The Orphan G Protein-Coupled Receptor Gpr175 (TPRA40) Enhances Hedgehog Signaling by Modulating cAMP Levels [Signal Transduction]

October 8th, 2015 by Singh, J., Wen, X., Scales, S. J.

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as TPRA1 or TPRA40: transmembrane protein, adipocyte associated 1 or of 40 kDa) that also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway.

Mechanism of assembly of a substrate-transfer complex during tail-anchored protein targeting [Membrane Biology]

October 7th, 2015 by

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET (Guided Entry of TA proteins) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3-Get4/5 complex. Here we report crystal structures of a Get3-Get4/5 complex from Saccharomyces cerevisiae (Sc) at 2.8 Å and 6.0 Å, which reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3-Get4/5 complex. Furthermore, we provide evidence that the Get3-Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limitd electrostatically driven Get3-Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.

Long Non-coding RNA Growth Arrest-specific Transcript 5 (GAS5) Inhibits Liver Fibrogenesis through a Mechanism of Competing Endogenous RNA [RNA]

October 7th, 2015 by

Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. Pull down assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs (ceRNAs), GAS5 had no effect on pri-miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. qRT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a ceRNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs crosstalk by competing for shared miRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.
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Ribosome Reinitiation Directs Gene-Specific Translation and Regulates the Integrated Stress Response [Gene Regulation]

October 7th, 2015 by Young, S. K., Willy, J. A., Wu, C., Sachs, M. S., Wek, R. C.

In the Integrated Stress Response, phosphorylation of eIF2α (eIF2α~P) reduces protein synthesis to conserve resources and facilitate preferential translation of transcripts that promote stress adaptation. Preferentially translated GADD34 (PPP1R15A) and constitutively expressed CReP (PPP1R15B) function to dephosphorylate eIF2α~P and restore protein synthesis. The 5′-leaders of GADD34 and CReP contain two upstream ORFs (uORFs). Using biochemical and genetic approaches we show that features of these uORFs are central for their differential expression. In the absence of stress, translation of an inhibitory uORF in GADD34 acts as a barrier that prevents reinitiation at the GADD34 coding region. Enhanced eIF2α~P during stress facilitates ribosome bypass of the uORF, facilitating translation of the GADD34 coding region. CReP expression occurs independent of eIF2α~P via an uORF that allows for translation reinitiation at the CReP coding region independent of stress. Importantly, alterations in the GADD34 uORF affect the status of eIF2α~P, translational control, and cell adaptation to stress. These results show that properties of uORFs that permit ribosome reinitiation are critical for directing gene-specific translational control in the Integrated Stress Response.

O-GlcNAcomic Profiling Identifies Widespread O-GlcNAcylation in Oxidative Phosphorylation System Regulating Cardiac Mitochondrial Function [Metabolism]

October 7th, 2015 by Ma, J., Liu, T., Wei, A.-C., Banerjee, P., O'Rourke, B., Hart, G. W.

Dynamic cycling of O-linked β-N-acetylglucosamine (O-GlcNAc) on nucleo-cytoplasmic proteins serves as a nutrient sensor to regulate numerous biological processes. However, mitochondrial protein O-GlcNAcylation and its affects on function are largely unexplored. In this study, we performed a comparative analysis of the proteome and O-GlcNAcome of cardiac mitochondria from rats acutely (12 h) treated without or with Thiamet G (TMG), a potent and specific inhibitor of O-GlcNAcase. We then determined the functional consequences in mitochondria isolated from the two groups. O-GlcNAcomic profiling finds that over 88 mitochondrial proteins are O-GlcNAcylated, with the oxidative phosphorylation system as a major target. Moreover, in comparison to controls, cardiac mitochondria from TMG treated rats did not exhibit altered protein abundance, but showed overall elevated O-GlcNAcylation of many proteins. However, O-GlcNAc was unexpectedly down-regulated at certain sites of specific proteins. Concomitantly, TMG treatment resulted in significantly increased mitochondrial oxygen consumption rates, ATP production rates, and enhanced threshold for permeability transition pore opening by Ca2+. Our data reveal widespread and dynamic mitochondrial protein O-GlcNAcylation, serving as a regulator to their function.
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Distinct acute lymphoblastic leukemia (ALL)-associated Janus Kinase 3 (JAK3) mutants exhibit different cytokine-receptor requirements and JAK-inhibitor specificities [Molecular Bases of Disease]

October 7th, 2015 by

JAK1 and JAK3 are recurrently mutated in acute lymphoblastic leukemia. These tyrosine kinases associate with heterodimeric cytokine receptors such as IL-7R or IL-9R, in which JAK1 is appended to the specific chain and JAK3 to the common gamma chain. Here, we studied the role of these receptor complexes in mediating the oncogenic activity of JAK3 mutants. While JAK3V674A and the majority of other JAK3 mutants needed to bind to a functional cytokine receptor complex in order to constitutively activate STAT5, JAK3L857P was unexpectedly found to not depend on such receptor complexes for its activity, which was induced without receptor or JAK1 co-expression. Introducing a mutation in the FERM domain that abolished JAK-receptor interaction did not affect JAK3L857P activity, while it inhibited the other receptor-dependent mutants. The same cytokine receptor independence as for JAK3L857P was observed for homologous L857 mutations of JAK1 and JAK2 and for JAK3L875H. This different cytokine receptor requirement correlated with different functional properties in vivo and with distinct sensitivity to JAK inhibitors. Transduction of murine hematopoietic cells with JAK3V674A led homogenously to lymphoblastic leukemias in BALB/c mice. In contrast, transduction with JAK3L857P induced various types of lymphoid and myeloid leukemias. Moreover, Ruxolitinib, which preferentially blocks JAK1 and JAK2, abolished the proliferation of cells transformed by the receptor-dependent JAK3V674A, yet proved much less potent on cells expressing JAK3L857P. These particular cells were, in contrast, more sensitive to JAK3-specific inhibitors. Altogether, our results showed that different JAK3 mutations induce constitutive activation through distinct mechanisms, pointing to specific therapeutic perspectives.
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The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes [Membrane Biology]

October 7th, 2015 by Dubuke, M. L., Maniatis, S., Shaffer, S. A., Munson, M.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes (MTCs) and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific MTC, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We previously demonstrated that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1:Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. In order to map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly, but rather binds both the binary Sec9:Sso1 and ternary Sec9:Sso1:Snc2 SNARE complex. Together, these results suggest a new model wherein Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.

Bakuchiol is a Phenolic Isoprenoid with Novel Enantiomer-Selective Anti-Influenza A Virus Activity Involving Nrf2 Activation [Microbiology]

October 7th, 2015 by

Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia Linn.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (-)-(R)-bakuchiol, inhibited influenza A viral infection and growth, and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β; and myxovirus resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (-)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and a Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase {gamma} by Organization of the Template DNA [Enzymology]

October 7th, 2015 by

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pol γs with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and are characterized by a similar organization of the template DNA for both Pol γs. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity, and the variation among systems.
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Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi [Molecular Bases of Disease]

October 7th, 2015 by

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing of Atg8 proteins near the carboxy-terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using Positional Scanning-Substrate Combinatorial Library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Although these substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2 and UCH-L3.