Pseudomonas aeruginosa ExoT induces mitochondrial apoptosis in target host cells in a manner that depends on its GAP domain activity. [Molecular Bases of Disease]

October 8th, 2015 by

Pseudomonas aeruginosa is the most common cause of hospital-acquired pneumonia and a killer of immunocompromised patients. Others and we have demonstrated that the type III Secretion System (T3SS) effector protein ExoT plays a pivotal role in facilitating P. aeruginosa pathogenesis. ExoT possesses an N-terminal GTPase activating protein (GAP) domain and a C-terminal ADP ribosyltransferase (ADPRT) domain. Because it targets multiple non-overlapping cellular targets, ExoT performs several distinct virulence functions for P. aeruginosa, including induction of apoptosis in a variety of target host cells. Both the ADPRT and the GAP domain activities contribute to ExoT-induced apoptosis. The ADPRT domain of ExoT induces atypical anoikis by transforming an innocuous cellular protein, Crk, into a cytotoxin, which interferes with integrin survival signaling. However, the mechanism underlying the GAP-induced apoptosis remains unknown. In this report, we demonstrate that the GAP domain activity is both necessary and sufficient to induce mitochondrial (intrinsic) apoptosis. We show that intoxication with GAP domain results in: (i) JNK1/2 activation; (ii) substantial increases in the mitochondrial levels of activated pro-apoptotic proteins Bax and Bid, and to a lesser extent Bim; (iii) loss of mitochondrial membrane potential and cytochrome c release; and (iv) activation of initiator caspase-9 and executioner caspase-3. Further, GAP-induced apoptosis is partially mediated by JNK1/2 but it is completely dependent on caspase-9 activity. Together, the ADPRT and the GAP domains make ExoT into a highly versatile and potent cytotoxin, capable of inducing multiple forms of apoptosis in target host cells.
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Neuropilin Function as an Essential Cell Surface Receptor [Signal Transduction]

October 8th, 2015 by Guo, H.-F., Vander Kooi, C. W.

The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.

Role of Janus Kinase 3 in Predisposition to Obesity associated Metabolic Syndrome [Signal Transduction]

October 8th, 2015 by Mishra, J., Verma, R. K., Alpini, G., Meng, F., Kumar, N.

Obesity, a worldwide epidemic, is a major risk factor for the development of metabolic syndrome (MetS) including diabetes and associated health complications. Recent studies indicate that chronic low-grade inflammation (CLGI) plays a key role in metabolic deterioration in the obese population. Previously, we reported that Jak3 was essential for mucosal differentiation and enhanced colonic barrier functions and its loss in mice resulted in basal CLGI and predisposition to DSS induced colitis. Since CLGI is associated with diabetes, obesity, and metabolic syndrome, present studies determined the role of Jak3 in development of such conditions. Our data show that loss of Jak3 resulted in increased body weight, basal systemic CLGI, compromised glycemic homeostasis, hyperinsulinemia, and early symptoms of liver steatosis. Lack of Jak3 also resulted in exaggerated symptoms of metabolic syndrome by western high-fat diet. Mechanistically, Jak3 was essential for reduced expression and activation of toll like receptors (TLRs) in murine intestinal mucosa and human intestinal epithelial cells where Jak3 interacted with and activated p85, the regulatory sub-unit of the PI3K, through tyrosine phosphorylation of adapter protein insulin receptor substrate (IRS1). These interactions resulted in activation of PI3K-Akt axis, which was essential for reduced TLR expression and TLR associated NFkB activation. Collectively, these results demonstrate the essential role of Jak3 in promoting mucosal tolerance through suppressed expression and limiting activation of TLRs thereby preventing intestinal and systemic CLGI and associated obesity and MetS.

Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria. [Cell Biology]

October 8th, 2015 by

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca2+]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca2+]i increases downstream of ATP administration may have a polarized effect on ENaC where apical application of ATP and subsequent [Ca2+]i increase has an inhibitory effect on the channel whereas basolateral ATP and [Ca2+]i have a stimulatory effect. We asked if this polarized effect of ATP is in fact reflective of a polarized effect of increased [Ca2+]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca2+]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca2+]i in a polarized fashion, with apical increases being inhibitory and basolateral stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca2+]i, creating [Ca2+]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca2+]i uptake destroyed the polarized response of ENaC to [Ca2+]i. Overall, our data suggest that ENaC is regulated by [Ca2+]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca2+]i sequestration.
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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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