APP binds to the EGFR ligands HB-EGF and EGF, acting synergistically with EGF to promote ERK signaling and neuritogenesis

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer’s disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders like Schizophrenia and AD. Here we show that APP interacts with these two EGFR ligands and characterize the effects of APP-EGF interaction in ERK activation and neuritogenesis. HB-EGF was identified as a novel APP interactor in a yeast two-hybrid screen of a human brain cDNA library. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Moreover, co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were also observed to synergistically activate the ERK signaling pathway, crucial for early neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in conditions of APP overexpression and EGF exposure, confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes per cell. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration.

Sp100 interacts with phage ΦC31 integrase to inhibit its recombination activity.

Phage ΦC31 integrase is a potential vector for the insertion of therapeutic genes into specific sites in the human genome. To understand the mechanism involved in ΦC31 integrase-mediated recombination, it is important to understand the interaction between the integrase and cellular proteins. Using a yeast two-hybrid system with pLexA-ΦC31 integrase as bait, we screened a pB42AD human fetal brain cDNA library for potential interacting cellular proteins. From the 10⁶ independent clones that were screened, 11 potential interacting clones were isolated, of which one encoded C-terminal fragment of Sp100. The interaction between Sp100 and ΦC31 integrase was further confirmed by yeast mating and co-immunoprecipitation assays. The hybridization between a ΦC31 integrase peptide array and an HEK293 cell extract revealed that residues 81RILN84 in the N-terminus of ΦC31 integrase are responsible for the interaction with Sp100. Knocking down endogenous Sp100 with Sp100-specific siRNA increased ΦC31 integrase-mediated recombination but did not impact reporter gene expression. Therefore, endogenous Sp100 may interact with ΦC31 integrase and inhibit the efficiency of ΦC31 integrase-mediated recombination.

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.

Functional Selection of Phagocytosis-Promoting Genes

Phagocytosis is a critical host defense mechanism that clears invading pathogens, apoptotic cells, and cell debris; it is an essential process for normal development, tissue remodeling, immune response, and inflammation. Here, a functional selection strategy was used to isolate novel phagocytosis-promoting genes. After the retroviral transfer of mouse brain cDNA library into NIH3T3 mouse fibroblast cells, cell sorting was used to select the cells that phagocytosed fluorescent zymosan particles. The cDNAs were retrieved from the selected cells and identified by DNA sequencing as eIF5A, Meg3, Tubb5, Sparcl-1, Uchl-1, Bsg (CD147), Ube2v1, and Pamr1. The phagocytosis-promoting activity for some of these cDNAs was confirmed by transient transfection in the independent phagocytosis assays. Thus, the unbiased selection procedure successfully identified multiple phagocytosis-promoting genes. The selection method can be applied to other cell-based assays where cells with a desired phenotype can be physically separated. Moreover, the new gene targets uncovered in this study could be relevant to biomolecule screening in search of phagocytosis-regulating agents. In a small-scale screen, a series of imidazopyridine compounds was tested to identify the small molecules that modulate eIF5A-mediated phagocytic activity. Several compounds that influenced the phagocytic activity can be further used as chemical-genetic tools to delineate the mechanisms of eIF5A action and be potential drug candidates that are capable of therapeutically modulating phagocytic activity.

A Novel Functional Screen in Human Cells Identifies MOCA as a Negative Regulator of Wnt Signaling

Aberrant Wnt signal transduction is involved in many human diseases such as cancer and neurodegenerative disorders. The key effector protein of the canonical Wnt pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate gene transcription that leads to expression of Wnt target genes. In this study we provide results obtained from a novel functional screen of a human brain cDNA library used to identify 63 genes that are putative negative Wnt regulators. These genes were divided into eight functional groups that include known canonical and noncanonical Wnt pathway components and genes that had not yet been assigned to the Wnt pathway. One of the groups, the presenilin-binding proteins, contains the modifier of cell adhesion (MOCA) gene. We show that MOCA is a novel inhibitor of Wnt/β-catenin signaling. MOCA forms a complex with β-catenin and inhibits transcription of known Wnt target genes. Epistasis experiments indicate that MOCA acts to reduce the levels of nuclear β-catenin, increase the levels of membrane-bound β-catenin, and enhances cell–cell adhesion. Therefore, our data indicate that MOCA is a novel Wnt negative regulator and demonstrate that this screening approach can be a rapid means for isolation of new Wnt regulators.