Inhibition of G-protein-coupled receptor kinase 2 prevents the dysfunctional cardiac substrate metabolism in fatty acid synthase-transgenic mice [Signal Transduction]

December 15th, 2015 by Abd Alla, J., Graemer, M., Fu, X., Quitterer, U.

Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN-transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN-transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN-transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK (extracellular signal-regulated kinase) axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine-273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart.
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Involvement of receptor activator of nuclear factor kappa-B ligand-induced incomplete cytokinesis in polyploidization of osteoclasts [Developmental Biology]

December 15th, 2015 by

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation.
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Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particle [Cell Biology]

December 15th, 2015 by Waite, K. A., De La Mota-Peynado, A., Vontz, G., Roelofs, J.

The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, while a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, while CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well-characterized transcriptional upregulation of genes encoding proteasome subunits, cells are also capable of down regulating cellular levels of proteasomes through proteaphagy.

A Novel Serpin Regulatory Mechanism: SERPINB9 is Reversibly Inhibited By Vicinal Disulfide Bond Formation in the Reactive Center Loop. [Immunology]

December 15th, 2015 by

The intracellular protease inhibitor, SERPINB9 (Sb9), is a regulator of the cytotoxic lymphocyte protease, granzyme B (GzmB). Although primarily involved in the destruction of compromised cells, recent evidence suggests that GzmB is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization, and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1′ in rodents; P1′-P2′ in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3′ reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5′ residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows for example that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.
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Leishmania donovani exploits myeloid cell leukemia 1 (MCL-1) protein to prevent mitochondria dependent host cell apoptosis [Cell Biology]

December 15th, 2015 by Giri, J., Srivastav, S., Basu, M., Palit, S., Gupta, P., Ukil, A.

Apoptosis is one of the mechanisms used by host cells to get rid of unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Amongst all the anti apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels, and compared to infected control, MCL-1 silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection induced MCL-1 expression was regulated by the transcription factor CREB and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70 silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited macrophage anti-apoptotic protein MCL-1 to prevent BAK mediated mitochondria dependent apoptosis thereby protecting its niche, which is essential for disease progression.

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erb{beta} Leads to its Degradation and Indirectly Regulates its Interaction with Nuclear Receptor Corepressor [Metabolism]

December 15th, 2015 by Carter, E. L., Gupta, N., Ragsdale, S. W.

Rev-erbα and Rev-erbβ are heme-binding nuclear receptors (NRs) that repress the transcription of genes involved in regulating metabolism, inflammation and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases affinity of the ligand-binding domain (LBD) of Rev-erbs for NCoR1 peptides. One explanation for this discrepancy is that the LBD and NCoR1 peptides used for in vitro studies cannot replicate key features of the full-length proteins used in cellular studies. However, combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbβ (FLRev-erbβ) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme- replete FLRev-erbβ:DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbβ-NCoR1 complex. The interaction between FLRev-erbβ and NCoR1 as well as Rev-erbβ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating degradation of Rev-erbβ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10-6 s-1) for heme dissociation from Rev-erbβ, rules out a prior proposal that Rev-erbβ acts as an intracellular heme sensor.
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Nerve Growth Factor is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs [Molecular Bases of Disease]

December 14th, 2015 by

Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity, but more recently were found to be activated by endogenous alarmins such as fragmented extra-cellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1β (control) treatment increased NGF, BDNF and IL-1β gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1β treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2 and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-κB, and to a lesser extent p38, but not ERK1/2 activity blocked TLR2-driven NGF upregulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-κB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-κB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.

Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 [Molecular Bases of Disease]

December 14th, 2015 by Chen, Q., Lu, M., Monks, B. R., Birnbaum, M. J.

Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, while the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was significantly decreased in livers with insulin signaling disrupted by ablation of insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

Low Level Pro-Inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide Mediated Protein Kinase C{delta} [Signal Transduction]

December 14th, 2015 by

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine cocktail containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of Connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.
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Inhibition of MAP Kinase-Interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing both a 5′-Terminal Cap and Hairpin [RNA]

December 14th, 2015 by Korneeva, N. L., Song, A., Gram, H., Edens, M. A., Rhoads, R. E.

The mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular-signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eIF4G and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5′-ends, we show that a MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5′-untranslated region (UTR). Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m7GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5′-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.
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