Sebastian Hogl

Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons.

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane‐protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome‐wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry‐mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates—seizure‐protein 6, L1, CHL1 and contactin‐2—were validated in brains of BACE1 inhibitor‐treated and BACE1 knock‐out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

Secretome Protein Enrichment with Click Sugars Identifies Physiological Substrates of the Alzheimer Protease BACE1 in Primary Neurons

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane‐protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome‐wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry‐mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates—seizure‐protein 6, L1, CHL1 and contactin‐2—were validated in brains of BACE1 inhibitor‐treated and BACE1 knock‐out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP‐43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule‐associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over‐expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant‐negative Rab7‐interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.

Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth

Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43+ inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS- and FTLD-TDP–associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Double-homozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.

Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and γ-Secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development.

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

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 Alzheimers 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 together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.

Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

QARIP: a web server for quantitative proteomic analysis of regulated intramembrane proteolysis

Regulated intramembrane proteolysis (RIP) is a critical mechanism for intercellular communication and regulates the function of membrane proteins through sequential proteolysis. RIP typically starts with ectodomain shedding of membrane proteins by extracellular membrane-bound proteases followed by intramembrane proteolysis of the resulting membrane-tethered fragment. However, for the majority of RIP proteases the corresponding substrates and thus, their functions, remain unknown. Proteome-wide identification of RIP protease substrates is possible by mass spectrometry-based quantitative comparison of RIP substrates or their cleavage products between different biological states. However, this requires quantification of peptides from only the ectodomain or cytoplasmic domain. Current analysis software does not allow matching peptides to either domain. Here we present the QARIP (Quantitative Analysis of Regulated Intramembrane Proteolysis) web server which matches identified peptides to the protein transmembrane topology. QARIP allows determination of quantitative ratios separately for the topological domains (cytoplasmic, ectodomain) of a given protein and is thus a powerful tool for quality control, improvement of quantitative ratios and identification of novel substrates in proteomic RIP datasets. To our knowledge, the QARIP web server is the first tool directly addressing the phenomenon of RIP. The web server is available at http://webclu.bio.wzw.tum.de/qarip/. This website is free and open to all users and there is no login requirement.

Label‐free quantitative analysis of the membrane proteome of Bace1 protease knock‐out zebrafish brains

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimers disease, where it acts as the β‐secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter‐aided sample preparation, strong‐anion exchange fractionation, and label‐free quantification. We used bace1‐deficient zebrafish and quantified differences in protein levels between wild‐type and bace1 −/− zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild‐type and bace1 −/− zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty‐four membrane proteins accumulated in the bace1 −/− brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican‐1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock‐out zebrafish tissue and demonstrates that combining gene knock‐out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.

Therapeutic potential of BACE1/beta-secretase: Proteome-wide identification of physiological BACE1 substrates in primary neurons and mouse brain

SECRETASE: PROTEOME-WIDE IDENTIFICATION OF PHYSIOLOGICAL BACE1 SUBSTRATES IN PRIMARY NEURONS AND MOUSE BRAIN Stefan Lichtenthaler, Bastian Dislich, Sebastian Hogl, Alessio Colombo, Ulrike Zeitschel, Michael Willem, Christian Haass, Christiane Volbracht, Steffen Roßner, Peer-Hendrik Kuhn, DZNE-University of Munich, Munich, Germany; University Leipzig, Leipzig, Germany; 3 Lundbeck, Copenhagen, Denmark.