共查询到20条相似文献,搜索用时 888 毫秒
1.
C Popov T Radic F Haasters W C Prall A Aszodi D Gullberg M Schieker D Docheva 《Cell death & disease》2011,2(7):e186
Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors – α1β1, α2β1 and α11β1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2β1- or α11β1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I. 相似文献
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3.
Can Zhang Andrew Browne Daniel Child Jason R. DiVito Jesse A. Stevenson Rudolph E. Tanzi 《The Journal of biological chemistry》2010,285(12):8515-8526
Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date ∼80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Aβ, the proteolytic product of β-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Aβ and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Aβ40 and Aβ42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Aβ levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Aβ levels is modulated via β-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating β-secretase cleavage of APP and Aβ levels. 相似文献
4.
Guorong Li Coralia Luna Jianming Qiu David L. Epstein Pedro Gonzalez 《The Journal of biological chemistry》2010,285(8):5461-5471
MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H2O2. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells. 相似文献
5.
Yuya Sato Toshihiko Uemura Keisuke Morimitsu Ryoko Sato-Nishiuchi Ri-ichiroh Manabe Junichi Takagi Masashi Yamada Kiyotoshi Sekiguchi 《The Journal of biological chemistry》2009,284(21):14524-14536
Integrin α8β1 interacts with a variety of Arg-Gly-Asp
(RGD)-containing ligands in the extracellular matrix. Here, we examined the
binding activities of α8β1 integrin toward a panel of
RGD-containing ligands. Integrin α8β1 bound specifically to
nephronectin with an apparent dissociation constant of 0.28 ± 0.01
nm, but showed only marginal affinities for fibronectin and other
RGD-containing ligands. The high-affinity binding to α8β1 integrin
was fully reproduced with a recombinant nephronectin fragment derived from the
RGD-containing central “linker” segment. A series of deletion
mutants of the recombinant fragment identified the LFEIFEIER sequence on the
C-terminal side of the RGD motif as an auxiliary site required for
high-affinity binding to α8β1 integrin. Alanine scanning
mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a
critical motif ensuring the high-affinity integrin-ligand interaction.
Although a synthetic LFEIFEIER peptide failed to inhibit the binding of
α8β1 integrin to nephronectin, a longer peptide containing both the
RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was
∼2,000-fold more potent than a peptide containing only the RGD motif.
Furthermore, trans-complementation assays using recombinant fragments
containing either the RGD motif or LFEIFEIER sequence revealed a clear
synergism in the binding to α8β1 integrin. Taken together, these
results indicate that the specific high-affinity binding of nephronectin to
α8β1 integrin is achieved by bipartite interaction of the integrin
with the RGD motif and LFEIFEIER sequence, with the latter serving as a
synergy site that greatly potentiates the RGD-driven integrin-ligand
interaction but has only marginal activity to secure the interaction by
itself.Integrins are a family of adhesion receptors that interact with a variety
of extracellular ligands, typically cell-adhesive proteins in the
extracellular matrix
(ECM).2 They play
mandatory roles in embryonic development and the maintenance of tissue
architectures by providing essential links between cells and the ECM
(1). Integrins are composed of
two non-covalently associated subunits, termed α and β. In mammals,
18 α and 8 β subunits have been identified, and combinations of
these subunits give rise to at least 24 distinct integrin heterodimers. Based
on their ligand-binding specificities, ECM-binding integrins are classified
into three groups, namely laminin-, collagen- and RGD-binding integrins
(2,
3), of which the RGD-binding
integrins have been most extensively investigated. The RGD-binding integrins
include α5β1, α8β1, αIIbβ3, and
αV-containing integrins, and have been shown to interact with a variety
of ECM ligands, such as fibronectin and vitronectin, with distinct binding
specificities.The α8 integrin subunit was originally identified in chick nerves
(4). Integrin α8β1
is expressed in the metanephric mesenchyme and plays a crucial role in
epithelial-mesenchymal interactions during the early stages of kidney
morphogenesis. Disruption of the α8 gene in mice was found to be
associated with severe defects in kidney morphogenesis
(5) and stereocilia development
(6). To date, α8β1
integrin has been shown to bind to fibronectin, vitronectin, osteopontin,
latency-associated peptide of transforming growth factor-β1, tenascin-W,
and nephronectin (also named POEM)
(7–13),
among which nephronectin is believed to be an α8β1 integrin ligand
involved in kidney development
(10).Nephronectin is one of the basement membrane proteins whose expression and
localization patterns are restricted in a tissue-specific and developmentally
regulated manner (10,
11). Nephronectin consists of
five epidermal growth factor-like repeats, a linker segment containing the RGD
cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor
protein-tyrosine phosphatase μ (MAM) domain (see
Fig. 3A). Although the
physiological functions of nephronectin remain only poorly understood, it is
thought to play a role in epithelial-mesenchymal interactions through binding
to α8β1 integrin, thereby transmitting signals from the epithelium
to the mesenchyme across the basement membrane
(10). Recently, mice deficient
in nephronectin expression were produced by homologous recombination
(14). These
nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype
reminiscent of α8 integrin knock-out mice
(14), despite the fact that
other RGD-containing ligands, including fibronectin and osteopontin, were
expressed in the embryonic kidneys
(9,
15). The failure of the other
RGD-containing ligands to compensate for the deficiency of nephronectin in the
developing kidneys suggests that nephronectin is an indispensable
α8β1 ligand that plays a mandatory role in epithelial-mesenchymal
interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its
fragments. A, schematic diagrams of full-length nephronectin
(NN) and its fragments. RGD-linker and RGD-linker
(GST), the central RGD-containing linker segments expressed in
mammalian and bacterial expression systems, respectively; PRGDV, a
short RGD-containing peptide modeled after nephronectin and expressed as a GST
fusion protein (see Fig.
4A for the peptide sequence). The arrowheads
indicate the positions of the RGD motif. B, purified recombinant
proteins were analyzed by SDS-PAGE in 7–15% gradient (left and
center panels) and 12% (right panels) gels, followed by
Coomassie Brilliant Blue (CBB) staining, immunoblotting with an
anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded
were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for
blotting with anti-FLAG and PNA) in the left and center
panels;1 μg in the right panel. C, recombinant proteins (10
nm) were coated on microtiter plates and assessed for their binding
activities toward α8β1 integrin (10 nm) in the presence
of 1 mm Mn2+. The backgrounds were subtracted as
described in the legend to Fig.
2. The results represent the mean ± S.D. of triplicate
determinations. D, titration curves of α8β1 integrin bound
to full-length nephronectin (NN, closed squares), the RGD-linker
segments expressed in 293F cells (RGD-linker, closed triangles) and
E. coli (RGD-linker (GST), open
triangles), the MAM domain (MAM, closed diamonds), and the PRGDV
peptide expressed as a GST fusion protein in E. coli (PRGDV
(GST), open circles). The assays were performed as described
in the legend to Fig.
2B. The results represent the means of duplicate
determinations.Although ligand recognition by RGD-binding integrins is primarily
determined by the RGD motif in the ligands, it is the residues outside the RGD
motif that define the binding specificities and affinities toward individual
integrins (16,
17). For example,
α5β1 integrin specifically binds to fibronectin among the many
RGD-containing ligands, and requires not only the RGD motif in the 10th type
III repeat but also the so-called “synergy site” within the
preceding 9th type III repeat for fibronectin recognition
(18). Recently, DiCara et
al. (19) demonstrated
that the high-affinity binding of αVβ6 integrin to its natural
ligands, e.g. foot-and-mouth disease virus, requires the RGD motif
immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to
align the two conserved hydrophobic residues along the length of the helix.
Given the presence of many naturally occurring RGD-containing ligands, it is
conceivable that the specificities of the RGD-binding integrins are dictated
by the sequences flanking the RGD motif or those in neighboring domains that
come into close proximity with the RGD motif in the intact ligand proteins.
However, the preferences of α8β1 integrin for RGD-containing
ligands and how it secures its high-affinity binding toward its preferred
ligands remain unknown.In the present study, we investigated the binding specificities of
α8β1 integrin toward a panel of RGD-containing cell-adhesive
proteins. Our data reveal that nephronectin is a preferred ligand for
α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side
of its RGD motif serves as a synergy site to ensure the specific high-affinity
binding of nephronectin to α8β1 integrin. 相似文献
6.
Shi-Bin Cheng Stephanie A. Amici Xiao-Qin Ren Susan B. McKay Magdalen W. Treuil Jon M. Lindstrom Jayaraman Rao Rene Anand 《The Journal of biological chemistry》2009,284(35):23251-23259
The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre- and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs)5 regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of α2–α10 and β2–β4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The α4β2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for α4β2 AChRs in nicotine addiction. α4β2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7, 8). Furthermore, β2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). α4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing α4β2 AChRs hypersensitive to nicotine demonstrate that α4β2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12). High resolution ultrastructural studies show that α4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13), and a sequence motif has been identified within the α4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14). However, the mechanisms underlying the targeting and clustering of α4β2 AChRs to presynaptic sites in neurons remain elusive.Recently, bi-directional interactions between neurexins and neuroligins have been shown to promote synapse assembly and maturation by fostering pre- and postsynaptic differentiation (reviewed in Refs. 15–17). The neurexins are encoded by three genes corresponding to neurexins I–III (18, 19), each encoding longer α-neurexins and shorter β-neurexins, because of differential promoter use. Neurexins recruit N- and P/Q-type calcium channels via scaffolding proteins, including calmodulin-associated serine/threonine kinase (20), to active zones of presynaptic terminals (21, 22). Recently, α-neurexins were shown to specifically induce GABAergic postsynaptic differentiation (23). Neuroligins, postsynaptic binding partners of neurexins, cluster N-methyl-d-aspartate receptors and GABAA receptors by recruiting the scaffolding proteins PSD-95 (post-synaptic density 95) and gephyrin, respectively (24, 25). Interestingly, neurexins and neuroligins also modulate the postsynaptic clustering of α3-containing AChRs in chick ciliary ganglia (26, 27). In this study, using multiple experimental strategies, we provide evidence for the formation of complexes between neurexin-1β and α4β2 AChRs and a role for neurexin in the targeting of α4β2 AChRs to presynaptic terminals of neurons. 相似文献
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We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases. 相似文献
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13.
Robin J. Marjoram Bryan Voss Yumei Pan S. Kent Dickeson Mary M. Zutter Heidi E. Hamm Samuel A. Santoro 《The Journal of biological chemistry》2009,284(50):34640-34647
Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites. 相似文献
14.
Yoshihiro Ishikawa Jackie Wirz Janice A. Vranka Kazuhiro Nagata Hans Peter B?chinger 《The Journal of biological chemistry》2009,284(26):17641-17647
The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. 相似文献
15.
Masahiro Nakajima Mamoru Nishimoto Motomitsu Kitaoka 《The Journal of biological chemistry》2009,284(29):19220-19227
We characterized three d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc), thus indicating that they are d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with l-rhamnose as the acceptor in the reverse reaction using α-d-galactose 1-phosphate as the donor. The reaction product was d-galactosyl-β1→4-l-rhamnose. The enzyme also showed activity on l-mannose, l-lyxose, d-glucose, 2-deoxy-d-glucose, and d-galactose in this order. When d-glucose derivatives were used as acceptors, reaction products were β-1,3-galactosides. Kinetic parameters of phosphorolytic activity on d-galactosyl-β1→4-l-rhamnose were kcat = 45 s−1 and Km = 7.9 mm, thus indicating that these values are common among other phosphorylases. We propose d-galactosyl-β1→4-l-rhamnose phosphorylase as the name for Cphy1920 protein.Phosphorylases are a group of enzymes involved in formation and cleavage of glycoside linkage together with glycoside hydrolases and glycosyl-nucleotide glycosyltransferases (synthases). Phosphorylases, which reversibly phosphorolyze oligosaccharides to produce monosaccharide 1-phosphate, are generally intracellular enzymes showing strict substrate specificity. Physiologically, such strict substrate specificity is considered to be closely related to the environment containing bacteria possessing them. For example, d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylase (GalHexNAcP2; EC 2.4.1.211) from Bifidobacterium longum, an intestinal bacterium, forms part of the pathway metabolizing galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) from mucin and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc) from human milk oligosaccharides, both of which are present in the intestinal environment, with GNB- and LNB-releasing enzymes and GNB/LNB transporter (1–8). Another example is cellobiose phosphorylase from Cellvibrio gilvus, which is a cellulolytic bacterium. Cellobiose phosphorylase forms an important cellulose metabolic pathway with an extracellular cellulase system producing cellobiose (9, 10).The reversible catalytic reaction of phosphorylases is one of the most remarkable features that make them suitable catalysts for practical syntheses of oligosaccharides. An oligosaccharide can be produced from inexpensive material by combining reactions of two phosphorylases, one for phosphorolyzing the material and the other for synthesizing the oligosaccharide, in one pot. Based on this idea, LNB is synthesized on a large (kg) scale using sucrose phosphorylase and GalHexNAcP (11). Practical synthesis methods of trehalose and cellobiose have also been developed (12, 13). However, only 14 kinds of substrate specificities have been reported among phosphorylases (13), thus restricting their use. Therefore, it would be useful to find a phosphorylase with novel activity.GalHexNAcP phosphorolyzes GNB and LNB to produce α-d-galactose 1-phosphate (Gal 1-P) and the corresponding N-acetyl-d-hexosamine. To date, GalHexNAcP is the only phosphorylase known to act on β-galactoside. This enzyme was first found in the cell-free extract of Bifidobacterium bifidum (14) and then in B. longum (1, 15), Clostridium perfringens (16), Propionibacterium acnes (17), and Vibrio vulnificus (18). These studies revealed that GalHexNAcPs were classified into three subgroups based on substrate preference between GNB and LNB. These subgroups are as follows: 1) galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP), showing similar activity on both GNB and LNB (B. longum and B. bifidum); 2) galacto-N-biose phosphorylase (GNBP), preferring GNB to LNB (C. perfringens and P. acnes); and 3) lacto-N-biose I phosphorylase (LNBP), preferring LNB to GNB (V. vulnificus) (18). The ternary structure of GLNBP from B. longum (GLNBPBl) has been revealed recently (19). Based on the similarity in ternary structures between GLNBPBl and β-galactosidase from Thermus thermophilus, which belongs to glycoside hydrolase family 42 (19, 20), GalHexNAcP homologs are classified as GH112 (glycoside hydrolase family 112), although phosphorylases are glycosyltransferases (21, 22).Clostridium phytofermentans is an anaerobic cellulolytic bacterium. It is found in soil and grows optimally at 37 °C (23). Its whole genome sequence has been revealed (GenBankTM accession number ). The bacterium possesses three GalHexNAcP homologous genes (cphy0577, cphy1920, and cphy3030 genes; GenBankTM accession numbers are CP000885, ABX40964.1, and ABX42289.1, respectively). C. phytofermentans has the ability to utilize a wide range of plant polysaccharides ( ABX43387.123), and substrate specificities of these three gene products (Cphy0577, Cphy1920, and Cphy3030 proteins) are considered to be responsible for this ability. Furthermore, the three proteins have not been clearly categorized as GLNBP, GNBP, or LNBP, based on the phylogenetic tree shown in Fig. 1.Open in a separate windowFIGURE 1.Phylogenetic tree of GalHexNAcP homologs in GH112. Multiple alignment was performed using ClustalW2 (available on the World Wide Web). A phylogenetic tree was constructed using Treeview version 1.6.6. The proteins characterized in this study are represented with boldface letters in boxes with a heavy outline. The other proteins are numbered serially in boxes. Characterized GLNBP, GNBP, and LNBP are represented with boldface black letters on a gray background, boldface white letters on a gray background, and boldface white letters on a black background, respectively. Organisms and GenBankTM accession numbers of numbered proteins are as follows: 1, CPF0553 (C. perfringens ATCC13124, ) ( ABG83511.116); 2, CPE0573 (C. perfringens str.13, ); 3, CPR0537 (C. perfringens BAB80279.1SM101, ); 4, LnpA2 (B. bifidum ABG86710.1JCM1254, ) ( BAE95374.114, 15); 5, LnpA1 (B. bifidum JCM1254, ) ( BAD80752.114, 15); 6, GLNBPBl (B. longum subsp. longum JCM 1217, ) ( BAD80751.11, 16); 7, Blon_2174 (B. longum subsp. infantis ATCC 15697, ); 8, BL1641 (B. longum ACJ53235.1NCC2705, ); 9, BLD_1765 (B. longum AAN25428.1DJO10A, ); 10, GnpA (P. acnes ACD99210.1JCM6473, ) ( AB46806517); 11, GnpA (P. acnes JCM6425, ) ( AB46806617); 12, PPA0083 (P. acnes KPA171202, ); 13, VV2_1091 (V. vulnificus CMCP6, AAT81843.1) ( AAO07997.118); 14, VVA1614 (V. vulnificus YJ016, ); 15, Oter_1377 (Opitutus terrae BAC97640.1PB90-1, ); 16, BCQ_1989 (B. cereus Q1, ACB74662.1); 17, BCAH187_A2105 (Bacillus cereus ACM12417.1AH187, ).In this study, we characterized the three proteins. We reported that two of them were GalHexNAcPs and that the other was a β-galactoside phosphorylase showing unique substrate specificity. ACJ78918.1相似文献
16.
17.
Eukaryotic elongation factor-2 (eEF-2) catalyses the motion of the growing peptide chain relative to the mRNA at the ribosomes during protein synthesis. This highly conserved G-protein is the specific target of two lethal bacterial toxins, Pseudomonas aeruginosa exotoxin A and diphtheria toxin. These toxins exert their detrimental action by ADP-ribosylating a biologically unique posttranslationally modified histidine residue (diphthamide(715)) within eEF-2, thus inactivating the enzyme. Diphthamide(715) is also the target of endogenous (mono) ADP-ribosyl transferase activity. In this article, we report the first known activator of endogenous ADP-ribosylation of eEF-2, interleukin-1β (IL-1β). Thereby, systemic inflammatory processes may link to protein synthesis regulation. 相似文献
18.
Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
19.
20.
Mi-Kyung Shin Luciano F. Drager Qiaoling Yao Shannon Bevans-Fonti Doo-Young Yoo Jonathan C. Jun Susan Aja Sanjay Bhanot Vsevolod Y. Polotsky 《PloS one》2012,7(10)
Obesity is associated with tissue hypoxia and the up-regulation of hypoxia inducible factor 1 alpha (HIF-1α). Prior studies in transgenic mice have shown that HIF-1α plays a role in the metabolic dysfunction associated with obesity. Therefore, we hypothesized that, after the development of diet-induced obesity (DIO), metabolic function could be improved by administration of HIF-1α antisense oligonucleotides (ASO). DIO mice were treated with HIF-1α ASO or with control ASO for 8 weeks and compared with an untreated group. We found that HIF-1α ASO markedly suppressed Hif-1α gene expression in adipose tissue and the liver. HIF-1α ASO administration induced weight loss. Final body weight was 41.6±1.4 g in the HIF-1α ASO group vs 46.7±0.9 g in the control ASO group and 47.9±0.8 g in untreated mice (p<0.001). HIF-1α ASO increased energy expenditure (13.3±0.6 vs 12±0.1 and 11.9±0.4 kcal/kg/hr, respectively, p<0.001) and decreased the respiratory exchange ratio (0.71±0.01 vs 0.75±0.01 and 0.76±0.01, respectively, p<0.001), which suggested switching metabolism to fat oxidation. In contrast, HIF-1a ASO had no effect on food intake or activity. HIF-1α ASO treatment decreased fasting blood glucose (195.5±8.4 mg/dl vs 239±7.8 mg/dl in the control ASO group and 222±8.2 mg/dl in untreated mice, p<0.01), plasma insulin, hepatic glucose output, and liver fat content. These findings demonstrate that the metabolic consequences of DIO are attenuated by HIF-1α ASO treatment. 相似文献