Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin ␤ is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin ␤ and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin ␤. Processing of APP by meprin ␤ was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin ␤ ؊/؊ mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin ␤ is a physiologically relevant enzyme in APP processing.

BackgroundThe metalloprotease meprin β cleaves the Alzheimer’s Disease (AD) relevant amyloid precursor protein (APP) as a β-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aβ2-x variants.ResultsHerein, we observed increased endogenous sAPPα levels in the brains of meprin β knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis. The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin β and that, interestingly, meprin β is unable to generate N-terminally truncated Aβ peptides from Swedish mutant APP (APPswe).ConclusionConcluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0084-5) contains supplementary material, which is available to authorized users.

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.

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The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important role in regulation of the function of the receptor. The impact of single and double inactivating knock-in mutations of these motifs on receptor maturation, cell surface expression, and ligand internalization was analyzed in mutant and control wild-type mice and MEFs. Single inactivation of the proximal NPXY or in combination with inactivation of the distal NPXYXXL motif are both shown to be associated with an impaired maturation and premature proteasomal degradation of full-length LRP1. Therefore, only a small mature LRP1 pool is able to reach the cell surface resulting indirectly in severe impairment of ligand internalization. Single inactivation of the NPXYXXL motif revealed normal maturation, but direct impairment of ligand internalization. In conclusion, the proximal NPXY motif proves to be essential for early steps in the LRP1 biosynthesis, whereas NPXYXXL appears rather relevant for internalization.

The enzyme silicatein has been bioengineered to carry a thiol-bearing Au-affinity tag (Cys-tag) for direct immobilization on gold carriers in shortest time without the need for prior surface functionalization. Through microcontact printing, defined silicatein micropatterns were created on gold surfaces, facilitating the subsequent enzymatically controlled synthesis of photocatalytically active TiO(2).

Survivin orchestrates intracellular pathways during cell division and apoptosis. Its central function as mitotic regulator and inhibitor of cell death has major implications for tumor cell proliferation. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution a complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulator, a survivin-like protein (SURVL) of one of the earliest-branching metazoan taxa was identified and functionally characterized. SURVL of the sponge Suberites domuncula shares considerable similarities with its metazoan homologs, ranging from conserved exon/intron structure to presence of protein-interaction domains. Whereas sponge tissue shows a low steady-state level, SURVL expression was significantly upregulated in rapidly proliferating primmorph cells. In addition, challenge of tissue and primmorphs with heavy metal or lipopeptide stimulated SURVL expression, concurrent with the expression of a newly discovered caspase. Complementary functional analyses in transfected HEK-293 cells revealed that heterologous expression of a SURVL-EFGP fusion not only promotes proliferation but also enhances resistance to cadmiuminduced cell death. Taken together, these results suggest both a deep evolutionary conserved dual role of survivin and an equally conserved central position in the interconnected pathways of cell cycle and apoptosis. Apoptosis has a crucial role not only in countless physiological processes (e.g., elimination of redundant cells during vertebrate embryonic development) but also during pathogenesis. As apoptosis relies on the clear-cut function of numerous proand antiapoptotic factors, any malfunction on their part or disruption of their respective interplay inevitably leads to dysfunctional cellular metabolism. The ensuing delayed or premature cell death often manifests in severe pathologies, including carcinogenesis and neurodegenerative diseases.The groundbreaking discovery of the Caenorhabditis elegans proteins cell death abnormality (Ced)-9, homolog of human antiapoptotic Bcl-2, and Ced-3, homolog of human caspase-1, has shown the molecular conservation of the apoptotic cell death program along a broad range of Metazoa, from nematodes to vertebrates.1 Since then, the fundamental role of apoptosis has been increasingly recognized in various physiological processes. In addition, several invertebrate model organisms revealed a significant potential for analyses of apoptotic mechanisms, not only under an evolutionary point of view. Thus, the identification of the human crumbs homolog 1 and its causative role in the manifestation of retinitis pigmentosa was based on the discovery of mutational variants of the Drosophila melanogaster protein crumbs.2 In addition, loss-of-function mutations of the C. elegans dystrophin-1 gene confirmed the role of human dystrophin in the manifestation...

Inspired by the intermolecular cross-linking of mussel foot proteins and their adhesive properties, tyrosinase has been used to modify recombinant silicatein. DOPA/DOPAquinone-mediated cross-linking and interfacial interactions enhanced both self-assembly of silicatein building blocks and templating of core-shell silica spheres, resulting in fluorescent biomimetic silicatein-silica hybrid mesofibers.