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41.
Qurra-tul-Ann Afza Gardner Hooria Younas Muhammad Akhtar 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):182-190
Human M-proinsulin was cleaved by trypsin at the R31R32–E33 and K64R65–G66 bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K59R60–G61 bond but at the B/C junction cleavage occurred at the R31R32–E33 as well as the R31–R32E33 bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the kcat./Km values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 105 s− 1 M− 1) and the cleavage of the K29–T30 bond of M-insulin-RR (1.3 ± 0.07 × 105 s− 1 M− 1). However, the K29–T30 bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (kcat./Km values around 1000 s− 1 M− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K29–T30 bond becomes shielded, exposed then shielded again respectively. 相似文献
42.
The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the ‘low affinity’ IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium+ uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself. 相似文献
43.
Leanne Stokes 《Purinergic signalling》2013,9(1):113-121
The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation. 相似文献
44.
Clare E Buckley Xiaoyun Ren Laura C Ward Gemma C Girdler Claudio Araya Mary J Green Brian S Clark Brian A Link Jonathan D W Clarke 《The EMBO journal》2013,32(1):30-44
By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror‐symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen. 相似文献
45.
46.
Robert H. C. Chen Sabine Wislet-Gendebien Filsy Samuel Naomi P. Visanji Gang Zhang Diana Marsilio Tammy Langman Paul E. Fraser Anurag Tandon 《The Journal of biological chemistry》2013,288(11):7438-7449
α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity. 相似文献
47.
Zhong Guo Xiaomin Hou Roger S. Goody Aymelt Itzen 《The Journal of biological chemistry》2013,288(45):32466-32474
Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange. 相似文献
48.
49.
The hERG (human ether-a-go-go-related gene) encodes the α subunit of the rapidly activating delayed rectifier potassium channel (IKr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling. 相似文献
50.
Daopeng Yuan Anthony H. Keeble Richard G. Hibbert Stella Fabiane Hannah J. Gould James M. McDonnell Andrew J. Beavil Brian J. Sutton Balvinder Dhaliwal 《The Journal of biological chemistry》2013,288(30):21667-21677
Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca2+-free and Ca2+-bound forms, as well as the crystal structure of the Ca2+-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca2+ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca2+ binds at the principal and evolutionarily conserved binding site in CD23. Ca2+ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca2+-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca2+ brings an extra degree of modulation to CD23 function. 相似文献