共查询到20条相似文献,搜索用时 15 毫秒
1.
Ganesan Senthil Kumar Heiko Zettl Rebecca Page Wolfgang Peti 《The Journal of biological chemistry》2013,288(39):28347-28356
Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins. 相似文献
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《The Journal of biological chemistry》2013,288(39):28357
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Maria A. Schumacher Nagababu Chinnam Tomoo Ohashi Riddhi Sanjay Shah Harold P. Erickson 《The Journal of biological chemistry》2013,288(47):33738-33744
Irisin was recently identified as a putative myokine that is induced by exercise. Studies suggest that it is produced by cleavage of the FNDC5 (fibronectin domain-containing protein 5) receptor; irisin corresponds to the extracellular receptor ectodomain. Data suggesting that irisin stimulates white-to-brown fat conversion have led to the hypothesis that it does so by binding an unknown receptor, thus functioning as a myokine. As brown fat promotes energy dissipation, myokines that elicit the transformation of white to brown fat have potentially profound benefits in the treatment of obesity and metabolic disorders. Understanding the molecular basis for such exercise-induced phenomena is thus of considerable interest. Moreover, FNDC5-like receptors are highly conserved and have been shown to be critical for neuronal development. However, the structural and molecular mechanisms utilized by these proteins are currently unknown. Here, we describe the crystal structure and biochemical characterization of the FNDC5 ectodomain, corresponding to the irisin myokine. The 2.28 Å structure shows that irisin consists of an N-terminal fibronectin III (FNIII)-like domain attached to a flexible C-terminal tail. Strikingly, the FNIII-like domain forms a continuous intersubunit β-sheet dimer, previously unobserved for any FNIII protein. Biochemical data confirm that irisin is a dimer and that dimerization is unaffected by glycosylation. This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors. 相似文献
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Shruthi Ravimohan Lucio Gama Sheila A. Barber Janice E. Clements 《The Journal of biological chemistry》2010,285(4):2258-2273
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Xianlong Gao Deepti Chaturvedi Tarun B. Patel 《The Journal of biological chemistry》2010,285(10):6970-6979
Previously we showed that the inactive form of p90 ribosomal S6 kinase 1 (RSK1) interacts with the regulatory subunit, PKARIα, of protein kinase A (PKA), whereas the active RSK1 interacts with the catalytic subunit (PKAc) of PKA. Herein, we demonstrate that the N-terminal kinase domain (NTK) of RSK1 is necessary for interactions with PKARIα. Substitution of the activation loop phosphorylation site (Ser-221) in the NTK with the negatively charged Asp residue abrogated the association between RSK1 and PKARIα. This explains the lack of an interaction between active RSK1 and PKARIα. Full-length RSK1 bound to PKARIα with an affinity of 0.8 nm. The NTK domain of RSK1 competed with PKAc for binding to the pseudosubstrate region (amino acids 93–99) of PKARIα. Overexpressed RSK1 dissociated PKAc from PKARIα, increasing PKAc activity, whereas silencing of RSK1 increased PKAc/PKARIα interactions and decreased PKAc activity. Unlike PKAc, which requires Arg-95 and -96 in the pseudosubstrate region of PKARIα for their interactions, RSK1/PKARIα association requires all four Arg residues (Arg-93–96) in the pseudosubstrate site of PKARIα. A peptide (Wt-PS) corresponding to residues 91–99 of PKARIα competed for binding of RSK1 with PKARIα both in vitro and in intact cells. Furthermore, peptide Wt-PS (but not control peptide Mut-PS), by dissociating RSK1 from PKARIα, activated RSK1 in the absence of any growth factors and protected cells from apoptosis. Thus, by competing for binding to the pseudosubstrate region of PKARIα, RSK1 regulates PKAc activity in a cAMP-independent manner, and PKARIα by associating with RSK1 regulates its activation and its biological functions. 相似文献
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Jennifer L. Meitzler Paul R. Ortiz de Montellano 《The Journal of biological chemistry》2009,284(28):18634-18643
The seven members of the NOX/DUOX family are responsible for generation of the superoxide and H2O2 required for a variety of host defense and cell signaling functions in nonphagocytic cells. Two members, the dual oxidase isozymes DUOX1 and DUOX2, share a structurally unique feature: an N-terminal peroxidase-like domain. Despite sequence similarity to the mammalian peroxidases, the absence of key active site residues makes their binding of heme and their catalytic function uncertain. To explore this domain we have expressed in a baculovirus system and purified the Caenorhabditis elegans (CeDUOX11–589) and human (hDUOX11–593) DUOX1 “peroxidase” domains. Evaluation of these proteins demonstrated that the isolated hDUOX11–593 does not bind heme and has no intrinsic peroxidase activity. In contrast, CeDUOX11–589 binds heme covalently, exhibits a modest peroxidase activity, but does not oxidize bromide ion. Surprisingly, the heme appears to have two covalent links to the protein despite the absence of a second conserved carboxyl group in the active site. Although the N-terminal dual oxidase motif has been proposed to directly convert superoxide to H2O2, neither DUOX1 domain demonstrated significant superoxide dismutase activity. These results strengthen the in vivo conclusion that the CeDUOX1 protein supports controlled peroxidative polymerization of tyrosine residues and indicate that the hDUOX1 protein either has a unique function or must interact with other protein factors to express its catalytic activity.The purposeful generation of reactive oxygen species within phagocytic cells has long been recognized as a component of their antibacterial defense system (1, 2). Reactive oxygen species generation is mediated by a membrane-bound NADPH oxidase (NOX)2 and is activated by a diverse number of stimuli. The NOX enzymes catalyze the NADPH-dependent one-electron reduction of oxygen to superoxide (O2−̇) (3). It has long been debated whether the generation of similar species in other cell types is also an intentional, physiologically controlled process or is an accident of aerobic respiration. This controversy has been clarified by identification of the NOX/DUOX family of NADPH oxidases. The seven members of this family (NOX 1–5 and DUOX1 and 2) have been shown to produce the reactive oxygen species utilized for functions as varied as cellular signaling, host defense, and thyroid hormone biosynthesis (4–8). The latter function is specifically attributed to the DUOX members of this family.DUOX1 and 2 (formerly also known as ThOX1 and 2 for thyroid oxidase) were first identified in the mammalian thyroid gland (9, 10). This localization is not exclusive because both can also be found in nonthyroid tissues; DUOX1 is prominent in airway epithelial cells (11) and DUOX2 in the salivary glands and gastrointestinal tract (4, 12, 13). Homologs of each DUOX have also been identified in lower organisms, including Caenorhabditis elegans and Drosophila melanogaster (14). The human isoforms are 83% homologous, ∼190 kDa in size (after glycosylation), and are located in close proximity, because they are configured head-to-head on human chromosome 15 (15, 16). The glycosylation of both DUOX1 and 2 is extensive, contributing ∼30 kDa to the total apparent protein mass (17). Recent investigation has uncovered that maturation factors DUOXA1 and DUOXA2 are required to achieve heterologous expression of each DUOX in full-length, active form (18).Structurally, DUOX1 and 2 are characterized by a defining N-terminal, extracellular domain exhibiting considerable sequence identity with the mammalian peroxidases, a transmembrane (TM) segment appended to an EF-hand calcium-binding cytosolic region and a NOX2 homologous structure (six TMs tethered to NADPH oxidase; see Fig. 1A) (10). Both isoforms have a conserved calcium-binding site in the N-terminal peroxidase domain, mimicking that found in MPO, LPO, EPO, and TPO. Interestingly, although homologous to these heme-containing peroxidases, the peroxidase-like domains of the DUOX proteins lack some of the highly conserved amino acid residues that are thought to be essential for heme binding and/or peroxidase catalysis (see Fig. 1B) (16).Open in a separate windowFIGURE 1.Comparison of sequence and structural features of hDUOX1 and CeDUOX1. A, schematic view of the domain structures of hDUOX1 and CeDUOX1. Each DUOX1 protein contains an N-terminal extracellular peroxidase domain (dark gray rectangle), putative TM domains (light gray tubes), and cytosolic EF-hand and NADPH oxidase domains (light gray rectangles). Both proteins were truncated to generate soluble expression constructions focusing on the peroxidase domain, as shown. B, sequence alignment of classical peroxidase domains with the DUOX1 proteins. Highlighted segments shown focus on regions of active site residues (bold face) including the distal histidine (hMPO His261), catalytic arginine (hMPO Arg405), proximal histidine (hMPO His502), and covalent heme-binding residues (hMPO Asp260 and Glu408). hEPO, human eosinophil peroxidase; bLPO, bovine lactoperoxidase; hTPO, human thyroid peroxidase; hVPO, human vascular peroxidase; hPxn, human peroxidasin.Functionally, mature DUOX enzymes appear to produce H2O2, in contrast to other NOX family members that produce superoxide. This activity is regulated by Ca2+ concentration through triggered dissociation of NOXA1 and possibly other as yet unidentified interacting proteins (19). Because the N-terminal peroxidase domain is the structural feature that differentiates the dual oxidases from the NOX proteins, it may be directly responsible for the conversion of superoxide to H2O2. To investigate this crucial domain, we report here the first expression, purification, and characterization of the Homo sapiens (hDUOX11–593) and C. elegans (CeDUOX11–589) DUOX1 peroxidase domains. We demonstrate that heme is covalently bound to CeDUOX11–589 (two covalent bonds are suggested by heme hydroxylation studies), whereas hDUOX11–593 does not stably bind this co-factor. Both domains share overall sequence similarity with the mammalian peroxidases (specifically LPO), but only CeDUOX11–589 exhibits peroxidase activity, as measured with either ABTS or tyrosine ethyl ester as the substrate. We also demonstrate that neither DUOX1 domain has significant superoxide dismutase or halide oxidizing activity. 相似文献
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Kristopher Clark Lorna Plater Mark Peggie Philip Cohen 《The Journal of biological chemistry》2009,284(21):14136-14146
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Pu Wang Fei Zhu Norman H. Lee Konstantinos Konstantopoulos 《The Journal of biological chemistry》2010,285(32):24793-24804
Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E2, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE2 positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE2 signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors. 相似文献
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Vishukumar Aimanianda C��cile Clavaud Catherine Simenel Thierry Fontaine Muriel Delepierre Jean-Paul Latg�� 《The Journal of biological chemistry》2009,284(20):13401-13412
Despite its essential role in the yeast cell wall, the exact composition of
the β-(1,6)-glucan component is not well characterized. While
solubilizing the cell wall alkali-insoluble fraction from a wild type strain
of Saccharomyces cerevisiae using a recombinant
β-(1,3)-glucanase followed by chromatographic characterization of the
digest on an anion exchange column, we observed a soluble polymer that eluted
at the end of the solvent gradient run. Further characterization indicated
this soluble polymer to have a molecular mass of ∼38 kDa and could be
hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry
and NMR (1H and 13C) analyses confirmed it to be a
β-(1,6)-glucan polymer with, on average, branching at every fifth residue
with one or two β-(1,3)-linked glucose units in the side chain. This
polymer peak was significantly reduced in the corresponding digests from
mutants of the kre genes (kre9 and kre5) that are
known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the
current study, we have developed a biochemical assay wherein incubation of
UDP-[14C]glucose with permeabilized S. cerevisiae yeasts
resulted in the synthesis of a polymer chemically identical to the branched
β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters
essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts
contains two types of β-glucans. In the former yeast, branched
β-(1,3)-glucan accounts for ∼50–55%, whereas
β-(1,6)-glucan represents 10–15% of the total yeast cell wall
polysaccharides, each chain of the latter extending up to 140–350
glucose residues in length. The amount of 3,6-branched glucose residues varies
with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida
albicans, and Schizosaccharomyces pombe, respectively
(1). β-(1,6)-Glucan
stabilizes the cell wall, since it plays a central role as a linker for
specific cell wall components, including β-(1,3)-glucan, chitin, and
mannoproteins (2,
3). However, the exact
structure of the β-(1,6)-glucan and the mode of biosynthesis of this
polymer are largely unknown. In S. pombe, immunodetection studies
suggested that synthesis of this polymer backbone begins in the endoplasmic
reticulum, with extension occurring in the Golgi
(4) and final processing at the
plasma membrane. In S. cerevisiae, Montijn and co-workers
(5), by immunogold labeling,
detected β-(1,6)-glucan at the plasma membrane, suggesting that the
synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and
their homologues, SKN1 and KNH1, have been reported to be
involved in β-(1,6)-glucan synthesis in S. cerevisiae, C.
albicans, and Candida glabrata
(6–10).
Among all of these genes, the ones that seem to play the major synthetic role
are KRE5 and KRE9, since their disruption caused significant
reduction (100 and 80%, respectively, relative to wild type) in the cell wall
β-(1,6)-glucan content
(11–13).To date, the biochemical reaction responsible for the synthesis of
β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in
most cases, when membrane preparations are incubated with UDP-glucose, only
linear β-(1,3)-glucan polymers are produced, although some studies have
reported the production of low amounts of β-(1,6)-glucans by membrane
preparations
(14–17).
These data suggest that disruption of the fungal cell prevents or at least has
a strong negative effect on β-(1,6)-glucan synthesis. The use of
permeabilized cells, which allows substrates, such as nucleotide sugar
precursors, to be readily transported across the plasma membrane, is an
alternative method to study in situ cell wall enzyme activities
(18–22).
A number of methods have been developed to permeabilize the yeast cell wall
(23), of which osmotic shock
was successfully used to demonstrate β-(1,3)-glucan and chitin synthase
activities (20,
24). Herein, we describe the
biochemical activity responsible for β-(1,6)-glucan synthesis using
permeabilized S. cerevisiae cells and UDP-[14C]glucose as
a substrate. We also have analyzed the physicochemical parameters of this
activity and chemically characterized the end product and its structural
organization within the mature yeast cell wall. 相似文献
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W. T. Ashton L. C. Meurer R. L. Tolman J. D. Karkas R. Liou H. C. Perryt 《Nucleosides, nucleotides & nucleic acids》2013,32(5-6):1157-1158
Abstract The title compound was prepared and found to be a potent and selective inhibitor of HSV-I thymidine kinase. This compound delayed the reactivation of latent virus from explanted mouse ganglia but exacerbated the primary HSV-I infection in mice. 相似文献
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James C. S. Ho Petter Storm Anna Rydstr?m Ben Bowen Fredrik Alsin Louise Sullivan Inès Ambite K. H. Mok Trent Northen Catharina Svanborg 《The Journal of biological chemistry》2013,288(24):17460-17471
Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance 13C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein. 相似文献
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Karim Abid Bertrand Rochat Paul-Gerhard Lassahn Reto St?cklin Sophie Michalet Noureddine Brakch Jean-Francois Aubert Bilgin Vatansever Patricia Tella Ingrid De Meester Eric Grouzmann 《The Journal of biological chemistry》2009,284(37):24715-24724
There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY1–36 upon incubation with human serum. Kinetic studies indicated that NPY1–36 is rapidly cleaved in serum into 3 main fragments with the following order of efficacy: NPY3–36 ≫ NPY3–35 > NPY2–36. Trace amounts of additional NPY forms were identified by accurate mass spectrometry. Specific inhibitors of dipeptidyl peptidase IV, kallikrein, and aminopeptidase P prevented the production of NPY3–36, NPY3–35, and NPY2–36, respectively. Plasma kallikrein at physiological concentrations converted NPY3–36 into NPY3–35. Receptor binding assays revealed that NPY3–35 is unable to bind to NPY Y1, Y2, and Y5 receptors; thus NPY3–35 may represent the major metabolic clearance product of the Y2/Y5 agonist, NPY3–36.Neuropeptide Y (NPY)2 is a 36-amino acid peptide involved in the central and peripheral control of blood pressure (1–4) and in feeding behavior and obesity (5–9). NPY stimulates at least 6 types of receptors, called Y1, Y2, Y3, Y4, Y5, and y6 (10–12). The Y1 receptor has high affinity for full-length NPY, while Y2 and Y5 receptors bind and are stimulated by full-length and N-terminally truncated NPY. The physiological effects associated to the Y1 and Y2 receptors are the best known; exposure to a Y1 agonist causes an increase in blood pressure and potentiates postsynaptically the action of other vasoactive substances (1, 4, 13), whereas Y2 receptors are mainly located presynaptically, and upon stimulation mediate the inhibition of neurotransmitter release (14, 15). NPY is a prototype of peptide whose function can be altered by proteases. Among peptidases displaying a high affinity for NPY, the primary role appears to be played by dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), a serine-type protease, also known as CD26, that releases an N-terminal dipeptide, Xaa-Xab- -Xac, preferentially when Xab is a proline or an alanine residue (16). By cleaving the Tyr-Pro dipeptide off the NPY N-terminal extremity, DPPIV generates NPY3–36, a truncated form that loses its affinity for the Y1 receptor and becomes a Y2/Y5 receptor agonist (17, 18).NPY can also be degraded by aminopeptidase P (AmP, EC 3.4.11.9), a metalloprotease that hydrolyzes the peptide bond between the first and the second amino acid residue at the N terminus of proteins, if the second amino acid is a proline (19). AmP removes the N-terminal tyrosine from NPY to generate NPY2–36, a selective Y2 agonist (18, 20). There is little information on how NPY cleavage by these enzymes occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive (NPYir) forms and also because the factors affecting the expression of these enzymes have been poorly studied. Recently, Frerker et al. (21) reported by MALDI-TOF mass spectrometry that NPY1–36 is exclusively degraded by DPPIV into NPY3–36 in EDTA-plasma but they did not provide kinetics of NPY cleavage efficiency of DPPIV. Beck-Sickinger and co-workers (22) studied with the same technique the metabolic stability of fluorescent N-terminally labeled NPY analogues incubated in human plasma and found that the 36th, 35th, and 33rd residues of NPY analogues may also be removed by unknown carboxypeptidases.We have set up a method using liquid chromatography coupled with tandem mass spectrometry (LC-MSn) to selectively quantify NPY and its C-terminal fragments NPY2–36 and NPY3–36 digested by human serum. The assays used the internal standard methodology with stable isotopes NPY1–36 (IDA) (23, 24) or porcine NPY1–36 as internal standard.The goal of this work was: 1) to determine to which extent NPY1–36 is degraded by proteases present in human serum and whether an inhibition of DPPIV and AmP by vildagliptin and apstatin (two specific protease inhibitors), respectively, may affect the metabolism of NPY in serum; 2) to assign kinetic values to the proteases involved in the cleavage process toward NPY; and 3) to characterize new NPY-truncated forms and to check for their possible binding capacities on NPY receptors. 相似文献