The regulatory domain of squalene monooxygenase contains a re-entrant loop and senses cholesterol via a conformational change [Lipids]

October 3rd, 2015 by Howe, V., Chua, N. K., Stevenson, J., Brown, A. J.

Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3 hydroxy 3 methylglutaryl CoA reductase (HMGCR). Although it is known to associate with the endoplasmic reticulum (ER), its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM, comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide (mPEG). We have identified a region integrally associated with the ER membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system (UPS), since degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N-terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis.

The role of phospholipase D in regulated exocytosis [Lipids]

October 2nd, 2015 by Rogasevskaia, T. P., Coorssen, J. R.

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca2+-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D -derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphaltidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca2+-sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.

Procollagen Lysyl Hydroxylase 2 Expression is Regulated by an Alternative Downstream Transforming Growth Factor Beta-1 Activation Mechanism [Gene Regulation]

October 2nd, 2015 by Gjaltema, R. A. F., de Rond, S., Rots, M. G., Bank, R. A.

PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) is a transforming growth factor beta-1 (TGFβ1) responsive gene that hydroxylates lysyl residues in collagen telopeptides and is essential for collagen pyridinoline cross-link formation in fibrotic pathologies. In this report we examined the molecular processes underlying TGFβ1-induced PLOD2 expression. We found that binding of the TGFβ1 pathway related transcription factors SMAD3 and SP1 mediated TGFβ1 enhanced PLOD2 expression and could be correlated to an increase of acetylated histone H3 and H4 at the PLOD2 promoter. Interestingly, the classical co-activators of SMAD3 complexes, p300 and CBP, were not responsible for the enhanced H3 and H4 acetylation. Depletion of SMAD3 reduced PLOD2 acetylated H3 and H4, indicating that another as of yet unidentified histone acetyltransferase binds to SMAD3 at PLOD2. Assessing histone methylation marks at the PLOD2 promoter depicted an increase of the active histone mark H3K79me2, a decrease of the repressive H4K20me3 mark, but no role for the generally strong transcription-related modifications: H3K4me3, H3K9me3 and H3K27me3. Collectively, our findings reveal that TGFβ1 induces a SP1- and SMAD3-dependent recruitment of histone modifying enzymes to the PLOD2 promoter other than the currently known TGFβ1 downstream co-activators and epigenetic modifications. This also suggests that additional activation strategies are used downstream of the TGFβ1 pathway, and hence their unraveling could be of great importance to fully understand TGFβ1 activation of genes.
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The phytosiderophore efflux transporter TOM2 is involved in metal transport in rice [Plant Biology]

October 2nd, 2015 by

Iron (Fe) is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire Fe in the soil. Phytosiderophores chelate and solubilize insoluble Fe hydroxide in the soil. Subsequently, plants take up Fe-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals including Fe. In the present study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-β-glucuronidase (GUS) revealed that TOM2 was expressed in tissues involved in metal translocation, while TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, while TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared to non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released 14C-labeled deoxymugineic acid (DMA), the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of DMA, which is required for normal plant growth.

TAZ protein accumulation is negatively regulated by YAP abundance in mammalian cells [Gene Regulation]

October 2nd, 2015 by

The mammalian Hippo signaling pathway regulates cell growth and survival and is frequently dysregulated in cancer. YAP and TAZ are transcriptional coactivators that function as effectors of this signaling pathway. Aberrant YAP and TAZ activity is reported in several human cancers, and normally the expression and nuclear localisation of these proteins is tightly regulated. We sought to establish whether a direct relationship exists between YAP and TAZ. Using knockdown and overexpression experiments we show YAP inversely regulates the abundance of TAZ protein by proteasomal degradation. Interestingly this phenomenon was uni-directional since TAZ expression did not affect YAP abundance. Structure/function analyses suggest that YAP-induced TAZ degradation is a consequence of YAP-targeted gene transcription involving TEAD factors. Subsequent investigation of known regulators of TAZ degradation using specific inhibitors revealed a role for heat shock protein 90 and glycogen synthase kinase 3 but not casein kinase 1 nor LATS in YAP-mediated TAZ loss. Importantly, this phenomenon is conserved from mouse to human, however interestingly, different YAP isoforms varied in their ability to degrade TAZ. Since shRNA-mediated TAZ depletion in HeLa and D645 cells caused apoptotic cell death, we propose that isoform specific YAP-mediated TAZ degradation may contribute to the contradicting roles reported for YAP overexpression. This study identifies a novel mechanism of TAZ regulation by YAP, which has significant implications for our understanding of Hippo pathway regulation, YAP-isoform specific signaling, and the role of these proteins in cell proliferation, apoptosis and tumorigenesis.

Finding Channels [Membrane Biology]

October 2nd, 2015 by Catterall, W. A.

Voltage-gated ion channels are responsible for action potential generation in nerve and muscle and other excitable cells, and they participate in many forms of cellular regulation in other cell types. In excitable cells, action potentials are typically initiated by activation of voltage-gated sodium channels, which conduct sodium rapidly into the cell and depolarize the cell membrane potential. Depolarization activates voltage-gated calcium channels, which conduct calcium into the cell. Calcium entry sustains the depolarization of the cell membrane and generates intracellular calcium transients that initiate many intracellular events, including contraction, secretion, synaptic transmission, regulation of enzymes, and regulation of gene expression. Action potentials are terminated by activation of voltage-gated potassium channels, which conduct potassium out of the cell, repolarize the membrane, and contribute to setting the resting membrane potential.

The Regulatory and Kinase Domains but not the Interdomain Linker Determine Human Double-Stranded RNA-Activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs [Protein Structure and Folding]

October 2nd, 2015 by S., S., Schwartz, S. L., Conn, G. L.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain (KD), interposed by an unstructured ~80 residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR), and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries, and that each exhibits comparable poly(rI:rC) dsRNA activation profiles and binding affinities for adenoviral VA RNAI and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR (rPKR) by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rPKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR.
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Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells [Cell Biology]

October 2nd, 2015 by

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. While at least in some fungi and plants mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells, still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the A. thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in mitochondria of human cells.

Phospholipase C{eta}2 Activation Re-directs Vesicle Trafficking By Regulating F-actin [Lipids]

October 2nd, 2015 by Yamaga, M., Kielar-Grevstad, D. M., Martin, T. F. J.

PI(4,5)P2 localizes to sites of dense-core vesicle exocytosis in neuroendocrine cells and is required for Ca2+-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously we reported that Ca2+-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (aka CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca2+-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 mM or 95 mM KCl buffers increased peak Ca2+ levels to ~400 nM or ~800 nM, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mM K+ preferentially elicited the exocytosis of plasma membrane-resident vesicles whereas 95 mM K+ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mM K+ but not with 56 mM K+ activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly-arriving vesicles. Consistent with its role as a Ca2+-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis, and reveal a new role for PLCη2 as a Ca2+-dependent regulator of F-actin dynamics and vesicle trafficking.

A Novel {alpha}2/{alpha}4 Subtype-Selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting From the C-tail of an {alpha} Subunit [Protein Structure and Folding]

October 2nd, 2015 by

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs, but is without effect on α3* or α6* nAChRs (′*′ indicates presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short-term desensitized nAChRs that have at least two α4 subunits, but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long-term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently, and overcomes short- and long-term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.
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