Two pore channels (TPC2s) and nicotinic acid adenine dinucleotide phosphate (NAADP) at lysosomal-sarcoplasmic reticular ȷunctions contribute to acute and chronic {beta}-adrenoceptor signaling in the heart [Cell Biology]

October 5th, 2015 by

Calcium-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP) evoked Ca2+ release in many diverse cell types. Here we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca2+ responses in cardiac myocytes from Tpcn2-/- mice, unlike myocytes from wild-type (WT) mice. CaMKII inhibitors suppressed actions of NAADP in myocytes. Ca2+ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoceptor stimulation were reduced in myocytes from Tpcn2-/- mice. Increases in amplitude of L-type Ca2+ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2-/- mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2-/- mice also showed reduced inotropic effects of isoproterenol and a reduced tendency to arrhythmias following acute β-adrenoceptor stimulation. Hearts from Tpcn2-/- mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared to WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation approximately 25 nm). We propose that Ca2+ signaling nanodomains between lysosomes and SR dependent on NAADP and TPC2 comprise an important element in β-adrenoceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/SR junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.
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Multipart Chaperone-effector Recognition in the Type III Secretion System of Chlamydia trachomatis [Gene Regulation]

October 5th, 2015 by

Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein , CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4·Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results combined with our previous findings indicating a role in transcription for Scc4 (Rao et al. (2009) Genes Dev 23, 1818), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.

Proton Matrix ENDOR Studies on Ca2+-Depleted and Sr2+-Substituted Mn Cluster in Photosystem II [Bioenergetics]

October 5th, 2015 by Nagashima, H., Nakajima, Y., Shen, J.-R., Mino, H.

Proton matrix ENDOR spectra were measured for Ca2+-depleted and Sr2+-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Thermosynechococcus vulcanus in the S2-state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca2+-containing and Sr2+-substituted PSII, indicating that the proton arrangements around the Mn-cluster in cyanobacterial and higher-plant PSII and Ca2+-containing and Sr2+-substituted PSII are similar in the S2 state, in agreement with the similarity of the crystal structure of both Ca2+-containing and Sr2+-substituted PSII in the S1 state. Nevertheless, slightly different hyperfine separations were found between Ca2+-containing and Sr2+-substituted PSII due to modifications of the water protons ligating to the Sr2+ ion. Importantly, Ca2+ depletion caused the loss of ENDOR signals with a 1.36 MHz separation due to the loss of the water proton W4 connecting Ca2+ and YZ directly. With respect to the crystal structure and the functions of Ca2+ in oxygen evolution, it was concluded that the roles of Ca2+ and Sr2+ involve the maintenance of the hydrogen-bond network near the Ca2+ site and electron transfer pathway to the Mn cluster.

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: Nucleotide Excision Repair, DNA Damage Checkpoint, and Apoptosis [Cell Biology]

October 5th, 2015 by Choi, J.-H., Kim, S.-Y., Kim, S.-K., Kemp, M. G., Sancar, A.

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.
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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.