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    ABSTRACT: The cyclic AMP sensor, EPAC1, activates AP1-mediated transcription in HUVECs. Correspondingly, induction of the SOCS3 minimal promoter by EPAC1 requires a single AP1 site that constitutively binds phosphorylated (Ser63) c-Jun in DNA-pull-down assays. c-Jun (Ser63) becomes further phosphorylated following cyclic AMP stimulation and specific activation of protein kinase A (PKA), but not through selective activation of EPAC1. Moreover, despite a requirement for c-Jun for SOCS3 induction in fibroblasts, phospho-null c-Jun (Ser63/73Ala) had little effect on SOCS3 induction by cyclic AMP in HUVECs. AP1 activation and SOCS3 induction by EPAC1 in HUVECs therefore occur independently of c-Jun phosphorylation on Ser63.
    FEBS letters 03/2014;
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    ABSTRACT: Over the past decade, confocal microscopy and the ever-expanding toolchest of fluorescent protein (xFP) markers and technologies have become routine methods for the biological laboratory. A common use of xFP fluorophores is in localizing proteins and the subcellular structures with which they associate, including analyzing their distribution and dynamics and the interactions of proteins in vivo. Additionally, a number of so-called optical highlighters have proven especially useful in analyzing the kinetics of these processes in pulse-chase studies of protein relocation(s) following an experimental challenge. Here we focus on exemplary methods in transformation and live-cell imaging in plant cells, with the expectation that researchers will find these and the accompanying resources useful as a starting point in developing their own expertise.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1062:487-507.
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    ABSTRACT: Evidence is provided from studies on natural and artificial biofluids that the sequestration of amorphous calcium phosphate by peptides or proteins to form nanocluster complexes is of general importance in the control of physiological calcification. A naturally occurring mixture of osteopontin peptides was shown, by light and neutron scattering, to form calcium phosphate nanoclusters with a core-shell structure. In blood serum and stimulated saliva, an invariant calcium phosphate ion activity product was found which corresponds closely in form and magnitude to the ion activity product observed in solutions of these osteopontin nanoclusters. This suggests that types of nanocluster complexes are present in these biofluids as well as in milk. Precipitation of amorphous calcium phosphate from artificial blood serum, urine and saliva was determined as a function of pH and the concentration of osteopontin or casein phosphopeptides. The position of the boundary between stability and precipitation was found to agree quantitatively with the theory of nanocluster formation. Artificial biofluids were prepared that closely matched their natural counterparts in calcium and phosphate concentrations, pH, saturation, ionic strength and osmolality. Such fluids, stabilised by a low concentration of sequestering phosphopeptides, were found to be highly stable and may have a number of beneficial applications in medicine.
    Journal of Structural Biology 12/2013;
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    ABSTRACT: Recently it has been shown that both recycling endosome and ESCRT components are required for cytokinesis, where they are thought to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes are targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of SNARE complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remains unclear. Here we demonstrate that Syntaxin 16 is a key regulator of cytokinesis as it is required for the recruitment of both recycling endosome-associated Exocyst and ESCRT machinery during late telophase and therefore that these two distinct facets of cytokinesis are inextricably linked.
    Molecular biology of the cell 10/2013;
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    ABSTRACT: Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.
    The Plant Cell 09/2013;
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    ABSTRACT: We introduce here a series of papers focused on Mitochondria from authors contributing to two FEBS courses: a FEBS/EMBO lecture course on "Mitochondria in life, death and disease" held in Crete, May 2012 (www.mito2012.gr) and a FEBS/MiP workshop on "Mitochondrial Biochemistry, Physiology and Pathology" in Cambridge, July 2012 (www.mitophysiology.org). Mitochondrial research has been diversifying, and this is reflected in the present series of papers (three reviews and five regular papers), which spans the scale from molecules to man. They are focused on two main themes: (i) mitochondrial biogenesis and turnover, and (ii) mitochondrial pathology. This article is protected by copyright. All rights reserved.
    FEBS Journal 08/2013;
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    ABSTRACT: The capa peptide family exists in a very wide range of insects including species of medical, veterinary and agricultural importance. Capa peptides act via a cognate G-protein coupled receptor (capaR) and have a diuretic action on the Malpighian tubules of Dipteran and Lepidopteran species. Capa signaling is critical for fluid homeostasis and has been associated with desiccation tolerance in the fly, D. melanogaster. The mode of capa signaling is highly complex, affecting calcium, nitric oxide and cyclic GMP pathways. Such complex physiological regulation by cell signaling pathways may occur ultimately for optimal organismal stress tolerance to multiple stressors. Here we show that D. melanogaster capa-1 (Drome-capa-1) acts via the Nuclear Factor kappa B (NF-kB) stress signaling network. Human PCR gene arrays of capaR-transfected Human Embryonic Kidney (HEK) 293 cells showed that Drome-capa-1 increases expression of NF-kB, NF-kB regulated genes including IL8, TNF and PTGS2, and NF-kB pathway-associated transcription factors i.e EGR1, FOS, cJUN. Furthermore, desiccated HEK293 cells show increased EGR1, EGR3 and PTGS2 - but not IL8 - expression. CapaR-transfected NF-kB reporter cells showed that Drome-capa-1 increased NF-kB promoter activity via increased calcium. In Malpighian tubules, both Drome-capa-1 stimulation and desiccation result in increased gene expression of the D. melanogaster NF-kB orthologue, Relish; as well as EGR-like stripe and klumpfuss. Drome-capa-1 also induces Relish translocation in tubule principal cells. Targeted knockdown of Relish in only tubule principal cells reduces desiccation stress tolerance of adult flies. Together, these data suggest that Drome-capa-1 acts in desiccation stress tolerance, by activating NF-kB signaling.
    Peptides 08/2013;
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    ABSTRACT: Previous models of glycolysis in the sleeping sickness parasite Trypanosoma brucei assumed that the core part of glycolysis in this unicellular parasite is tightly compartimentalized within an organelle, the glycosome, which had earlier been shown to contain most of the glycolytic enzymes. The glycosomes were assumed to be largely impermeable, and exchange of metabolites between the cytosol and the glycosome was assumed to be regulated by specific transporters in the glycosomal membrane. This tight compartmentalization was considered essential for parasite viability. Recently, size-specific metabolite pores were discovered in the membrane of glycosomes. These channels are proposed to allow smaller metabolites to diffuse across the membrane but not larger ones. In light of this new finding, we re-analysed the model taking into account uncertainty about the topology of the metabolic system in Trypanosoma brucei, as well as uncertainty about the values of all parameters of individual enzymatic reactions. Our analysis shows that these newly discovered nonspecific pores are not necessarily incompatible with our current knowledge of the glycosomal metabolic system, provided that the known cytosolic activities of the glycosomal enzymes play an important role in the regulation of glycolytic fluxes and the concentration of metabolic intermediates of the pathway. This article is protected by copyright. All rights reserved.
    FEBS Journal 07/2013;
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    ABSTRACT: Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins, utilise the Sec translocon as the major facilitator of insertion, however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion, this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F0c (subunit c of the F0F1 ATPsynthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F0c and (iii) show that incubation of F0c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F0c through Blue Native PAGE and fluorescence quenching reveals a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain.
    Biochimica et Biophysica Acta 07/2013;
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    ABSTRACT: ERdj5 is a member of the protein disulfide isomerase family of proteins localized to the endoplasmic reticulum (ER) of mammalian cells. To date, only a limited number of substrates for ERdj5 are known. Here we identify a number of endogenous substrates that form mixed disulfides with ERdj5, greatly expanding its client repertoire. ERdj5 previously had been thought to exclusively reduce disulfides in proteins destined for dislocation to the cytosol for degradation. However, we demonstrate here that for one of the identified substrates, the low-density lipoprotein receptor (LDLR), ERdj5 is required not for degradation, but rather for effi- cient folding. Our results demonstrate that the crucial role of ERdj5 is to reduce non-native disul- fides formed during productive folding and that this requirement is dependent on its interaction with BiP. Hence, ERdj5 acts as the ER reductase, both preparing misfolded proteins for degradation and catalyzing the folding of proteins that form obligatory non-native disulfides.
    Molecular cell 06/2013;
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