196,538 research outputs found
Expression, localization, structural, and functional characterization of pFGE, the paralog of the C alpha-formylglycine-generating enzyme
No full textMariappan M, Preusser-Kunze A, Balleininger M, et al. Expression, localization, structural, and functional characterization of pFGE, the paralog of the C alpha-formylglycine-generating enzyme. JOURNAL OF BIOLOGICAL CHEMISTRY. 2005;280(15):15173-15179.pFGE is the paralog of the formylglycine-generating enzyme (FGE), which catalyzes the oxidation of a specific cysteine to C alpha-formylglycine, the catalytic residue in the active site of sulfatases. The enzymatic activity of sulfatases depends on this posttranslational modification, and the genetic defect of FGE causes multiple sulfatase deficiency. The structural and functional properties of pFGE were analyzed. The comparison with FGE demonstrates that both share a tissue-specific expression pattern and the localization in the lumen of the endoplasmic reticulum. Both are retained in the endoplasmic reticulum by a saturable mechanism. Limited proteolytic cleavage at similar sites indicates that both also share a similar three-dimensional structure. pFGE, however, is lacking the formylglycine-generating activity of FGE. Although overexpression of FGE stimulates the generation of catalytically active sulfatases, overexpression of pFGE has an inhibitory effect. In vitro pFGE interacts with sulfatase-derived peptides but not with FGE. The inhibitory effect of pFGE on the generation of active sulfatases may therefore be caused by a competition of pFGE and FGE for newly synthesized sulfatase polypeptides
ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum
No full textMariappan M, Radhakrishnan K, Dierks T, Schmidt B, von Figura K. ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum. JOURNAL OF BIOLOGICAL CHEMISTRY. 2008;283(10):6375-6383.Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation
Molecular characterization of the human C alpha-formylglycine-generating enzyme
No full textPreusser-Kunze A, Mariappan M, Schmidt B, et al. Molecular characterization of the human C alpha-formylglycine-generating enzyme. JOURNAL OF BIOLOGICAL CHEMISTRY. 2005;280(15):14900-14910.alpha-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calcium-binding protein containing an N-terminal (residues 86 168) and a C-terminal (residues 178-374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the N-terminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocross-linking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species
The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum
No full textMariappan M, Gande SL, Radhakrishnan K, Schmidt B, Dierks T, von Figura K. The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum. JOURNAL OF BIOLOGICAL CHEMISTRY. 2008;283(17):11556-11564.Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension ( residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER
Paralog of the formylglycine-generating enzyme - retention in the endoplasmic reticulum by canonical and noncanonical signals
No full textGande SL, Mariappan M, Schmidt B, Pringle TH, von Figura K, Dierks T. Paralog of the formylglycine-generating enzyme - retention in the endoplasmic reticulum by canonical and noncanonical signals. FEBS JOURNAL. 2008;275(6):1118-1130.Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the oxidation of a specific cysteine residue to formylglycine, which is the catalytic residue required for sulfate ester hydrolysis. This post-translational modification occurs in the endoplasmic reticulum (ER), and is an essential step in the biogenesis of this enzyme family. A paralog of FGE (pFGE) also localizes to the ER. It shares many properties with FGE, but lacks formylglycine-generating activity. There is evidence that FGE and pFGE act in concert, possibly by forming complexes with sulfatases and one another. Here we show that human pFGE, but not FGE, is retained in the ER through its C-terminal tetrapeptide PGEL, a noncanonical variant of the classic KDEL ER-retention signal. Surprisingly, PGEL, although having two nonconsensus residues (PG), confers efficient ER retention when fused to a secretory protein. Inducible coexpression of pFGE at different levels in FGE-expressing cells did not significantly influence the kinetics of FGE secretion, suggesting that pFGE is not a retention factor for FGE in vivo. PGEL is accessible at the surface of the pFGE structure. It is found in 21 mammalian species with available pFGE sequences. Other species carry either canonical signals (eight mammals and 26 nonmammals) or different noncanonical variants (six mammals and six nonmammals). Among the latter, SGEL was tested and found to also confer ER retention. Although evolutionarily conserved for mammalian pFGE, the PGEL signal is found only in one further human protein entering the ER. Its consequences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved
Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme
No full textEnnemann E, Radhakrishnan K, Mariappan M, et al. Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme. Journal of Biological Chemistry. 2013;288(8):5828-5839.Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34-72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR72 down arrow, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum
A precision desktop plate-to-roll apparatus for development of advanced flexographic printing processes
No full textFlexographic printing, which involves high-speed contact of an inked stamp against a substrate, is of increasing interest for scalable manufacturing of electronics in new formats. However, the adaptation of flexography to thinner, finer features which are generally required for printed electronics requires improved understanding of stamp-substrate contact mechanics. Here, we present a desktop plate-to-roll (P2R) printing apparatus which enables the study of flexographic printing in a semi-continuous format that mimics industrial printing. In particular, we tailor the specifications of the machine to use nanoporous stamps which have been shown to enable flexographic printing of ultrathin ink features with micron-scale linewidth. Printing with nanoporous stamps requires precise control of stamp-substrate contact force (2-250 mN) and elimination of shear force at the interface among others; these are accomplished using a flexure-supported substrate, and by coordinated rotary-linear motion of the system. We detail the design and evaluation of the P2R machine and demonstrate printing of high-resolution features (<3 mu m line width) with nanopomus stamps at speeds of up to 0.2 m/s.
Dr. Duane M. Jackson, Morehouse College, July 2011
No full textThis video is a conversation with Dr. Duane M. Jackson. Dr. Jackson talks about his paper, "Recall and the Serial Position Effect: The Role of Primacy and Recency on Accounting Students' Performance." Jackie Daniel, AUC Woodruff Library, is the interviewer
Increased fat oxidation in 3T3-L1 adipocytes through forced expression of UCP 1
No full textObesity is a chronic condition that primarily develops from an
increase in body fat in the form of white adipose tissue (WAT) mass. The
resulting adiposity is a risk factor for many diseases, including type 2
diabetes (T2D), cardiovascular diseases, and some forms of cancer. Reducing
WAT mass by targeted modulation of metabolic enzymes in fat cell
metabolism is an attractive molecular therapeutic alternative to dietary
approaches. In the present study, we exogenously up-regulate a novel
respiratory uncoupling protein to increase substrate oxidation, and thereby
control adipocyte fatty acid content.
Increasing molecular evidence points to a family of uncoupling
proteins (UCPs) playing an important role in adipocyte fat metabolism. Of
specific interest is UCP1, which in brown adipocytes mediates energy
dissipation as heat by de-coupling respiration and ATP synthesis. UCP1 is
minimally expressed in white adipose tissue (WAT). We hypothesize that
controlled expression of UCP1 in WAT will result in enhanced fatty acid oxidation to compensate for reduced ATP synthesis. We used a Tet-Off
retroviral transfection system to express UCP1, with doxycycline being used
to control the extent of expression. UCP1 cDNA was cloned into pRevTRE
and was stably transfected into 3T3-L1 preadipocytes prior to differentiating
them into adipocytes. A reporter gene (EGFP) was also transfected in
parallel to optimize the transfection and preadipocyte differentiation
conditions as well as to demonstrate regulated expression. Metabolite
measurements showed that the UCP1-expressing adipocytes accumulated
83% less triglyceride and 85 % free fatty acids while maintaining constant
ATP levels. These results suggest UCP1 and other metabolic enzymes as
potential targets for development of pharmacological agents for the
treatment of obesity and related disorders
"Reflections on the subject of Emigration from Europe with a view to Settlement in the United States" By M. Carey.
No full text"Reflections on the subject of Emigration from Europe with a view to Settlement in the United States: containing bried sketches of the moral and political character of those states.
By M. Carey, member of the American philosophical, and of the American Antiquarian Society, and author of The Olive Branch, Cindiciae Hibernicae, essays on banking, on political economy, and on internal improvement.
To which are now added the English editor's comments on the subject; together with Important Advice to Emigrants, and Cautions Against Impositions Practiced in the Outports
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