Ute Schepers

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.

Bright coppertunities: multinuclear Cu(I) complexes with N-P ligands and their applications.

Easy come, easy go: the great structural diversity of Cu(I) complexes is an ambivalent trait. Apart from the well-known catalytic properties of Cu(I), a great number of potent luminescent complexes have been found in the last ten years featuring a plethora of structural motifs. The downside of this variety is the undesired formation of other species upon processing. In here, strategies to avoid this behavior are presented: Only one favorable structural unit often exists for multinuclear Cu(I) complexes with bridging ligands. In addition, these complexes exhibit favorable photophysical properties due to cooperative effects of the metal halide core. Furthermore, we demonstrate the broad range of applications of emitting Cu(I) compounds.

Luminescent cell-penetrating pentadecanuclear lanthanide clusters.

A novel pentadecanuclear lanthanide hydroxy cluster [{Ln15(μ3-OH)20(PepCO2)10(DBM)10Cl}Cl4] (Ln = Eu (1), Tb (2)) featuring the first example with peptoids as supporting ligands was prepared and fully characterized. The solid-state structures of 1 and 2 were established via single-crystal X-ray crystallography. ESI-MS experiments revealed the retention of the cluster core in solution. Although OH groups are present, 1 showed intense red fluorescence with 11(1)% absolute quantum yield, whereas the emission intensity and the quantum yield of 2 were significantly weaker. In vitro investigations on 1 and 2 with HeLa tumor cells revealed an accumulation of the clusters in the endosomal-lyosomal system, as confirmed by confocal microscopy in the TRLLM mode. The cytotoxicity of 1 and 2 toward the HeLa cells is moderate.

Schlank, a member of the ceramide synthase family controls growth and body fat in Drosophila

Ceramide synthases are highly conserved transmembrane proteins involved in the biosynthesis of sphingolipids, which are essential structural components of eukaryotic membranes and can act as second messengers regulating tissue homeostasis. However, the role of these enzymes in development is poorly understood due to the lack of animal models. We identified schlank as a new Drosophila member of the ceramide synthase family. We demonstrate that schlank is involved in the de novo synthesis of a broad range of ceramides, the key metabolites of sphingolipid biosynthesis. Unexpectedly, schlank mutants also show reduction of storage fat, which is deposited as triacylglyerols in the fat body. We found that schlank can positively regulate fatty acid synthesis by promoting the expression of sterol‐responsive element‐binding protein (SREBP) and SREBP‐target genes. It further prevents lipolysis by downregulating the expression of triacylglycerol lipase. Our results identify schlank as a new regulator of the balance between lipogenesis and lipolysis in Drosophila. Furthermore, our studies of schlank and the mammalian Lass2 family member suggest a novel role for ceramide synthases in regulating body fat metabolism.

Long Endogenous dsRNAs Can Induce Complete Gene Silencing in Mammalian Cells and Primary Cultures

Recently, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has rapidly developed to a powerful instrument for specific silencing of gene expression in several organisms, including Caenorhabditis elegans, Drosophila melanogaster, and plants. The finding that synthetic small interfering RNAs (siRNAs) of 21 nt as well as stable, endogenously expressed, large dsRNA are suited to specifically induce gene silencing in mammalian cells offered the possibility of expanding this technique to mammalian systems. In this work, we engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes. We used this technique to specifically silence genes coding for glucosylceramide synthase (GCS), the sphingolipid activator protein precursor (SAP), and glucocerebrosidase (GBA), all implicated in glycosphingolipid metabolism. From a 1600-bp inverted repeat DNA template, a dsRNA of 800 bp is expressed and predicted to mediate the specific suppression of the corresponding gene by RNAi. Remarkably, we were able to use this method to achieve complete inhibition of those genes we targeted in different cultured human cell lists. These findings testify to the generality of RNAi application in suppressing gene expression in mammalian cells.

Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function

Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimers disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain. DOI: http://dx.doi.org/10.7554/eLife.12748.001

Tin Tungstate Nanoparticles: A Photosensitizer for Photodynamic Tumor Therapy

The nanoparticulate inorganic photosensitizer β-SnWO4 is suggested for photodynamic therapy (PDT) of near-surface tumors via reiterated 5 min blue-light LED illumination. β-SnWO4 nanoparticles are obtained via water-based synthesis and comprise excellent colloidal stability under physiological conditions and high biocompatibility at low material complexity. Antitumor and antimetastatic effects were investigated with a spontaneously metastasizing (4T1 cells) orthotopic breast cancer BALB/c mouse model. Besides protamine-functionalized β-SnWO4 (23 mg/kg of body weight, in PBS buffer), chemotherapeutic doxorubicin was used as positive control (2.5 mg/kg of body weight, in PBS buffer) and physiological saline (DPBS) as a negative control. After 21 days, treatment with β-SnWO4 resulted in a clearly inhibited growth of the primary tumor (all tumor volumes below 3 cm(3)) as compared to the doxorubicin and DPBS control groups (volumes up to 6 cm(3)). Histological evaluations of lymph nodes and lungs as well as the volume of ipsilateral lymph nodes show a remarkable antimetastatic effect being similar to chemotherapeutic doxorubicin but-according to blood counts-at significantly reduced side effects. On the basis of low material complexity, high cytotoxicity under blue-light LED illumination at low dark and long-term toxicity, β-SnWO4 can be an interesting addition to PDT and the treatment of near-surface tumors, including skin cancer, esophageal/gastric/colon tumors as well as certain types of breast cancer.

Chemical Synthesis of Glycosaminoglycans

Glycosaminoglycans (GAGs) as one major part of the glycocalyx are involved in many essential biological cell processes, as well as in many courses of diseases. Because of the potential therapeutic application of GAG polymers, fragments, and also derivatives toward different diseases (e.g., heparin derivatives against Alzheimers disease), there is a continual growing demand for new chemical syntheses, which suffice the high claim to stereoselectivity and chemoselectivity. This Review summarizes the progress of chemical syntheses of GAGs over the last 10 years. For each class of the glycosaminoglycans-hyaluronan (HA), heparan sulfate/heparin (HS/HP), chondroitin/dermatan sulfate (CS/DS), and keratan sulfate (KS)-mainly novel glycosylation strategies, elongation sequences, and protecting group patterns are discussed, but also (semi)automated syntheses, enzymatic approaches, and functionalizations of synthesized or isolated GAGs are considered.

Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry

The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme beta-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZ/V5-His, which was used for the constitutive expression in stably transformed Sf21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by sequential chromatography on Ni-NTA-agarose and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the structure (GlcNAc)(2)Fuc(Man)(3).