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71.
Estela Puchulu-Campanella Haiyan Chu David J. Anstee Jacob A. Galan W. Andy Tao Philip S. Low 《The Journal of biological chemistry》2013,288(2):848-858
Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction. 相似文献
72.
Tatyana A. Shelkovnikova Owen M. Peters Alexey V. Deykin Natalie Connor-Robson Hannah Robinson Alexey A. Ustyugov Sergey O. Bachurin Tatyana G. Ermolkevich Igor L. Goldman Elena R. Sadchikova Elena A. Kovrazhkina Veronica I. Skvortsova Shuo-Chien Ling Sandrine Da Cruz Philippe A. Parone Vladimir L. Buchman Natalia N. Ninkina 《The Journal of biological chemistry》2013,288(35):25266-25274
Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice. 相似文献
73.
Jimmy Stalin Karim Harhouri Lucas Hubert Caroline Subrini Daniel Lafitte Jean-Claude Lissitzky Nadia Elganfoud Stéphane Robert Alexandrine Foucault-Bertaud Elise Kaspi Florence Sabatier Michel Aurrand-Lions Nathalie Bardin Lars Holmgren Fran?oise Dignat-George Marcel Blot-Chabaud 《The Journal of biological chemistry》2013,288(13):8991-9000
The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases. 相似文献
74.
The budding yeast G-tail binding complex CST (Cdc13-Stn1-Ten1) is crucial for both telomere protection and replication. Previous studies revealed a family of Cdc13 orthologues (Cdc13A) in Candida species that are unusually small but are nevertheless responsible for G-tail binding and the regulation of telomere lengths and structures. Here we report the identification and characterization of a second family of Cdc13-like proteins in the Candida clade, named Cdc13B. Phylogenetic analysis and sequence alignment indicate that Cdc13B probably arose through gene duplication prior to Candida speciation. Like Cdc13A, Cdc13B appears to be essential. Deleting one copy each of the CDC13A and CDC13B genes caused a synergistic effect on aberrant telomere elongation and t-circle accumulation, suggesting that the two paralogues mediate overlapping and nonredundant functions in telomere regulation. Interestingly, Cdc13B utilizes its C-terminal OB-fold domain (OB4) to mediate self-association and binding to Cdc13A. Moreover, the stability of the heterodimer is evidently greater than that of either homodimer. Both the Cdc13 A/A homodimer and A/B heterodimer, but not the B/B homodimer, recognized the telomere G-tail repeat with high affinity and sequence specificity. Our results reveal novel evolutionary elaborations of the G-tail-binding protein in Saccharomycotina yeast, suggesting a drastic remodeling of CDC13 that entails gene duplication, fusion, and functional specialization. The repeated and independent duplication of G-tail-binding proteins such as Cdc13 and Pot1 hints at the evolutionary advantage of having multiple G-tail-binding proteins. 相似文献
75.
Christopher P. Vellano Nicole E. Brown Joe B. Blumer John R. Hepler 《The Journal of biological chemistry》2013,288(5):3620-3631
Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gαi/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gαi1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gαi1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gαi1-GDP but not active Gαi1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gαi1 complex in brain lysates, and co-expression of RGS14 and Gαi1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gαi-linked α2A-adrenergic receptor induces a conformational change in the Gαi1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gαi1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs. 相似文献
76.
Jelena Djokic Christine Fagotto-Kaufmann Rainer Bartels Valentin Nelea Dieter P. Reinhardt 《The Journal of biological chemistry》2013,288(31):22821-22835
Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10–15, ∼20–25, and ∼30–50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate. 相似文献
77.
Sandro Vivona Daniel J. Cipriano Seán O'Leary Ye Henry Li Timothy D. Fenn Axel T. Brunger 《The Journal of biological chemistry》2013,288(34):24984-24991
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism. 相似文献
78.
Monifa Fahie Fabian B. Romano Christina Chisholm Alejandro P. Heuck Mark Zbinden Min Chen 《The Journal of biological chemistry》2013,288(43):31042-31051
Cytolysin A (ClyA) is an α-pore forming toxin from pathogenic Escherichia coli (E. coli) and Salmonella enterica. Here, we report that E. coli ClyA assembles into an oligomeric structure in solution in the absence of either bilayer membranes or detergents at physiological temperature. These oligomers can rearrange to create transmembrane pores when in contact with detergents or biological membranes. Intrinsic fluorescence measurements revealed that oligomers adopted an intermediate state found during the transition between monomer and transmembrane pore. These results indicate that the water-soluble oligomer represents a prepore intermediate state. Furthermore, we show that ClyA does not form transmembrane pores on E. coli lipid membranes. Because ClyA is delivered to the target host cell in an oligomeric conformation within outer membrane vesicles (OMVs), our findings suggest ClyA forms a prepore oligomeric structure independently of the lipid membrane within the OMV. The proposed model for ClyA represents a non-classical pathway to attack eukaryotic host cells. 相似文献
79.
Ucheor B. Choi Rashek Kazi Natalie Stenzoski Lonnie P. Wollmuth Vladimir N. Uversky Mark E. Bowen 《The Journal of biological chemistry》2013,288(31):22506-22515
The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating. 相似文献
80.
Cécile Breyton Ali Flayhan Frank Gabel Mathilde Lethier Grégory Durand Pascale Boulanger Mohamed Chami Christine Ebel 《The Journal of biological chemistry》2013,288(42):30763-30772
Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available. 相似文献