Integrins ��2��1 and ��11��1 regulate the survival of mesenchymal stem cells on collagen I

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.     

Loss of Function of ATXN1 Increases Amyloid ��-Protein Levels by Potentiating ��-Secretase Processing of ��-Amyloid Precursor Protein

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.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

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.     

Molecular Basis of the Recognition of Nephronectin by Integrin ��8��1

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.     

Presynaptic Targeting of ��4��2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1��

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 GABA_A 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.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

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.     

Suboptimal Activation of Protease-activated Receptors Enhances ��2��1 Integrin-mediated Platelet Adhesion to Collagen

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 G_q-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 α_IIbβ₃ 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 G_q-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α₂β₁ binding to collagens containing GFOGER sites.     

Biochemical Characterization of the Prolyl 3-Hydroxylase 1��Cartilage-associated Protein��Cyclophilin B Complex

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.     

Characterization of Three ��-Galactoside Phosphorylases from Clostridium phytofermentans: DISCOVERY OF d-GALACTOSYL-��1��4-l-RHAMNOSE PHOSPHORYLASE*

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 k_cat = 45 s⁻¹ and K_m = 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 (GLNBP_Bl) has been revealed recently (19). Based on the similarity in ternary structures between GLNBP_Bl 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 (GenBank^TM accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000885","term_id":"160426828","term_text":"CP000885"}}CP000885). The bacterium possesses three GalHexNAcP homologous genes (cphy0577, cphy1920, and cphy3030 genes; GenBank^TM accession numbers are {"type":"entrez-protein","attrs":{"text":"ABX40964.1","term_id":"160427401","term_text":"ABX40964.1"}}ABX40964.1, {"type":"entrez-protein","attrs":{"text":"ABX42289.1","term_id":"160428726","term_text":"ABX42289.1"}}ABX42289.1, and {"type":"entrez-protein","attrs":{"text":"ABX43387.1","term_id":"160429824","term_text":"ABX43387.1"}}ABX43387.1, respectively). C. phytofermentans has the ability to utilize a wide range of plant polysaccharides (23), 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 GenBank^TM accession numbers of numbered proteins are as follows: 1, CPF0553 (C. perfringens ATCC13124, {"type":"entrez-protein","attrs":{"text":"ABG83511.1","term_id":"110674524","term_text":"ABG83511.1"}}ABG83511.1) (16); 2, CPE0573 (C. perfringens str.13, {"type":"entrez-protein","attrs":{"text":"BAB80279.1","term_id":"18144232","term_text":"BAB80279.1"}}BAB80279.1); 3, CPR0537 (C. perfringens SM101, {"type":"entrez-protein","attrs":{"text":"ABG86710.1","term_id":"110683340","term_text":"ABG86710.1"}}ABG86710.1); 4, LnpA2 (B. bifidum JCM1254, {"type":"entrez-protein","attrs":{"text":"BAE95374.1","term_id":"107597820","term_text":"BAE95374.1"}}BAE95374.1) (14, 15); 5, LnpA1 (B. bifidum JCM1254, {"type":"entrez-protein","attrs":{"text":"BAD80752.1","term_id":"56676017","term_text":"BAD80752.1"}}BAD80752.1) (14, 15); 6, GLNBP_Bl (B. longum subsp. longum JCM 1217, {"type":"entrez-protein","attrs":{"text":"BAD80751.1","term_id":"56676015","term_text":"BAD80751.1"}}BAD80751.1) (1, 16); 7, Blon_2174 (B. longum subsp. infantis ATCC 15697, {"type":"entrez-protein","attrs":{"text":"ACJ53235.1","term_id":"213524488","term_text":"ACJ53235.1"}}ACJ53235.1); 8, BL1641 (B. longum NCC2705, {"type":"entrez-protein","attrs":{"text":"AAN25428.1","term_id":"23326930","term_text":"AAN25428.1"}}AAN25428.1); 9, BLD_1765 (B. longum DJO10A, {"type":"entrez-protein","attrs":{"text":"ACD99210.1","term_id":"189429062","term_text":"ACD99210.1"}}ACD99210.1); 10, GnpA (P. acnes JCM6473, {"type":"entrez-nucleotide","attrs":{"text":"AB468065","term_id":"219807264","term_text":"AB468065"}}AB468065) (17); 11, GnpA (P. acnes JCM6425, {"type":"entrez-nucleotide","attrs":{"text":"AB468066","term_id":"219807262","term_text":"AB468066"}}AB468066) (17); 12, PPA0083 (P. acnes KPA171202, {"type":"entrez-protein","attrs":{"text":"AAT81843.1","term_id":"50839176","term_text":"AAT81843.1"}}AAT81843.1); 13, VV2_1091 (V. vulnificus CMCP6, {"type":"entrez-protein","attrs":{"text":"AAO07997.1","term_id":"27359049","term_text":"AAO07997.1"}}AAO07997.1) (18); 14, VVA1614 (V. vulnificus YJ016, {"type":"entrez-protein","attrs":{"text":"BAC97640.1","term_id":"37201820","term_text":"BAC97640.1"}}BAC97640.1); 15, Oter_1377 (Opitutus terrae PB90-1, {"type":"entrez-protein","attrs":{"text":"ACB74662.1","term_id":"177840410","term_text":"ACB74662.1"}}ACB74662.1); 16, BCQ_1989 (B. cereus Q1, {"type":"entrez-protein","attrs":{"text":"ACM12417.1","term_id":"221239707","term_text":"ACM12417.1"}}ACM12417.1); 17, BCAH187_A2105 (Bacillus cereus AH187, {"type":"entrez-protein","attrs":{"text":"ACJ78918.1","term_id":"217064668","term_text":"ACJ78918.1"}}ACJ78918.1).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.     

Endogenous ADP-ribosylation of elongation factor-2 by interleukin-1��

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.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

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.     

Transforming Growth Factor-�� (TGF-��1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-�� Receptor Kinase Activity in Mesangial Cells

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI.Members of the transforming growth factor-β (TGF-β)³ superfamily are key regulators of various biological processes such as cellular differentiation, proliferation, apoptosis, and wound healing (1, 2). TGF-β1, the prototype of TGF-β family, is a potent inducer of extracellular matrix synthesis and is well established as a central mediator in the final common pathway of fibrosis associated with progressive kidney diseases (3, 4). Upon ligand stimulation, TGF-β type I (TβRI) and type II (TβRII) receptors form heterotetrameric complexes, by which TβRI is phosphorylated in the GS domain and activated. Smad signaling pathway is well established as a canonical pathway induced by TGF-β1 (5, 6). Receptor-regulated Smads (Smad2 and Smad3) are recruited and activated by the activated TβRI. The phosphorylation in the GS domain (7) and L45 loop (8) of TβRI are crucial for its interaction with receptor-regulated Smads. After phosphorylation, receptor-regulated Smads are rapidly dissociated from TβRI and interact with common Smad (Smad4) followed by nuclear translocation. In addition to the Smad pathway, a recently emerging body of evidence has demonstrated that TGF-β1 also induces various Smad-independent signaling pathways (9–17) by which mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) (18, 19), p38 MAPK (20–22), and extracellular signal-regulated kinase 1/2 (23, 24) can be activated by TGF-β1.TAK1, initially identified as a MAPK kinase kinase 7 (MKKK7 or MAP3K7) in the TGF-β signaling pathway (11, 12), also can be activated by environmental stress (25), proinflammatory cytokines such as IL-1 and TNF-α (26, 27) and lipopolysaccharide (28). For TAK1 activation, phosphorylation at Thr-187 and Ser-192 in the activation loop of TAK1 is essentially required (29–31). TAK1 can transduce signals to several downstream signaling cascades, including the MAPK kinase (MKK) 4/7-JNK cascade, MKK3/6-p38 MAPK cascade, and nuclear factor κB (NF-κB)-inducing kinase-IκB kinase cascade (26–28). A recent report has shown that TAK1 is also activated by agonists of AMP-activated kinase (AMPK) and ischemia, which in turn activates the LKB1/AMPK pathway, a pivotal energy-sensor pathway (32). TAK1 is also involved in Wnt signaling (33). We and others have previously demonstrated that TAK1 is a major mediator of TGF-β1-induced type I collagen and fibronectin expression through activation of the MKK3-p38 MAPK and MKK4-JNK signaling cascades, respectively (34–37). Furthermore, increased expression and activation of TAK1 enhance p38 phosphorylation and promote interstitial fibrosis in the myocardium from 9-day-old TAK1 transgenic mice (37). These data implicate a crucial role of TAK1 in extracellular matrix production and tissue fibrosis. TAK1 is also implicated in regulation of cell cycle (38), cell apoptosis (39–41), and the Smad signaling pathway (42–44). Thus, TAK1 may function as an important regulator and mediator of TGF-β1-induced Smad-dependent and Smad-independent signaling pathways.It has been demonstrated that TAK1 can be activated by the interaction with TAK1-binding protein 1 (TAB1) by in vitro binding assays and in overexpression studies (29–31); however, it is not clear whether TAB1 plays a crucial role in ligand-induced TAK1 activation. In embryonic fibroblasts from TAB1 null mice, IL-1 and TNF-α could induce TAK1-mediated NF-κB and JNK activation (45). TAK1 activation induced by TNF-α, IL-1, and T-cell receptor requires TAB2 or its homologous protein TAB3 (46–50). Although many questions still remain, much progress has been made in understanding the activation mechanism of TAK1 by inflammatory cytokines (46, 47, 51–53). Ligand binding of IL-1 receptor (IL-1R) results in recruitment of MyD88, which serves as an adaptor for IL-1 receptor-associated kinase (IRAK) 1 and 4. Subsequently IRAK1 is hyperphosphorylated and induces interaction with TNF-α receptor-associated factor 6 (TRAF6), resulting in TRAF6 oligomerization. After oligomerization of TRAF6, IRAK1-TRAF6 complex is dissociated from the receptor and associated with TAK1, which is mediated by TAB2 (or TAB3). In this process polyubiquitination of TRAF6 by Ubc13/Uev1A is thought to be critical for the association with TAB2 (or TAB3), which links TAK1 activation (46, 54, 55). In the case of TNF-α stimulation, TNF-α receptors form trimers and recruit adaptor proteins, TRAF2/5, and receptor-interacting protein 1 on the membrane. Ubc13/Uev1A- and TRAF2-dependent polyubiquitination of receptor-interacting protein 1 induce association of TAB2 (or TAB3), which then activates TAK1. Thus, TAB2 is required for ubiquitin-dependent activation of TAK1 by TRAFs. On the other hand, it has been demonstrated that hematopoietic progenitor kinase 1 plays a role as an upstream mediator of TGF-β-induced TAK1 activation, which in turn activates the MKK4-JNK signaling cascade in 293T cells (56, 57). Besides hematopoietic progenitor kinase 1, it has been also suggested that X-linked inhibitor of apoptosis (XIAP) might link TAK1 to TGF-β/BMP receptors through the capability of XIAP to interact with TGF-β/BMP receptors and TAB1 (58). Thus, although various molecules participate in the activation of TAK1, the precise mechanism by which TGF-β1 induces TAK1 activation is incompletely understood. Here, we provide evidence that the association of TAK1 with TGF-β receptors is important for TGF-β1-induced activation of TAK1 in mouse mesangial cells. TGF-β1 stimulation induces interaction of TβRI and TβRII, triggering dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation, independent of receptor kinase activity of TβRI.     

Metabolic Consequences of High-Fat Diet Are Attenuated by Suppression of HIF-1��

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.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

para-Nitrophenyl Sulfate Activation of Human Sulfotransferase 1A1 Is Consistent with Intercepting the E��PAP Complex and Reformation of E��PAPS

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biological activities of various biosignaling molecules and metabolizes hydroxyl-containing drugs and xenobiotics. The universal sulfuryl group donor for SULT-catalyzed sulfation is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), whereas the reaction products are a sulfated product and adenosine 3′,5′-diphosphate (PAP). Although SULT-catalyzed kinetic mechanisms have been studied since the 1980s, they remain unclear. Human SULT1A1 is an important phase II drug-metabolizing enzyme. Previously, isotope exchange at equilibrium indicated steady-state ordered mechanism with PAPS and PAP binding to the free SULT1A1 (Tyapochkin, E., Cook, P. F., and Chen, G. (2008) Biochemistry 47, 11894–11899). On the basis of activation of SULT1A1 by para-nitrophenyl sulfate (pNPS), an ordered bypass mechanism has been proposed where pNPS sulfates PAP prior to its release from the E·PAP complex regenerating E·PAPS. Data are consistent with uncompetitive substrate inhibition by naphthol as a result of formation of the E·PAP·naphthol dead-end complex; formation of the complex is corroborated by naphthol/PAP double inhibition experiments. pNPS activation data demonstrate an apparent ping-pong behavior with pNPS adding to E·PAP, and competitive inhibition by naphthol consistent with formation of the E·PAP·naphthol complex. Exchange against forward reaction flux (PAPS plus naphthol) beginning with [³⁵S]PAPS and generating [³⁵S]naphthyl sulfate is also consistent with pNPS intercepting the E·PAP complex. Overall, data are consistent with the proposed ordered bypass mechanism.Sulfotransferases (SULTs)³ are phase II drug-metabolizing enzymes that catalyze the sulfation (sulfonation) of various hydroxyl-containing compounds: biosignaling molecules such as hydroxysteroid hormones, thyroid hormones, glucocorticoid hormones, bile acids, neurotransmitters, and hydroxyl-containing xenobiotics (1–8). The sulfation proceeds as shown in reaction 1, where the sulfuryl group donor is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), and the reaction products are adenosine 3′,5′-diphosphate (PAP) and a sulfated product. One of the main biological functions of SULTs is the regulation of various hormones (9). Sulfation of xenobiotics is mainly associated with detoxification, biotransformation of a relatively hydrophobic xenobiotic into a more water-soluble sulfuric ester that is readily excreted. However, in some cases sulfation can also cause bioactivation of procarcinogens and promutagens, leading to possible toxic effects (10, 11).Studies of the SULTs kinetic mechanisms began to appear in the early 1980s (12). Although many SULT isoforms have been isolated and characterized, their biological functions and catalytic mechanisms are still not well understood. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics; it also catalyzes the sulfation of endogenous hydroxyl biosignaling molecules. It has very broad substrate specificity and high activity toward most phenolic compounds. SULT1A1 is also widely distributed in the human body. On the basis of isotope exchange at equilibrium, we showed that the kinetic mechanism for human SULT1A1 is steady-state-ordered with PAPS binding to the protein first, and PAP released last (13).Substrate inhibition by the hydroxyl substrate (sulfate acceptor) is a common feature of most cytosolic SULTs (14, 15). Inhibition of SULT1A1 has been observed by the substrate, naphthol. There are a number of different mechanisms that have been proposed for substrate inhibition, but the mechanism remains unclear. A ternary complex formed between substrate and the enzyme·PAP complex is the most likely possibility in an ordered mechanism, but binding to free enzyme is also possible (12, 16). It is also possible, but unlikely, that substrate could bind to central complexes. In addition, binding of two substrate molecules to the active site has been proposed (4, 14). A SULT1A1 crystal structure was solved that showed two molecules of p-nitrophenol (pNP) in the same active site. However, computer modeling of this structure indicated that the active site could not easily accommodate even one molecule of a larger substrate such as β-estradiol (17). Other SULT crystal structures solved with the bound substrate indicated that only one substrate is possible in the crystal structure (18–23).para-Nitrophenyl sulfate (pNPS) has been used for phenol SULTs enzyme activity assays (24–27). Recently, we have been interested in the mechanisms for pNPS activation of SULT1A1-catalyzed sulfation of other phenol substrates, such as naphthols. On the basis of this activation by pNPS, a mechanism was proposed that requires sulfation of PAP prior to its release, from the E·PAP complex (Scheme 1), i.e. pNPS binds to E·PAP and generates the E·PAPS·pNP complex, which dissociates pNP and generates the E·PAPS complex.Open in a separate windowSCHEME 1.Proposed ordered bypass mechanism with substrate inhibition by B binding to E·PAP. In the scheme, B, B₂, and P₂ are naphthol, pNP, and pNPS, respectively, whereas A and Q are PAPS and PAP, respectively. An additional EAP dead-end complex is allowed but not shown.In this work, the proposed mechanism was tested using the double inhibition method of Yonetani and Theorell (28), which provides information on whether binding of two inhibitors is mutually exclusive. Double inhibition experiments have been successful in demonstrating whether the binding of two inhibitors is mutually exclusive, or whether they show interference or synergism in binding (28–32). In addition, substrate inhibition by the hydroxyl substrate and exchange against forward reaction flux were used as probes of the mechanism. Data are discussed in terms of the overall mechanism of SULT1A1.