Subunits of ADA-Two-A-Containing (ATAC) or Spt-Ada-Gcn5-Acetyltrasferase (SAGA) Coactivator Complexes Enhance the Acetyltransferase Activity of GCN5 [Enzymology]

October 14th, 2015 by

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT family of HATs. In metazoans, this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 Acetyltransferase) and ATAC (Ada Two A Containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, while in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) GCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it is part of purified recombinant hATAC or hSAGA HAT modules, or endogenous hATAC or hSAGA complexes, using histone tail peptides and full-length histones as substrates. We demonstrate that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest stronger influence of ADA2b as compared to ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones is not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules, and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes.
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The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains is Governed by their NH2-terminal and Internal Protein Regions [Metabolism]

October 14th, 2015 by Park, J. W., Reed, J. R., Backes, W. L.

In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our lab has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N-terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302 peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations but was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo.
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JNK associated leucine zipper protein functions as a docking platform for Polo like kinase 1 and regulation of the associating transcription factor Forkhead box protein K1 [Signal Transduction]

October 14th, 2015 by

JLP (JNK associated Leucine zipper protein) is a scaffolding protein that interacts with various signaling proteins associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here we identified Polo like kinase 1 (PLK1) as a novel interaction partner of JLP through mass spectrometric approaches. Our results indicate that JLP is phospho-primed by PLK1 on Thr 351, which is recognized by the PBD of PLK1 leading to phosphorylation of JLP at additional sites. SILAC and quantitative LC-MS/MS analysis was performed to identify PLK1 dependent JLP interacting proteins. Treatment of cells with the PLK1 kinase inhibitor BI2536 suppressed binding of the Forkhead box protein K1 (FOXK1) transcriptional repressor to JLP. JLP was found to interact with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 affected the interaction between JLP and FOXK1. FOXK1 is a known transcriptional repressor of the CDK inhibitor p21/WAF1 and knockdown of JLP resulted in increased FOXK1 protein levels and a reduction of p21 transcript levels. Our results suggest a novel mechanism by which FOXK1 protein levels and activity are regulated by associating with JLP and PLK1.
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cAMP-Dependent Protein Kinase (PKA) Signaling is Impaired in the Diabetic Heart [Signal Transduction]

October 14th, 2015 by Bockus, L. B., Humphries, K. M.

Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed β-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Controls and streptozotocin-induced diabetic mice were therefore administered 8Br-cAMP acutely to activate PKA in a receptor-independent manner and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator, PFK-2, was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed remarkably similar changes in PKA signaling, suggesting lipotoxicity is a major contributor to diabetes induced β-adrenergic signaling dysfunction. This work demonstrates PKA signaling responds to metabolic conditions and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function.

Hypoxia inhibits myogenic differentiation through p53-dependent induction of Bhlhe40 [Cell Biology]

October 14th, 2015 by Wang, C., Liu, W., Liu, Z., Chen, L., Liu, X., Kuang, S.

Satellite cells are muscle resident stem cells capable of self-renewal and differentiation to repair injured muscles. However, muscle injury often leads to an ischemic hypoxia environment that impedes satellite cell differentiation and reduces the efficiency of muscle regeneration. Here, we performed microarray analysis and identified the basic Helix-Loop-Helix family transcription factor Bhlhe40 as a candidate mediator of the myogenic inhibitory effect of hypoxia. Bhlhe40 is strongly induced by hypoxia in satellite cell-derived primary myoblasts. Overexpression of Bhlhe40 inhibits Myogenin expression and mimics the effect of hypoxia on myogenesis. Inhibition of Bhlhe40 conversely upregulates Myogenin expression and promotes myogenic differentiation. Importantly, Bhlhe40 knockdown rescues myogenic differentiation under hypoxia. Mechanistically, Bhlhe40 binds to the proximal E-boxes of Myogenin promoter and reduces the binding affinity and transcriptional activity of MyoD on Myogenin. Interestingly, hypoxia induces Bhlhe40 expression independent of HIF1α, but through a novel p53-dependent signaling pathway. Together, our study establishes a crucial role of Bhlhe40 in mediating the repressive effect of hypoxia on myogenic differentiation and suggests that inhibition of Bhlhe40 or p53 may facilitate muscle regeneration after ischemic injuries.

Transthyretin Binding Heterogeneity and Anti-amyloidogenic Activity of Natural Polyphenols and their Metabolites [Molecular Bases of Disease]

October 14th, 2015 by

Transthyretin (TTR) is an amyloidogenic protein, whose amyloidogenic potential is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of X-ray analysis of TTR in complex with both ligands. While revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, while genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites, and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulphate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by X-ray analysis of their complexes with TTR.
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AztD, a Periplasmic Zinc Metallochaperone to an ATP Binding Cassette (ABC) Transporter System in Paracoccus denitrificans [Molecular Biophysics]

October 14th, 2015 by Handali, M., Roychowdhury, H., Neupane, D. P., Yukl, E. T.

Bacterial ATP binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of gram-negative bacteria including human pathogens have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase and solute binding protein (SBP) genes of the AztABC Zn transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties and interaction with the SBP (AztC). Transcription of the aztD gene is significantly upregulated under conditions of Zn starvation. Recombinantly expressed AztD purifies with slightly substoichiometric Zn from the periplasm of E. coli and is capable of binding up to 3 Zn ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound Zn nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a Zn chaperone to AztC and likely functions to maintain Zn homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic Zn trafficking and the function of chaperones in this process.
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Ca2+ influx through store-operated calcium channels replenishes the functional phosphatidylinositol 4,5-bisphosphate pool used by cysteinyl leukotriene type I receptors [Membrane Biology]

October 14th, 2015 by Alswied, A., Parekh, A. B.

Oscillations in cytoplasmic Ca2+ concentration are a universal mode of signalling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca2+ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5bisphosphate (PIP2), the source of the Ca2+-releasing second messenger inositol trisphosphate (InsP3). Here we show that cytoplasmic Ca2+ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pre-treated with Li+, an inhibitor of inositol monophosphatases and which prevents PIP2 resynthesis. In Li+-treated cells, cytoplasmic Ca2+ signals evoked by agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knock down of phosphatidylinositol 4-phosphate 5- kinases (PIP5 kinases) α and γ resulted in rapid loss of the intracellular Ca2+ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca2+ oscillations and these could not be rescued by inositol or PI4P. In Li+-treated cells, recovery of the cytoplasmic Ca2+ oscillations in the presence of inositol or PI4P was suppressed when Ca2+ influx through store-operated Ca2+ channels was inhibited. After rundown of the Ca2+ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent InsP3-dependent Ca2+ release. Hence leukotriene and P2Y receptors utilise distinct membrane PIP2 pools. Our findings show that store-operated Ca2+ entry is needed to sustain cytoplasmic Ca2+ signalling following leukotriene receptor activation both by refilling the Ca2+ stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.
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Metal Ion-Dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix [Protein Structure and Folding]

October 14th, 2015 by

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a crosslinked network by the cooperative action of three proteins, inter-alpha-inhibitor (IalphaI), pentraxin-3 and TNF-induced protein-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. While it is known that matrix stabilization involves the TSG-6-mediated transfer of IalphaI heavy chains (HC) onto hyaluronan (to form covalent HC-HA complexes that are crosslinked by pentraxin-3), and that this occurs via the formation of covalent HC-TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC-TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of a HC-associated magnesium ion. In addition, we have found that the well-characterised hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with either impaired transferase and/or hyaluronan-binding functions), revealed that while the TSG-6-mediated formation of HC-HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

A2E Accumulation and the Maintenance of the Visual Cycle are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium [Cell Biology]

October 14th, 2015 by

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7flox/flox;VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7 deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7 deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7flox/flox;VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E deposition. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4-/- and Abca4+/+ backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found upregulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.
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