| 1. |
- Abdian, Patricia L., et al.
(author)
-
RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides
- 2013
-
In: Journal of Biological Chemistry. - 1083-351X. ; 288:4, s. 2893-2904
-
Journal article (peer-reviewed)abstract
- In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like beta-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins.
|
|
| 2. |
- Abelein, Axel, et al.
(author)
-
Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates
- 2013
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:32, s. 23518-23528
-
Journal article (peer-reviewed)abstract
- Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.
|
|
| 3. |
- Aboulaich, Nabila, et al.
(author)
-
Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes
- 2006
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:17, s. 11446-11449
-
Journal article (peer-reviewed)abstract
- In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.
|
|
| 4. |
- Abrahamson, Magnus, et al.
(author)
-
Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin
- 1987
-
In: Journal of Biological Chemistry. - 1083-351X. ; 262:20, s. 9688-9694
-
Journal article (peer-reviewed)abstract
- When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy- trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate- like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.
|
|
| 5. |
- Abrahamson, Magnus, et al.
(author)
-
Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids
- 1986
-
In: Journal of Biological Chemistry. - 1083-351X. ; 261:24, s. 11282-11289
-
Journal article (peer-reviewed)abstract
- Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
- Adlerz, Linda, et al.
(author)
-
IGF-1-induced Processing of the Amyloid Precursor Protein Family Is Mediated by Different Signaling Pathways
- 2007
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:14, s. 10203-10209
-
Journal article (peer-reviewed)abstract
- The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.
|
|
| 10. |
- Agarwal, Pallavi, et al.
(author)
-
Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure
- 2012
-
In: Journal of Biological Chemistry. - 1083-351X. ; 287:27, s. 22549-22559
-
Journal article (peer-reviewed)abstract
- The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.
|
|
| 11. |
- Agarwal, Shruti, et al.
(author)
-
Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation.
- 2013
-
In: Journal of Biological Chemistry. - 1083-351X. ; 288:31, s. 22460-22468
-
Journal article (peer-reviewed)abstract
- The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors (GISTs), acute myeloid leukemia (AML), testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Y823 has been demonstrated to be a late event that is not required for kinase activation. However, since phosphorylation of Y823 is a ligand-activated event, we sought to investigate the functional consequences of Y823 phosphorylation. By using a tyrosine to phenylalanine mutant of tyrosine 823 we investigated the impact of Y823 on c-Kit signaling. We here demonstrate that Y823 is crucial for cell survival and proliferation and mutation of Y823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared to the wild-type receptor. Furthermore, the mutated receptor was upon ligand-stimulation quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase, Cbl was transient followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, Shc and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells.
|
|
| 12. |
- Agarwal, Vaibhav, et al.
(author)
-
A novel interaction between complement inhibitor C4b-binding protein and plasminogen that enhances plasminogen activation.
- 2015
-
In: Journal of Biological Chemistry. - 1083-351X. ; 290:30, s. 18333-18342
-
Journal article (peer-reviewed)abstract
- The complement, coagulation and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to S. pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, while the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation.
|
|
| 13. |
- Agarwal, Vaibhav, et al.
(author)
-
Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.
- 2014
-
In: Journal of Biological Chemistry. - 1083-351X. ; 289:22, s. 15833-15844
-
Journal article (peer-reviewed)abstract
- The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is an ubiquitously expressed, multifunctional plasminogen and fibronectin binding protein facilitating host cell invasion and evasion of innate immunity. In this study we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, while its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.
|
|
| 14. |
- Agarwal, Vaibhav, et al.
(author)
-
Streptococcus pneumoniae endopeptidase O (PepO): a multifunctional plasminogen and fibronectin binding protein, facilitating evasion of innate immunity and invasion of host cells.
- 2013
-
In: Journal of Biological Chemistry. - 1083-351X. ; 288:10, s. 6849-6863
-
Journal article (peer-reviewed)abstract
- Streptococcus pneumoniae infections remain a major cause of morbidity and mortality worldwide. Therefore a detailed understanding and characterization of the mechanism of host cell colonization and dissemination is critical in order to gain control over this versatile pathogen. Here we identified a novel 72 kDa pneumococcal protein endopeptidase O (PepO), as a plasminogen and fibronectin binding protein. Using a collection of clinical isolates, representing different serotypes, we found PepO to be ubiquitously present both at the gene and at the protein level. In addition, PepO protein was secreted in a growth-phase dependent manner to the culture supernatants of the pneumococcal isolates. Recombinant PepO bound human plasminogen and fibronectin in a dose-dependent manner and plasminogen did not compete with fibronectin for binding PepO. PepO bound plasminogen via lysine residues and the interaction was influenced by ionic strength. Moreover, upon activation of PepO bound plasminogen by urokinase-type plasminogen activator, generated plasmin cleaved complement protein C3b thus assisting in complement control. Furthermore, direct binding assays demonstrated the interaction of PepO with epithelial and endothelial cells that in turn blocked pneumococcal adherence. Moreover, a pepO-mutant strain showed impaired adherence to and invasion of host cells compared to their isogenic wild-type strains. Taken together, the results demonstrated that PepO is ubiquitously expressed plasminogen and fibronectin binding protein, which plays role in pneumococcal invasion of host cells and aids in immune evasion.
|
|
| 15. |
- Ahmad, Faiyaz, et al.
(author)
-
Regulation of SERCA2 activity by PDE3A in human myocardium: Phosphorylation-dependent interaction of PDE3A1 with SERCA2.
- 2015
-
In: Journal of Biological Chemistry. - 1083-351X. ; 290:11, s. 6763-6776
-
Journal article (peer-reviewed)abstract
- PDE3 regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects, and that murine PDE3A1 associates with SERCA2, PLB and AKAP18 in a multi-protein signalosome in human SR. Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel-filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct HMW and LMW peaks. HMW peaks contained PDE3A1 and PDE3A2, while LMW peaks contained PDE3A1, PDE3A2 and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immumoprecipitation of SERCA2, cav3, PKARII, PP2A and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of rhPDE3A isoforms by rPKAc increased co-immumoprecipitation with rSERCA2 and rAKAP18. Deletion of the rhPDE3A1/PDE3A2 N-terminus blocked interactions with rSERCA2. Serine-to-alanine substitutions identified S292/S293, a site unique to hPDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of hPDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation and SERCA2 activity.
|
|
| 16. |
|
|
| 17. |
- Akhatib, Bashar, et al.
(author)
-
Chondroadherin Fragmentation Mediated by the Protease HTRA1 Distinguishes Human Intervertebral Disc Degeneration from Normal Aging
- 2013
-
In: Journal of Biological Chemistry. - 1083-351X. ; 288:26, s. 19280-19287
-
Journal article (peer-reviewed)abstract
- Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.
|
|
| 18. |
- Albinsson, Sebastian, et al.
(author)
-
Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization
- 2004
-
In: Journal of Biological Chemistry. - 1083-351X. ; 279:33, s. 34849-34855
-
Journal article (peer-reviewed)abstract
- Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins beta-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, > 15 min; cofilin-P, > 1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.
|
|
| 19. |
- Alenius, Mattias, et al.
(author)
-
Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection
- 1997
-
In: Journal of Biological Chemistry. - : The American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 272:42, s. 26083-26086
-
Journal article (peer-reviewed)abstract
- We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin Ca-type domains followed by two fibronectin type III domains, Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane, These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II, In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections, The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.
|
|
| 20. |
- Aleshkov, S B, et al.
(author)
-
Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.
- 1996
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 271:35, s. 21231-8
-
Journal article (peer-reviewed)abstract
- Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.
|
|
| 21. |
- Alonso, A, et al.
(author)
-
VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX
- 2004
-
In: Journal of Biological Chemistry. - 1083-351X. ; 279:31, s. 32586-32591
-
Journal article (peer-reviewed)abstract
- The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells ( somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.
|
|
| 22. |
- Altgärde, Noomi, 1983, et al.
(author)
-
Mucin-like region of herpes simplex virus type 1 attachment protein gC modulates the virus-glycosaminoglycan interaction.
- 2015
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:35, s. 21473-21485
-
Journal article (peer-reviewed)abstract
- Glycoprotein C (gC) mediates the attachment of herpes simplex virus type 1 (HSV-1) to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying an HSV-1 mutant lacking the mucin- like domain in gC and the corresponding purified mutant protein (gCΔmuc), in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared to native HSV-1, i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells, and reduced release of viral particles from the surface of infected cells. Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared to native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.
|
|
| 23. |
|
|
| 24. |
|
|
| 25. |
- Alvarado-Kristensson, Maria, et al.
(author)
-
Protein phosphatase 2A regulates apoptosis in neutrophils by dephosphorylating both p38 MAPK and its substrate caspase 3.
- 2005
-
In: Journal of Biological Chemistry. - 1083-351X. ; 280:7, s. 6238-6244
-
Journal article (peer-reviewed)abstract
- The induction of apoptosis in neutrophils is an essential event in the resolution of an inflammatory process. We found recently that the reduction of the activity of the neutrophil survival factor p38 MAPK and dephosphorylation and thus activation of caspases must occur to initiate such cell death in these leukocytes. Here, we report a previously undetected early and transient activation of protein phosphatase 2A WPM in neutrophils undergoing apoptosis. The pharmacological inhibition of this phosphatase during Fas-induced apoptosis augmented the levels of phosphorylation of both p38 MAPK and caspase 3, resulting in a decreased activity of caspase 3 and an increased neutrophil survival. The complementary finding of a time-dependent association among PP2A, p38 MAPK, and caspase 3 in intact neutrophils indicated that there is a direct regulatory link among these signaling enzymes during Fas-provoked apoptosis. Moreover, immunoprecipitated active p38 MAPK and recombinant phosphorylated caspase 3 were dephosphorylated by exposure to purified PP2A in vitro. Consequently, the early and temporary activation of PP2A in neutrophils impaired not only the p38 MAPK-mediated inhibition of caspase 3 but also restored the activity to caspase 3 that had already been phosphorylated and thereby inactivated. These findings indicate that PP2A plays a pivotal dual role in the induction of neutrophil apoptosis and therefore also in the resolution of inflammation.
|
|
