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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Regulating Arp2/3-Dependent Actin Assembly by Collapsin Response Mediator Protein-1 [Cell Biology]

November 23rd, 2015 by Yu-Kemp, H.-C., Brieher, W. M.

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3 dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3 dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own nor does it directly activate the Arp2/3 complex. Rather, CRMP- 1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.

Runx1 Phosphorylation by Src Increases Trans-Activation via Augmented Stability, Reduced HDAC Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis [Signal Transduction]

November 23rd, 2015 by

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification - increased stability, reduced HDAC interaction, and increased DNA-binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA-binding, in gel shift assays and as assessed by chromatin immunoprecipitation with the -14 kb Pu.1 or +37 kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.
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Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy [Molecular Biophysics]

November 22nd, 2015 by Guler, G., Gartner, R. M., Ziegler, C., Mantele, W.

The Na+-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K+ during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behaviour of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidyl glycerol lipids to functional important parts of BetP suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K+ binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions especially with the C-terminal domain and the functional important gating helices TMH3 and TMH12 confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point towards a more general mechanism how active transport can be modified by dynamic lipid-protein interactions.

Optimal neutralization of Centruroides noxius venom is understood through a structural complex between two antibody fragments and the Cn2 toxin [Immunology]

November 20th, 2015 by

Nowadays, the trends in the research devoted to develop novel antidotes against scorpion stings have showed excellent results with the use of recombinant antibody fragments. The polyclonal character of commercial anti-venoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals we aimed to develop an equivalent recombinant anti-venom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpionCentruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously envenomed animals. The crystallographic structure of the ternary complex (scFv LR-Cn2-scFv RU1) allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.
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MicroRNA-424 Predicts a Role for {beta}-1,4 Branched Glycosylation in Cell Cycle Progression [Cell Biology]

November 20th, 2015 by Vaiana, C. A., Kurcon, T., Mahal, L. K.

MicroRNA (miRNA) regulation of protein expression plays an important role in mediating many cellular processes, from cell proliferation to cell death. The human microRNA hsa-miR-424 is upregulated in response to anti-proliferative cytokines, and directly represses cell cycle progression. Our lab recently established that miRNA can be used as a proxy to identify biological roles of glycosylation enzymes (glycogenes). Herein, we identify MGAT4A, OGT and GALNT13 as targets of miR-424. MGAT4A, but not the other two glycogenes, is regulated by miR-424 in the mammary epithelial cancer cell line MCF-7. Knockdown of MGAT4A in MCF-7 induces cell cycle arrest through decreasing CCND1 and CDC25A levels. Loss of MGAT4A does not affect levels of β-(1,6) branched N-glycans, arguing that this effect is not due to gross changes in N-glycan branching but rather is specific to the β-(1,4) branch. This is the first example of miR-424 regulation of glycosylation related genes, lending insight into one possible mechanism by which cells use specific branched glycosylation patterns to regulate cell cycle progression.