Interleukin-1{beta} processing is dependent upon a calcium-mediated interaction with calmodulin [Molecular Bases of Disease]

November 11th, 2015 by Ainscough, J. S., Gerberick, G. F., Kimber, I., Dearman, R. J.

The secretion of IL-1β is a central event in the initiation of inflammation. Unlike most other cytokines, the secretion of IL-1β requires two signals; one signal to induce the intracellular up-regulation of pro-IL-1β, and a second signal to drive secretion of the bioactive molecule. The release of pro-IL-1β is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here, we sought to identify novel proteins involved in IL-1β secretion and intracellular processing in order to gain further insight into the mechanism of IL-1 release. A human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1β. Probes with a signal to noise ration of >3 were defined as relevant biologically. In these analyses, calmodulin was identified as a particularly strong hit, with a SNR of ~11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1β, but not mature IL-1β, was confirmed and shown to be calcium dependent. Finally, using small molecule inhibitors it was demonstrated that both calcium and calmodulin were required for nigericin induced IL-1β secretion in THP-1 cells and primary human monocytes. Together, these data suggest that following calcium influx into the cell, pro-IL-1β interacts with calmodulin and that this interaction is important for IL-1β processing and release.

Nuclear Compartmentalization of Serine Racemase Regulates D-serine Production: Implications for N-methyl-D-aspartate (NMDA) Receptor Activation [Signal Transduction]

November 9th, 2015 by

D-serine is a physiological co-agonist that activates N-methyl D-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity and behavior. D-serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. D-serine is synthesized by the enzyme serine racemase (SR), which directly converts L-serine to D-serine. However, many aspects concerning the regulation of D-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of D-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling, and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular D-serine concentration is reduced, even though extracellular glutamate concentration increases several fold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.
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A genome-wide CRISPR screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. [Molecular Bases of Disease]

November 9th, 2015 by

Inflammasomes are high-molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1 dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense but at the same time its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer's disease or gout. While it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide gRNA library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. While the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.

Acid Ceramidase in Melanoma: Expression, Localization and Effects of Pharmacological Inhibition [Molecular Bases of Disease]

November 9th, 2015 by

Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine-1-phosphate (S1P). In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared to other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 =12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased S1P levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.

Unexpected Allosteric Network Contributes to LRH-1 Coregulator Selectivity [Computational Biology]

November 9th, 2015 by

Phospholipids (PLs) are unusual signaling hormones sensed by the nuclear receptor liver receptor homolog 1 (LRH 1), which has evolved a novel allosteric pathway to support appropriate interaction with coregulators depending on ligand status. LRH-1 plays an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of metabolic and neoplastic diseases. While the prospect of modulating LRH-1 via small molecules is exciting, the molecular mechanism linking PL structure to transcriptional coregulator preference is unknown. Previous studies showed that binding to an activating PL ligand, such as dilauroylphosphatidylcholine (DLPC), favors LRH-1s interaction with transcriptional coactivators to upregulate gene expression. Both crystallographic and solution based structural studies showed that DLPC binding drives unanticipated structural fluctuations outside of the canonical activation surface in an alternate activation function (AF) region, encompassing the beta-sheet-H6 region of the protein. However, the mechanism by which dynamics in the alternate AF influences coregulator selectivity remains elusive. Here we pair x-ray crystallography with molecular modeling to identify an unexpected allosteric network that traverses the protein ligand binding pocket and links these two elements to dictate selectivity. We show that communication between the alternate AF region and classical AF2 dictates the strength of the coregulator interaction. This work offers the first glimpse into the conformational dynamics that drive this unusual PL-mediated nuclear hormone receptor activation.

Spectroscopic and Kinetic Properties of the Molybdenum-Containing, NAD+-Dependent Formate Dehydrogenase from Ralstonia eutropha. [Molecular Biophysics]

November 9th, 2015 by Niks, D., Duvvuru, J., Escalona, M., Hille, R.

We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD+-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the EPR signal of the molybdenum center in its MoV state and demonstrated the direct transfer of the substrate Ca hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power and level of enzyme reduction, we are able to clearly identify the EPR signals for four of the iron-sulfur clusters of the enzyme, and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron-sulfur centers, permitting assignment of this signal to a specific iron-sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a Mo=S group of the molybdneum center.
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Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signalling and Prion Formation [Cell Biology]

November 9th, 2015 by Bate, C., Nolan, W., Williams, A.

The prion diseases occur following the conversion of the cellular prion protein (PrPC) into disease-related isoforms (PrPSc). In this study the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrPC in prion formation was examined using a cell painting technique. PrPSc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells), and in scrapie-infected primary cortical neurons, was increased following the introduction of PrPC. In contrast, PrPC containing a GPI anchor from which the sialic acid had been removed (desialylated PrPC) was not converted to PrPSc. Furthermore, the presence of desialylated PrPC inhibited the production of PrPSc within prion-infected cortical neurons, ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrPC contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrPC. Desialylated PrPC was less sensitive to cholesterol depletion than PrPC and was not released from cells by treatment with glimepiride The presence of desialylated PrPC in neurons caused the dissociation of cytoplasmic phospholipase A2 (cPLA2) from PrP-containing membrane rafts and reduced the activation of cPLA2. These findings show that the sialic acid moiety of the GPI attached to PrPC modifies local membrane microenvironments that are important in PrP-mediated cell signalling and PrPSc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.

DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites [Molecular Bases of Disease]

November 9th, 2015 by

The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but effects in dendrites have yet to be studied. Further, the mechanisms of this regulation, and its role in neuronal development and brain function are poorly understood. Here we demonstrate that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane (OMM) GTPase proteins, Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins Mitofusins. Using live cell imaging, we show that disruption of the DISC1 Miro/TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localises to mitochondria where it similarly disrupts mitochondrial dynamics and decreases ER-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro/TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate transport, fusion and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function, leading to abnormal neuronal development.

Exosomes from HIV-1 infected cells stimulate production of pro-inflammatory cytokines through TAR RNA [Cell Biology]

November 9th, 2015 by

HIV-1 infection results in a chronic illness since long-term HAART can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under HAART frequently develop various metabolic disorders, neurocognitive abnormalities and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. The current study indicates that exosomes from HIV-1 infected primary cells are highly abundant with TAR RNA as detected by RT-real-time PCR. Interestingly, up to a million copies of TAR RNA per microliter were also detected in the serum from HIV-1 infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1 infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-beta, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas 5-prime and 3-prime stems (TAR miRNAs) bound best to TLR 7 and 8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation and, therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR miRNA potentially can activate the NF-κB pathway and regulates cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1 infected patients under cART.

Molecular mechanism responsible for fibronectin-controlled alterations in tissue stiffness in advanced chronic liver fibrogenesis [Glycobiology and Extracellular Matrices]

November 9th, 2015 by

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to non-cancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver tissue stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes, and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-β activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-β1 through canonical and non-canonical Smad such as PI3 kinase-mediated pathways. TGF-β1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared to wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrshosis in negatively regulating TGF-β bioavailability, which in turn modulates ECM remodeling and stiffening, and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in broader variety of chronic fibrotic diseases.
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