Systematic Proteomic Analysis Identifies β-Site Amyloid Precursor Protein Cleaving Enzyme 2 and 1 (BACE2 and BACE1) Substrates in Pancreatic β-Cells

Stützer, Ina; Selevsek, Nathalie; Esterházy, Daria; Schmidt, Alexander; Aebersold, Ruedi; Stoffel, Markus

doi:10.1074/jbc.m112.444703

Cited by 79 publications

(108 citation statements)

References 64 publications

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“…Some of the previously identified proteins might only be detectable in murine CSF on previous immunodepletion or extensive fractionation, because of their low expression levels or the presence of highly abundant proteins such as albumin. This could explain why the overlap between the different studies is not complete and why some well-known BACE1 substrates such as seizure protein 6 were not quantified in our analysis (27,28). Our simplified workflow minimizes the variance during sample processing, which is helpful for the accuracy of label-free quantification.…”

Section: Discussionmentioning

confidence: 94%

“…PTPRN2 has been first described as a BACE1 substrate in the murine endocrine pancreas. This was demonstrated by a reduction of PTPRN2 protein levels in the conditioned media and the according increase of protein levels in the cell lysate of BACE1 deficient islets using SRM/MRM-based mass spectrometry (28). In addition, PTPRN2 has been shown to regulate insulin secretion (46).…”

Section: Discussionmentioning

confidence: 99%

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Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

Dislich

Wohlrab

Bachhuber

et al. 2015

Molecular & Cellular Proteomics

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Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1؊/؊ and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1؊/؊ and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1؊/؊ mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken Cerebrospinal fluid (CSF) 1 consists of interstitial fluid that is in continuous exchange with the central nervous system and the peripheral blood system. It represents the only body fluid in humans that is in direct contact with brain tissue and accessible in a routine clinical setting. Thus, the easy accessibility from the periphery renders CSF perfectly suited to study pathologic neurological processes (1). Human CSF has a relatively low protein content (ϳ 0.4 mg/ml), but features a highly diverse proteome. It is thus increasingly studied by modern mass spectrometry based proteomics (2). The proteomic analysis of human CSF typically involves various protein concentration and fractionation steps as well as the depletion of highly abundant proteins, such as serum albumin. This allows the identification of several hundred up to 2600 proteins from several milliliters of human CSF (3).Mice are the most popular animal model in preclinical research, because of their similarity to humans in genetics and physiology, their unlimited supply and their ease of genetic engineering. The study of their CSF can provide valuable insights into disease mechanisms and biomarker discovery Author Contributions: The manuscript was written by Bastian Dislich and Stefan F. Lichtenthaler. The study was guided and planned by Bastian Dislich, Sebastian Hogl, Melanie Meyer-Luehmann and Stefan F. Lichtenthaler. Bastian Dislich established the mass spectrometry of murine CSF and performed the proteomic analysis of the BACE1Ϫ/Ϫ CSF. Felix Wohlrab performed all other experiments, except for the CSF extraction, which was performed by Teresa Bachhuber, and the analysis of...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

Dislich

Wohlrab

Bachhuber

et al. 2015

Molecular & Cellular Proteomics

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“…The molecular basis for BACE subtype specificity in PMEL cleavage is not totally clear, but likely in part reflects the differential expression (17) and subcellular compartmentalization of BACE1 and BACE2 in pigment cells. Although substrate recognition also likely differs between BACE1 and BACE2, BACE2 has no known consensus recognition site (35), and the sheddase cleavage site for PMEL that is likely targeted by BACE2 in MNT1 cells (13) shares only a single leucine with other known BACE2 substrates like TMEM27 (32), or seizure 6-like protein (35).…”

Section: Discussionmentioning

confidence: 99%

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells

Rochin

Hurbain

Serneels

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

145

166

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Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aβ peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2 −/− but not Bace1 −/− mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APPderived pathological amyloids.

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“…Cathepsin D is a major lysosomal protease, and decreasing its activity would compromise the overall cellular degradation machinery. 100 Whilst the function of BACE2 remains unclear, a recent publication of its substrate proteome suggests that it may play an important role in glucose metabolism in the pancreas, 101 and this information should be taken into consideration when selecting a BACE1 inhibitor for clinical trials. Nonconventional inhibitors that work in an allosteric mechanism by displacing binding of the substrate have also been described.…”

Section: Bace1 Inhibitors In Preclinical and Clinical Trialsmentioning

confidence: 99%

Critical analysis of the use of β-site amyloid precursor protein-cleaving enzyme 1 inhibitors in the treatment of Alzheimer's disease

Evin

Barakat

2014

DNND

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Alzheimer's disease (AD) is the major cause of dementia in the elderly and an unmet clinical challenge. A variety of therapies that are currently under development are directed to the amyloid cascade. Indeed, the accumulation and toxicity of amyloid-β (Aβ) is believed to play a central role in the etiology of the disease, and thus rational interventions are aimed at reducing the levels of Aβ in the brain. Targeting β-site amyloid precursor protein-cleaving enzyme (BACE)-1 represents an attractive strategy, as this enzyme catalyzes the initial and rate-limiting step in Aβ production. Observation of increased levels of BACE1 and enzymatic activity in the brain, cerebrospinal fluid, and platelets of patients with AD and mild cognitive impairment supports the potential benefits of BACE1 inhibition. Numerous potent inhibitors have been generated, and many of these have been proved to lower Aβ levels in the brain of animal models. Over 10 years of intensive research on BACE1 inhibitors has now culminated in advancing half a dozen of these drugs into human trials, yet translating the in vitro and cellular efficacy of BACE1 inhibitors into preclinical and clinical trials represents a challenge. This review addresses the promises and also the potential problems associated with BACE1 inhibitors for AD therapy, as the complex biological function of BACE1 in the brain is becoming unraveled.

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Systematic Proteomic Analysis Identifies β-Site Amyloid Precursor Protein Cleaving Enzyme 2 and 1 (BACE2 and BACE1) Substrates in Pancreatic β-Cells

Cited by 79 publications

References 64 publications

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells

Critical analysis of the use of β-site amyloid precursor protein-cleaving enzyme 1 inhibitors in the treatment of Alzheimer's disease

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Systematic Proteomic Analysis Identifies β-Site Amyloid Precursor Protein Cleaving Enzyme 2 and 1 (BACE2 and BACE1) Substrates in Pancreatic β-Cells

Cited by 79 publications

References 64 publications

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells

Critical analysis of the use of &beta;-site amyloid precursor protein-cleaving enzyme 1 inhibitors in the treatment of Alzheimer&#39;s disease

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Critical analysis of the use of β-site amyloid precursor protein-cleaving enzyme 1 inhibitors in the treatment of Alzheimer's disease