Dataset Information

Identification of beta-secretase (BACE1) substrates using quantitative proteomics.

ABSTRACT: Beta-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the beta-secretase responsible for generating the amyloid-beta protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few beta-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative beta-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.

SUBMITTER: Hemming ML

PROVIDER: S-EPMC2793532 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Identification of beta-secretase (BACE1) substrates using quantitative proteomics.

Hemming Matthew L ML Elias Joshua E JE Gygi Steven P SP Selkoe Dennis J DJ

PloS one 20091229 12

Beta-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the beta-secretase responsible for generating the amyloid-beta protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to re ...[more]

PMID: 20041192

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Project description:Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires endoplasmic reticulum (ER)-resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation. Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic activation of fungal sterol regulatory element-binding protein transcription factors. The Dsc E3 ligase contains five integral membrane subunits and structurally resembles ER-associated degradation E3 ligases. Saccharomyces cerevisiae codes for homologs of Dsc E3 ligase subunits, including the Dsc1 E3 ligase homolog Tul1 that functions in Golgi protein quality control. Interestingly, S. cerevisiae lacks sterol regulatory element-binding protein homologs, indicating that novel Tul1 E3 ligase substrates exist. Here, we show that the S. cerevisiae Tul1 E3 ligase consists of Tul1, Dsc2, Dsc3, and Ubx3 and define Tul1 complex architecture. Tul1 E3 ligase function required each subunit as judged by vacuolar sorting of the artificial substrate Pep12D. Genetic studies demonstrated that Tul1 E3 ligase was required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we performed quantitative diGly proteomics using stable isotope labeling by amino acids in cell culture to survey ubiquitylation in wild-type and tul1? cells. We identified 3116 non-redundant ubiquitylation sites, including 10 sites in candidate substrates. Quantitative proteomics found 4.5% of quantified proteins (53/1172) to be differentially expressed in tul1? cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, our data demonstrate that the Tul1 E3 ligase functions in protein homeostasis under non-stress conditions and support a role in protein quality control. This quantitative diGly proteomics methodology will serve as a robust platform for screening for stress conditions that require Tul1 E3 ligase activity.

Dataset Information

Identification of beta-secretase (BACE1) substrates using quantitative proteomics.

Publications

Identification of beta-secretase (BACE1) substrates using quantitative proteomics.

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