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991.
Stella E. Weidauer Monika Beerbaum Hartmut Oschkinat 《Biochemical and biophysical research communications》2009,380(1):160-165
Sclerostin has been identified as a negative regulator of bone growth. Initially it was considered that Sclerostin performs its regulatory function via acting as a modulator of bone morphogenetic proteins (BMPs) similar to known examples such as Noggin, Chordin, and members of the DAN family. Recent findings, however, show that Sclerostin interferes with the Wnt signaling pathway due to binding to the Wnt co-receptor LRP5 thereby modulating bone growth. As Sclerostin is exclusively produced by osteocytes located in bones, neutralization of its bone-inhibiting functions makes it a highly interesting target for an osteoanabolic therapeutic approach in diseases characterized by bone loss, such as osteoporosis. Despite the huge interest in Sclerostin inhibitors the molecular basis of its function and its interaction with components of the Wnt signaling cascade has remained unclear. Here, we present the NMR structure of murine Sclerostin providing the first insights how Sclerostin might bind to LRP5. 相似文献
992.
Anita Johswich Benjamin Kraft Manfred Wuhrer Monika Berger Andr M. Deelder Cornelis H. Hokke Rita Gerardy-Schahn Hans Bakker 《The Journal of cell biology》2009,184(1):173-183
Drosophila melanogaster β4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcβ1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated β4GalNAcTB together with β4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive β4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of β4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with β4GalNAcTB and, when expressed with an ER retention signal, holds active β4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs β4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of β4GalNAcTB. 相似文献
993.
994.
Riikka Hynynen Monika Suchanek Johanna Spandl Nils B?ck Christoph Thiele Vesa M. Olkkonen 《Journal of lipid research》2009,50(7):1305-1315
Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (Kd 1.4 × 10−8 M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 μM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [14C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol. 相似文献
995.
Hans Bakker Takuji Oka Angel Ashikov Ajit Yadav Monika Berger Nadia A. Rana Xiaomei Bai Yoshifumi Jigami Robert S. Haltiwanger Jeffrey D. Esko Rita Gerardy-Schahn 《The Journal of biological chemistry》2009,284(4):2576-2583
In mammals, xylose is found as the first sugar residue of the
tetrasaccharide
GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the
formation of the glycosaminoglycans heparin/heparan sulfate and
chondroitin/dermatan sulfate. It is also found in the trisaccharide
Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor
repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes
the formation of the UDP-xylose substrate for the different
xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in
the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place
in these organelles, no obvious requirement exists for membrane transport of
UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has
been documented, and we recently succeeded with the cloning of a human
UDP-xylose transporter (SLC25B4). Here we provide new evidence for a
functional role of UDP-xylose transport by characterization of a new Chinese
hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The
mutant fails to initiate glycosaminoglycan synthesis and is not capable of
xylosylating Notch. Complementation was achieved by expression of a
cytoplasmic variant of UXS, which proves the existence of a functional Golgi
UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid
occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated
control of the cytoplasmically localized UDP-glucose dehydrogenase in the
mutant. The data presented in this study suggest the bidirectional transport
of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in
controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First,
xylose is the starting sugar residue of the common tetrasaccharide,
GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to
proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans
(GAGs)2
(1). Second, xylose is found in
the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in
epidermal growth factor (EGF)-like repeats of proteins, such as blood
coagulation factors VII and IX
(2) and Notch
(3)
(Fig. 1). Two variants of
O-xylosyltransferases (XylT1 and XylT2) are responsible for the
initiation of glycosaminoglycan biosynthesis, which differ in terms of
acceptor specificity and tissue distribution
(4-7),
and two different enzymatic activities have been identified that catalyze
xylosylation of O-glucose residues added to EGF repeats
(8-10).
On Notch, O-glucose occurs on EGF repeats in a similar fashion as
O-fucose, which modifications have been shown to influence
ligand-mediated Notch signaling
(11-16).
Recently, rumi, the gene encoding the Notch
O-glucosyltransferase in Drosophila, has been identified,
and inactivation of the gene was found to cause a temperature-sensitive
Notch phenotype (17).
Although this finding clearly demonstrated that O-glucosylation is
essential for Notch signaling, the importance of xylosylation for Notch
functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is
synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA,
and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is
inhibited by the product of the second enzyme, UDP-Xyl
(42). B, in mammals,
UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate
for different xylosyltransferases incorporating xylose in the
glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked
glycans. The nucleotide sugar transporter SLC35D1
(52) has been shown to
transport UDP-GlcA over the ER membrane and SLC35B4
(29) to transport UDP-Xyl over
the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in
GAG biosynthesis have been isolated by screening for reduced incorporation of
sulfate (18) and reduced
binding of fibroblast growth factor 2 (FGF-2)
(19,
20) and by direct selection
with FGF-2 conjugated to the plant cytotoxin saporin
(21). Isolated cells (called
pgs, for proteoglycan synthesis mutants)
(21) exhibited defects in
various stages of GAG biosynthesis, ranging from the initiating
xylosyltransferase to specific sulfation reactions
(18,
19,
21-25).
Mutants that affect overall GAG biosynthesis were shown to have a defect in
the assembly of the common core tetrasaccharide. Interestingly, these latter
mutants could be separated into clones in which GAG biosynthesis can be
restored by the external addition of xylosides as artificial primers and those
that cannot (18). The two
mutants belonging to the first group are pgsA-745 and pgsB-761. Although
pgs-745 is defective in XylT2
(4-6,
18), pgsB-761 exhibits a
defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the
first step in the elongation of the xylosylated protein (25 (see
Fig. 1B). Restoration
of GAG biosynthesis in the latter mutant presumably occurs through a second
β1-4-galactosyltransferase, able to act on xylosides when provided at
high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the
xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase
(UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes
the synthesis of UDP-Xyl, the common donor substrate for the different
xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly,
UXS in the animal cell is localized in the lumen of the ER and/or Golgi
(26-28),
superseding at first sight the need for the Golgi UDP-xylose transporter,
which has been recently cloned and characterized
(29). Using this cell variant,
experiments were designed that establish the functional significance of
UDP-Xyl transport with respect to UDP-glucuronic acid production and
xylosylation. 相似文献
996.
Harald W. Krenn Monika J. B. Eberhard Stefan H. Eberhard Anna-Laetitia Hikl Werner Huber Lawrence E. Gilbert 《Arthropod-Plant Interactions》2009,3(4):203-208
Neotropical Heliconius and Laparus butterflies actively collect pollen onto the proboscis and extract nutrients from it. This study investigates the impact of the processing behaviour on the condition of the pollen grains. Pollen samples (n = 72) were collected from proboscides of various Heliconius species and Laparus doris in surrounding habitats of the Tropical Research Station La Gamba (Costa Rica). Examination using a light microscope revealed that pollen loads contained 74.88 ± 53.67% of damaged Psychotria pollen, 72.04 ± 23.4% of damaged Psiguria/Gurania pollen, and 21.35 ± 14.5% of damaged Lantana pollen (numbers represent median ± first quartile). Damaged pollen grains showed deformed contours, inhomogeneous and/or leaking contents, or they were empty. Experiments with Heliconius and Laparus doris from a natural population in Costa Rica demonstrated that 200 min of pollen processing behaviour significantly increased the percentage of damaged pollen of Psychotria compared to pollen from anthers (P = 0.015, Z = ?2.44, Mann–Whitney U-test). Examination of pollen loads from green house reared Heliconius butterflies resulted in significantly greater amounts of damaged Psiguria pollen after 200 min of processing behaviour compared to pollen from flowers (P < 0.001, Z = ?4.583, Mann–Whitney U-test). These results indicate that pollen processing functions as extra oral digestion whereby pollen grains are ruptured to make the content available for ingestion. 相似文献
997.
Claas Wodarczyk Isaline Rowe Marco Chiaravalli Monika Pema Feng Qian Alessandra Boletta 《PloS one》2009,4(9)
Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (∼520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases. 相似文献
998.
Julia Hirschfeld Julia Maurer Diana Jung Monika Kwiecinski Al Karim Khimji Hans Peter Dienes Jochen W. U. Fries Margarete Odenthal 《Molecular biotechnology》2009,43(2):121-129
Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore,
they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases.
An artificial promoter containing the −702 bp regulatory sequence of the α-smooth muscle actin (SMA) gene linked to the first
intron enhancer sequence of the β-actin gene and the β-globin intron-exon junction was constructed and tested for myofibroblast-dependent
gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function
in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-β-actin promoter
hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound
healing experiments with SMA-β-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion,
the −702 bp regulatory region of the SMA promoter linked to enhancing β-actin and β-globin sequences benefits from its small
size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes. 相似文献
999.
Renata Myk-Kope Monika Bzowska Krystyna Staliska Tomasz Chemicki Micha Podkalicki Jarosaw Jucha Katarzyna Kowalczyk Pawe Mak Joanna Bereta 《Cytokine》2009,46(3):309-315
ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala76:Val77 within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity. 相似文献
1000.
Brinker M Brosché M Vinocur B Abo-Ogiala A Fayyaz P Janz D Ottow EA Cullmann AD Saborowski J Kangasjärvi J Altman A Polle A 《Plant physiology》2010,154(4):1697-1709