated degradation of BACE1.

Publications

Ubiquitin regulates GGA3-mediated degradation of BACE1.

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