The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.

Aberrant activation of the canonical Wnt signal transduction pathway is involved in many diseases including cancer and is especially implicated in the development and progression of colorectal cancer. The key effector protein of the canonical Wnt pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor to activate expression of Wnt target genes. In this study, we used a new functional screen based on cell survival in the presence of cDNAs encoding proteins that activate the Wnt pathway thus identifying novel Wnt signaling components. Here we identify carboxypeptidase E (|CPE) and its splice variant, ΔN-CPE, as novel regulators of the Wnt pathway. We show that whereas ΔN-CPE activates the Wnt signal, the full-length CPE (F-CPE) protein is an inhibitor of Wnt/β-catenin signaling. F-CPE forms a complex with the Wnt3a ligand and the Frizzled receptor. Moreover, F-CPE disrupts disheveled-induced signalosomes that are important for transducing the Wnt signal and reduces β-catenin protein levels and activity. Taken together, our data indicate that F-CPE and ΔN-CPE regulate the canonical Wnt signaling pathway negatively and positively, respectively, and demonstrate that this screening approach can be a rapid means for isolation of novel Wnt signaling components.

The Wnt signaling pathway is an evolutionary conserved system, having pivotal roles during animal development. When over-activated, this signaling pathway is involved in cancer initiation and progression. The canonical Wnt pathway regulates the stability of β-catenin primarily by a destruction complex containing a number of different proteins, including Glycogen synthase kinase 3β (GSK-3β) and Axin, that promote proteasomal degradation of β-catenin. As this signaling cascade is modified by various proteins, novel screens aimed at identifying new Wnt signaling regulators were conducted in our laboratory. One of the different genes that were identified as Wnt signaling activators was Aldolase C (ALDOC). Here we report that ALDOC, Aldolase A (ALDOA) and Aldolase B (ALDOB) activate Wnt signaling in a GSK-3β-dependent mechanism, by disrupting the GSK-3β-Axin interaction and targeting Axin to the dishevelled (Dvl)-induced signalosomes that positively regulate the Wnt pathway thus placing the Aldolase proteins as novel Wnt signaling regulators.

Klotho is an anti-aging transmembrane protein, which can be shed and function as a hormone. Accumulating data indicate klotho as a tumor suppressor in a wide array of malignancies and indicate the subdomain KL1 as the active region of the protein. We aimed to study the role of klotho as a tumor suppressor in colorectal cancer. Bioinformatics analyses of TCGA datasets indicated reduced klotho mRNA levels in human colorectal cancer, along with negative regulation of klotho expression by hypermethylation of the promoter and 1st exon, and hypomethylation of an area within the gene. Overexpression or treatment with klotho or KL1 inhibited proliferation of colorectal cancer cells in vitro. The in vivo activity of klotho and KL1 was examined using two models recapitulating development of tumors in the normal colonic environment of immune-competent mice. Treatment with klotho inhibited formation of colon polyps induced by the carcinogen azoxymethane, and KL1 treatment slowed growth of orthotopically-implanted colorectal tumors. Gene expression array revealed that klotho and KL1 expression enhanced the unfolded protein response (UPR) and this was further established by increased levels of spliced XBP1, GRP78 and phosphorylated-eIF2α. Furthermore, attenuation of the UPR partially abrogated klotho tumor suppressor activity. In conclusion, this study indicates klotho as a tumor suppressor in colorectal cancer and identifies, for the first time, the UPR as a pathway mediating klotho activities in cancer. These data suggest that administration of exogenous klotho or KL1 may serve as a novel strategy for prevention and treatment of colorectal cancer.

Establishment of a flow cytometry-based reporter assay to identify nonsense mutation read-through agents. Macrolide antibiotics can induce read-through of disease-causing stop codons. Macrolide-induced protein restoration can alleviate disease-like phenotypes.

Background Sarcopoterium spinosum (S. spinosum) is used by Bedouin medicinal practitioners for the treatment of diabetes. While the anti-diabetic activity of S. spinosum root extract was validated in previous studies, the activity of aerial parts of the same plants has not been elucidated yet. The aim of this study was to clarify the glucose lowering properties of the aerial parts of the shrub.MethodsAnti-diabetic properties were evaluated by measuring the activity of carbohydrate digesting enzymes, glucose uptake into 3 T3-L1 adipocytes, and insulin secretion. Insulin signaling cascade was followed in L6 myotubes using Western blot and PathScan analysis.ResultsActivity of α-amylase and α-glucosidase was inhibited by extracts of all S. spinosum organs. Basal and glucose-induced insulin secretion was measured in Min6 cells and found to be enhanced as well. Glucose uptake was induced by all S. spinosum extracts, with roots found to be the most effective and fruits the least.The effect of S. spinosum on Akt phosphorylation was minor compared to insulin effect. However, GSK3β and PRAS40, which are downstream elements of the insulin cascade, were found to be highly phosphorylated by S. spinosum extracts. Inhibition of PI3K and Akt, but not AMPK and ERK, abrogated the induction of glucose uptake by the aerial parts of the shrub.ConclusionThe aerial organs of S. spinosum have anti-diabetic properties and may be used as a basis for the development of dietary supplements or to identify new agents for the treatment of type 2 diabetes.

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Over the last two decades, a growing number of scientific evidences highlighted the potential therapeutic value of several structures of aminoglycoside antibiotics (including gentamicin and G418) for the treatment of various genetic diseases caused by nonsense mutations. These findings resulted in a fast evolvement of synthetic derivatives of aminoglycosides which were shown to be more target specific and less toxic than the clinically used antibiotics. The emerging progress in drug design and development has necessitated the urge to develop a fast, easy and accurate procedure for the determination of these potential therapeutic agents in various biologically derived matrices. Here we describe the preparation of a generic polyclonal antibody that was used for the development of homologous and heterologous immunoassays for the detection of a wide range of natural and synthetic aminoglycoside derivatives, highlighted today as potential therapeutic agents for the treatment of various genetic diseases. A common two-ring scaffold, NB82, present in the majority of compounds exhibiting potent biological activity, was used as a generic immunization hapten for the immunization of two rabbits. By using a series of chemical steps, NB82 was selectively conjugated via the N-1 position through glutaric acid linker to a carrier protein. Sensitivity (I50) values for the recognition of three representative compounds NB82, NB84 and NB124 were determined to be 10±3 ng mL−1, 0.5±0.04 μg mL−1 and 1±0.12 μg mL−1, respectively. Limits of detection were determined to be 1±0.3 ng mL−1 for NB82, 20±7 ng mL−1 for NB84 and 15±8 ng mL−1 for NB124. The developed assays were further exploited for the in-vivo monitoring of the therapeutic compounds in mice serum. Serum experimentations exhibited similar detection limits as observed for the PBS calibration experiments, demonstrating no interference with assays sensitivity, with rather high recovery ratios ranging from 92–107% in whole blood samples.