What can the kinetics of amyloid fibril formation tell about off-pathway aggregation? [Molecular Biophysics]

November 24th, 2015 by

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. While the pathogenic role of these fibrils is not totally established, increasing evidences suggest off-pathway aggregation (OPA) as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step ahead towards the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of OPA relatively to amyloid fibrillization; these include non-linear semi-log plots of amyloid progress curves, highly variable endpoint signals and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of Dynamic Light Scattering (DLS), Circular Dichroism (CD) and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

Nuclear factor of activated T cells-dependent downregulation of the transcription factor GLI1 underlies the growth inhibitory properties of arachidonic acid* [Cell Biology]

November 24th, 2015 by

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis, and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1 and 4-1BB, in AA-treated cells. We demonstrated that downregulation of the transcription factor GLI1 in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1 and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules, and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and downregulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating NFATc1 nuclear translocation, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants reduced the AA-induced apoptosis, downregulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for the future PUFA-based therapeutic approaches.
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Oxysterol binding protein activation at endoplasmic reticulum-golgi contact sites reorganizes phosphatidylinositol 4-phosphate pools [Cell Biology]

November 23rd, 2015 by Goto, A., Charman, M., Ridgway, N. D.

Oxysterol binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and trans-Golgi/trans-Golgi network. 25-hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro, and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology (PH) domain probe. The cellular mass of phosphatidylinositol mono-phosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) were unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of SM synthesis, suggesting that 25OH must be exchanged for PI-4P in order to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P pool specifically recruits PH domain containing proteins involved in lipid transfer and metabolism, such as CERT.
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Regulation of c-Myc expression by Ahnak promotes iPSC generation [Signal Transduction]

November 23rd, 2015 by

We have previously reported that Ahnak-mediated TGFb signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak-/- MEF) show an increased level of c-Myc expression compared to wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2 and Klf4 (hereafter 3F) was significantly enhanced in Ahnak-/- MEF. Similar results were obtained when Ahnak-specific siRNA was applied to wild type MEF. Of note, expression of Ahnak was significantly induced during the formation of embryoid bodies (EB) from embryonic stem (ES) cells suggesting that Ahnak-mediated c-Myc inhibition is involved in EB formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak-/- MEF cells (Ahnak-/--iPSC-3F) showed expression of all stem cell markers examined and capability to form three primary germ layers. Moreover, injection of Ahnak-/--iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers indicating that iPSC from Ahnak-/- MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide means to regulate iPSC generation.

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) acts as a glucose sensor to epigenetically regulate the insulin gene in pancreatic beta cells [Gene Regulation]

November 23rd, 2015 by Durning, S. P., Flanagan-Steet, H., Prasad, N., Wells, L.

The post-translational protein modification O-linked β-N-acetylglucosamine (O-GlcNAc) is a proposed nutrient sensor that has been shown to regulate multiple biological pathways. This dynamic and inducible enzymatic modification to intracellular proteins utilizes the end product of the nutrient sensing hexosamine biosynthetic pathway (HBP), UDP-GlcNAc, as its substrate-donor. Type II diabetic patients have elevated O-GlcNAc modified proteins within pancreatic beta-cells due to chronic hyperglycemia-induced glucose overload, but a molecular role for O-GlcNAc within beta cells remains unclear. Using directed pharmacological approaches in the mouse insulinoma-6 (Min6) cell line, we demonstrate that elevating nuclear O-GlcNAc increases intracellular insulin levels and preserves glucose stimulated insulin secretion during chronic hyperglycemia. The molecular mechanism for these observed changes appears to be, at least in part, due to elevated O-GlcNAc-dependent increases in Ins1 and Ins2 mRNA levels via elevations in histone H3 transcriptional activation marks. Further, RNA deep sequencing reveals that this mechanism of altered gene transcription is restricted and that the majority of genes regulated by elevated O-GlcNAc levels are similarly regulated by a shift from euglycemic to hyperglycemic conditions. These findings implicate the O-GlcNAc modification as a potential mechanism for hyperglycemic-regulated gene expression in the beta cell.
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Loss of Gs{alpha} in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy [Signal Transduction]

November 23rd, 2015 by

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTHR1) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTHR1 exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-GsαOsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-GsαOsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-GsαOsxKO mice. In mice that express a mutated PTHR1 that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C (PLC) via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that PLC activation is not required for increased bone turnover in response to PTH. Therefore while the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.
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Autoinhibitory Interdomain Interactions and Subfamily-Specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3 [Protein Structure and Folding]

November 23rd, 2015 by

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 A resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.
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The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of Transforming Growth Factor-{beta} (TGF{beta})-Induced Autophagy [Signal Transduction]

November 23rd, 2015 by Korah, J., Canaff, L., Lebrun, J.-J.

Transforming growth factor-β (TGFβ) is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Though autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes, and further highlights the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.
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Low-density lipoprotein receptor-related proteins in a novel mechanism of axon guidance and peripheral nerve regeneration [Neurobiology]

November 23rd, 2015 by

The low-density lipoprotein receptor-related protein receptors, 1 and 2 (LRP1, LRP2) are emerging as important cell signalling mediators in modulating neuronal growth and repair. We examined whether LRP1 or LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were Chemorepulsive: α2-macroglobulin, tissue plasminogen activator and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction towards a gradient of metallothionein II was calcium dependent and required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fibre regeneration, such that it was complete within 7 days, compared to 14 days in the saline treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction, and may form part of a signalling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signalling system, which has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.

The physiological characterization of Connexin41.8 and Connexin39.4, which are involved in the stripe pattern formation of zebrafish [Developmental Biology]

November 23rd, 2015 by Watanabe, M., Sawada, R., Aramaki, T., Skerrett, I. M., Kondo, S.

The zebrafish has a stripe skin pattern on its body, and Connexin41.8 (Cx41.8) and Cx39.4 are involved in stripe pattern formation. Mutations in these connexins change the stripe pattern to a spot or labyrinth pattern. In this study, we characterized Cx41.8 and Cx39.4 after expression in Xenopus oocytes. In addition, we analyzed Cx41.8 mutants Cx41.8I203F and Cx41.8M7, which caused spot or labyrinth skin patterns respectively in transgenic zebrafish. In the electrophysiological analysis, the gap junctions formed by Cx41.8 and Cx39.4 showed distinct sensitivity to transjunctional voltage. Analysis of non-junctional (hemichannel) currents revealed a large voltage-dependent current in Cx39.4-expressing oocytes that was absent in cells expressing Cx41.8. Junctional currents induced by both Cx41.8 and Cx39.4 were reduced by co-expression of Cx41.8I203F and abolished by co-expression of Cx41.8M7. In the transgenic experiment, Cx41.8I203F partially rescued the Cx41.8 null mutant phenotype, whereas Cx41.8M7 failed to rescue the null mutant, and it elicited a more severe phenotype than the Cx41.8 null mutant, as evidenced by a smaller spot pattern. Our results provide evidence that gap junctions formed by Cx41.8 play an important role in stripe/spot patterning and suggest that mutations in Cx41.8 can effect patterning by way of reduced function (I203F) and dominant negative effects (M7). Our results suggest that functional differences in Cx41.8 and Cx39.4 relate to spot or labyrinth mutant phenotypes and also provide evidence for these two connexins interact in vivo and in vitro.
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