Macrophage-specific de novo synthesis of ceramide is dispensable for inflammasome-driven inflammation and insulin-resistance in obesity [Metabolism]

October 5th, 2015 by

Dietary lipid overload and calorie excess during obesity is a low grade chronic inflammatory state with diminished ability to appropriately metabolize glucose or lipids. Macrophages are critical in maintaining adipose tissue homeostasis, in part by regulating lipid metabolism, energy homeostasis and tissue remodeling. During high fat diet-induced obesity, macrophages are activated by lipid derived "danger signals" such as ceramides and palmitate and promote the adipose tissue inflammation in an Nlrp3 inflammasome-dependent manner. Given that the metabolic fate of fatty acids in macrophages is not entirely elucidated, we have hypothesized that de novo synthesis of ceramide, through the rate-limiting enzyme serine palmitoyltransferase long chain (Sptlc)-2, is required for saturated fatty acid driven Nlrp3 inflammasome activation in macrophages. Here we report that mitochondrial targeted overexpression of catalase which is established to mitigate oxidative stress controls ceramide-induced Nlrp3 inflammasome activation but does not affect the ATP-mediated caspase-1 cleavage. Surprisingly, myeloid cell-specific deletion of Sptlc2 is not required for palmitate driven Nlrp3 inflammasome activation. Furthermore, the ablation of Sptlc2 in macrophages did not impact macrophage polarization or obesity-induced adipose tissue leukocytosis. Consistent with these data, investigation of insulin-resistance using hyperinsulinemic-euglycemic clamps revealed no significant differences in obese mice lacking ceramide de novo synthesis machinery in macrophages. These data suggest that alternate metabolic pathways control fatty acid derived ceramide synthesis in macrophage and the Nlrp3 inflammasome activation in obesity.
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Structural insights into the high-efficiency catalytic mechanism of the sterile {alpha}-motif/histidineaspartate domain-containing protein [Enzymology]

October 5th, 2015 by Li, Y., Kong, J., Peng, X., Hou, W., Qin, X., Yu, X.-F.

Sterile α-motif/histidine-aspartate domain-containing protein (SAMHD1), a homo-tetrameric GTP/dGTP-dependent dNTP triphosphohydrolase, catalyzes the conversion of dNTP into deoxynucleoside and triphosphate. As the only characterized dNTP triphosphohydrolase in human cells, SAMHD1 plays an important role in human innate immunity, autoimmunity, and cell cycle control. Previous biochemical studies and crystal structures have revealed that SAMHD1 interconverts between an inactive monomeric or dimeric form and a dGTP/GTP-induced active tetrameric form. Here, we describe a novel state of SAMHD1 (109-626 amino acids, SAMHD1C) that is characterized by a rapid initial hydrolysis rate. Interestingly, the crystal structure showed that this novel SAMHD1 tetramer contains only GTP and has structural features distinct from the GTP/dNTP-bound SAMHD1 tetramer. Our work thus reveals structural features of SAMHD1 that may represent one of its biological assembly states in cells. The biochemical and structural information generated by the present study not only provides an ordered pathway for the assembly and activation of SAMHD1 but also provides insights into the potential mechanisms of the high-efficiency catalytic activity of this enzyme family in vivo.
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PAK kinases mediate the phosphorylation of PREX2 to initiate feedback inhibition of Rac1 [Signal Transduction]

October 5th, 2015 by

Phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP bound Rac1 then activates its downstream effectors, including p21 activated kinases (PAK). PREX2 and Rac1 are frequently mutated in cancer, and have key roles within the insulin signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2 mediated activation of Rac1 by the second messengers PIP3 or Gβγ, we found that PREX2 was phosphorylated through a PAK dependent mechanism. PAK mediated phosphorylation of PREX2 reduced GEF activity towards Rac1 by inhibiting PREX2 binding to PIP3 and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Further, the onset of insulin induced phosphorylation of PREX2 was delayed compared to AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAK kinases phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal, and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.

High glucose-induced retinal pericyte apoptosis depends on association of GAPDH and Siah1 [Cell Biology]

October 5th, 2015 by Suarez, S., McCollum, G. W., Jayagopal, A., Penn, J. S.

Diabetic Retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive and are of limited effectiveness. Retinal pericyte death is an early pathologic
feature of DR. Though it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this manuscript we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5mM normal glucose, 25mM L- or D-glucose for 48hrs (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to downregulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1 directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this manuscript can provide a foundation to identify novel targets for early treatment of DR.

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.