The Rosetta Stone Hypothesis-Based Interaction of the Tumor Suppressor Proteins Nit1 and Fhit.

In previous studies, we have identified the tumor suppressor proteins Fhit (fragile histidine triad) and Nit1 (Nitrilase1) as interaction partners of ß-catenin both acting as repressors of the canonical Wnt pathway. Interestingly, in D. melanogaster and C. elegans these proteins are expressed as NitFhit fusion proteins. According to the Rosetta Stone hypothesis, if proteins are expressed as fusion proteins in one organism and as single proteins in others, the latter should interact physically and show common signaling function. Here, we tested this hypothesis and provide the first biochemical evidence for a direct association between Nit1 and Fhit. In addition, size exclusion chromatography of purified recombinant human Nit1 showed a tetrameric structure as also previously observed for the NitFhit Rosetta Stone fusion protein Nft-1 in C. elegans. Finally, in line with the Rosetta Stone hypothesis we identified Hsp60 and Ubc9 as other common interaction partners of Nit1 and Fhit. The interaction of Nit1 and Fhit may affect their enzymatic activities as well as interaction with other binding partners.

Hyperforin and Myrtucommulone Derivatives Act as Natural Modulators of Wnt/ß-Catenin Signaling in HCT116 Colon Cancer Cells.

The therapeutic activities of natural plant extracts have been well known for centuries. Many of them, in addition to antiviral and antibiotic effects, turned out to have anti-tumor activities by targeting different signaling pathways. The canonical Wnt pathway represents a major tumorigenic pathway deregulated in numerous tumor entities, including colon cancer. Here, we investigated the acylphloroglucinols hyperforin (HF) from St. John's wort (Hypericum perforatum L.) and myrtucommulone A (MC A) from myrtle (Myrtus communis) and semi-synthetic derivatives thereof (HM 177, HM 297, HM298) for their effects on Wnt/ß-catenin signaling. None of these substances revealed major cytotoxicity on STF293 embryonic kidney and HCT116 colon carcinoma cells at concentrations up to 10 µM. At this concentration, HF and HM 177 showed the strongest effect on cell proliferation, whereas MC A and HM 177 most prominently inhibited anchorage-independent growth of HCT116 cells. Western blot analyses of active ß-catenin and ß-catenin/TCF reporter gene assays in STF293 cells revealed inhibitory activities of HF, MC A and HM 177. In line with this, the expression of endogenous Wnt target genes, Axin and Sp5, in HCT116 cells was significantly reduced. Our data suggest that the acylphloroglucinols hyperforin, myrtucommulone A and its derivative HM 177 represent potential new therapeutic agents to inhibit Wnt/ß-catenin signaling in colon cancer.

GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.

Wnt Glycation Inhibits Canonical Signaling.

Glycation occurs as a non-enzymatic reaction between amino and thiol groups of proteins, lipids, and nucleotides with reducing sugars or α-dicarbonyl metabolites. The chemical reaction underlying is the Maillard reaction leading to the formation of a heterogeneous group of compounds named advanced glycation end products (AGEs). Deleterious effects have been observed to accompany glycation such as alterations of protein structure and function resulting in crosslinking and accumulation of insoluble protein aggregates. A substantial body of evidence associates glycation with aging. Wnt signaling plays a fundamental role in stem cell biology as well as in regeneration and repair mechanisms. Emerging evidence implicates that changes in Wnt/ß-catenin pathway activity contribute to the aging process. Here, we investigated the effect of glycation of Wnt3a on its signaling activity. METHODS: Glycation was induced by treatment of Wnt3a-conditioned medium (CM) with glyoxal (GO). Effects on Wnt3a signaling activity were analyzed by Topflash/Fopflash reporter gene assay, co-immunoprecipitation, and quantitative RT-PCR. RESULTS: Our data show that GO-treatment results in glycation of Wnt3a. Glycated Wnt3a suppresses ß-catenin transcriptional activity in reporter gene assays, reduced binding of ß-catenin to T-cell factor 4 (TCF-4) and extenuated transcription of Wnt/ß-catenin target genes. CONCLUSIONS: GO-induced glycation impairs Wnt3a signaling function.

Apoptotic Fragmentation of Tricellulin.

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell-cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. METHODS: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. RESULTS: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. CONCLUSIONS: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

Tricellulin is a target of the ubiquitin ligase Itch.

Tricellulin, a member of the tight junction-associated MAGUK protein family, preferentially localizes to tricellular junctions in confluent polarized epithelial cell layers and is downregulated during the epithelial-mesenchymal transition. Posttranslational modifications are assumed to play critical roles in the process of downregulation of tricellulin at the protein level. Here, we report that the E3 ubiquitin ligase Itch forms a complex with tricellulin and thereby enhances its ubiquitination. Pull-down assays confirmed a direct interaction between tricellulin and Itch, which is mediated by the Itch WW domain and the N-terminus of tricellulin. Experiments in the presence of the proteasome inhibitor MG-132 did not show major changes in the levels of ubiquitinated tricellulin in epithelial cells, suggesting that ubiquitination is not primarily involved in proteasomal degradation of tricellulin, but it appears to be important for endocytosis or recycling. In contrast, in HEK-293 cells, MG-132 caused polyubiquitination. Moreover, we observed that well-differentiated RT-112 and de-differentiated Cal-29 bladder cancer cells show an inverse expression of tricellulin and Itch. We postulate that ubiquitination is an important posttranslational modification involved in the determination of the intracellular fate of tricellulin deserving of more detailed further investigations into the underlying molecular mechanisms and their regulation.

SUMOylation regulates the intracellular fate of ZO-2.

The zonula occludens (ZO)-2 protein links tight junctional transmembrane proteins to the actin cytoskeleton and associates with splicing and transcription factors in the nucleus. Multiple posttranslational modifications control the intracellular distribution of ZO-2. Here, we report that ZO-2 is a target of the SUMOylation machinery and provide evidence on how this modification may affect its cellular distribution and function. We show that ZO-2 associates with the E2 SUMO-conjugating enzyme Ubc9 and with SUMO-deconjugating proteases SENP1 and SENP3. In line with this, modification of ZO-2 by endogenous SUMO1 was detectable. Ubc9 fusion-directed SUMOylation confirmed SUMOylation of ZO-2 and was inhibited in the presence of SENP1 but not by an enzymatic-dead SENP1 protein. Moreover, lysine 730 in human ZO-2 was identified as a potential modification site. Mutation of this site to arginine resulted in prolonged nuclear localization of ZO-2 in nuclear recruitment assays. In contrast, a construct mimicking constitutive SUMOylation of ZO-2 (SUMO1ΔGG-ZO-2) was preferentially localized in the cytoplasm. Based on previous findings the differential localization of these ZO-2 constructs may affect glycogen-synthase-kinase-3ß (GSK3ß) activity and ß-catenin/TCF-4-mediated transcription. In this context we observed that ZO-2 directly binds to GSK3ß and SUMO1ΔGG-ZO-2 modulates its kinase activity. Moreover, we show that ZO-2 forms a complex with ß-catenin. Wild-type ZO-2 and ZO-2-K730R inhibited transcriptional activity in reporter gene assays, whereas the cytosolic SUMO1ΔGG-ZO-2 did not. From these data we conclude that SUMOylation affects the intracellular localization of ZO-2 and its regulatory role on GSK3ß and ß-catenin signaling activity.

A novel role for the tumour suppressor Nitrilase1 modulating the Wnt/ß-catenin signalling pathway.

Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with ß-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with ß-catenin and LEF-1/TCF-4, represses ß-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that ß-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.

Berberine acts as a natural inhibitor of Wnt/ß-catenin signaling--identification of more active 13-arylalkyl derivatives.

Aberrant activation of the canonical Wnt/ß-catenin signaling pathway has been reported for numerous tumors of different origins. In most cases, mutations in components of the Wnt signaling pathway or in ß-catenin itself were detected which ultimately induce a genetic program that promotes cell proliferation and attenuates apoptosis. Thus, targeting of Wnt/ß-catenin signaling is of specific therapeutic interest. Herein, we investigated the plant-derived isoquinoline alkaloid berberine, which has been reported to have anticancer activity, and synthetic 13-arylalkyl derivatives thereof for their effects on Wnt/ß-catenin signaling. Berberine did not show major effects on viability of HEK-293 embryonic kidney and HCT116 colon carcinoma cells and was not toxic in concentrations up to 20 µM. Berberine inhibited ß-catenin transcriptional activity and attenuated anchorage-independent growth. As a result of berberine treatment, cellular levels of active ß-catenin were reduced concomitant with an increase in the expression of E-cadherin. However, in unstimulated cells, the effects on ß-catenin levels were low. A screen of synthetic 13-arylalkyl berberine derivatives identified compounds exhibiting activities superior to those of the naturally occurring parent substance with more than 100-fold lower EC50 values for Wnt-repression. Thus, berberine and its synthetic derivatives represent potential therapeutic agents to inhibit Wnt/ß-catenin signaling in tumorigenesis.

CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.

BACKGROUND: Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown. RESULTS: Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, whereas, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca2+-switch experiments. Stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance. CONCLUSIONS: These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400-408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ.