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Contents
19(10): Pp. 1054 - 1063
Harindarpal S. Gill
[Open Access Plus] |
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NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3 - ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane.
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19(9): Pp. 997 - 1004
Yuko Tagaya, Aya Osaki, Atsuko Miura, Shuichi Okada, Kihachi Ohshima, Koshi Hashimoto, Masanobu Yamada, Tetsurou Satoh, Hiroyuki Shimizu and Masatomo Mori
[Open Access Plus] |
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Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.
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19(6): Pp. 663 - 672
Liz M.B. Teles, Elaine N. Aquino, Anne C.D. Neves, Carlos H.S. Garcia, Peter Roepstorff, Belchor Fontes, Mariana S. Castro and Wagner Fontes
[Open Access Plus] |
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Background: Neutrophils have an impressive array of microbicidal weapons, and in the presence of a pathogen, progress from a quiescent state in the bloodstream to a completely activated state. Failure to regulate this activation, for example, when the blood is flooded with cytokines after severe trauma, causes inappropriate neutrophil activation that paradoxically, is associated with tissue and organ damage. Acidic proteomic maps of quiescent human neutrophils were analyzed and compared to those of activated neutrophils from severe trauma patients. The analysis revealed 114 spots whose measured volumes differed between activated and quiescent neutrophils, with 27 upregulated and 87 downregulated in trauma conditions. Among the identified proteins, grancalcin, S100-A9 and CACNB2 reinforce observed correlations between motility and ion flux, ANXA3, SNAP, FGD1 and Zfyve19 are involved in vesicular transport and exocytosis, and GSTP1, HSPA1 HSPA1L, MAOB, UCH-L5, and PPA1 presented evidence that activated neutrophils may have diminished protection against oxidative damage and are prone to apoptosis. These are discussed, along with proteins involved in cytoskeleton reorganization, reactive oxygen species production, and ion flux. Proteins such as Zfyve19, MAOB and albumin- like protein were described for the first time in the neutrophil. In this work we achieved the identification of several proteins potentially involved in inflammatory signaling after trauma, as well as proteins described for the first time in neutrophils.
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16(11): Pp. 1399 - 1406
Jack Ho Wong and Tzi Bun Ng
[Open Access Plus] |
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An antifungal protein with multiple stable biological activities unaffected by chemical modification of tyrosine and tryptophan, and a protein with N-terminal sequence and molecular mass resemblance to the antifungal protein but no identifiable activities were isolated from culture broth of Bacillus amyloliquefaciens. The findings are analogous to previous reports on thaumatin and thaumatin-like proteins.
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16(9): Pp. 1053 - 1056
Aliaksei Shymanets, Mohammad Reza Ahmadian and Bernd Nurnberg
[Open Access Plus] |
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G-protein βγ dimers are prime regulators transmitting extracellular signals to wide-ranging cellular effectors including phosphoinositide 3-kinase (PI3K) isoforms β and γ. Recombinant Gβ?? purified from Sf9 cells via metal-affinity and anion exchange chromatography exhibited a wortmannin-insensitive phospholipid kinase activity that copurified from the insect cells. To exclude false-positive results of Gβγ-dependent lipid kinase activity, the elimination of insect phospholipid kinase from Gβγ protein samples is necessary to avoid interference with the intrinsic lipid kinase activity of PI3K isoforms in reconstitution experiments. Here we describe an improved procedure of Gβ1γ2 purification from cell membranes that separates the contaminating phospholipid kinase.
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16(7): Pp. 766 - 778
Karen J. Cross, William A. Langley, Rupert J. Russell, John J. Skehel and David A. Steinhauer
[Open Access Plus] |
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Fusion of the influenza virus envelope with the endosomal membrane of host cells is mediated by the hemagglutinin glycoprotein (HA). The most conserved region of HA is at the N-terminus of the HA2 subunit, a relatively hydrophobic sequence of amino acids referred to as the fusion peptide. This domain is critical both for setting the trigger for fusion and for destabilizing target membranes during the fusion process. The “trigger” is set by cleavage of the HA precursor polypeptide, when the newly-generated HA2 N-terminal fusion peptide positions itself into the trimer interior and makes contacts with ionizable residues to generate a fusion competent neutral pH structure. This essentially “primes” the HA such that subsequent acidification of the endosomal environment can induce the irreversible conformational changes that result in membrane fusion. A key component of these acid-induced structural rearrangements involves the extrusion of the fusion peptide from its buried position and its relocation to interact with the target membrane. The role of the fusion peptide for both priming the neutral pH structure and interacting with cellular membranes during the fusion process is discussed.
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14(3): Pp. 265 - 268
Jikui Song, John L. Markley, Jikui Song and John L. Markley
[Open Access Plus] |
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As part of structural investigations of components of the molecular motor, dynein, we prepared the light chain, Robl1_mouse, with and without an N-terminal His-tag. We found that the His-tag introduced a spurious binding site for a second protein, IC74. We propose a molecular mechanism for functional interference by the His-tag.
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