Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.     

A novel haem-binding interface in the 22 kDa haem-binding protein p22HBP

The 22 kDa haem-binding protein, p22HBP, is highly expressed in erythropoietic tissues and binds to a range of metallo- and non-metalloporphyrin molecules with similar affinities, suggesting a role in haem regulation or synthesis. We have determined the three-dimensional solution structure of p22HBP and mapped the porphyrin-binding site, which comprises a number of loops and a alpha-helix all located on a single face of the molecule. The structure of p22HBP is related to the bacterial multi-drug resistance protein BmrR, and is the first protein with this fold to be identified in eukaryotes. Strikingly, the porphyrin-binding site in p22HBP is located in a similar position to the drug-binding site of BmrR. These similarities suggest that the broad ligand specificity observed for both BmrR and p22HBP may result from a conserved ligand interaction mechanism. Taken together, these data suggest that the both the fold and its associated function, that of binding to a broad range of small hydrophobic molecules, are ancient, and have been adapted throughout evolution for a variety of purposes.     

The structure of mixed cholesterol-phospholipid monolayers spread at the air-water interface as probed by interactions with band 3 protein from erythrocyte membranes

Solubilized band 3 protein from human erythrocyte membranes (the anion transport protein) interacts strongly and specifically with monolayers of cholesterol spread at the air-water interface whereas, at pH 7–10, it shows only moderate interactions with phospholipid monolayers (Klappauf, E. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1225–1235). When band 3 protein, at pH 7 and an ionic strength of approx. 100 mM, is added to the subphase of mixed cholesterol-glycerophospholipid monolayers, the changes Δπ in monolayer surface pressure induced by the protein depend on the mole fraction X of sterol in the mixture. However, Δπ(X) only increases with increasing X towards the high values of Δπ that are characteristic of cholesterol monolayers if X>0.67±0.04; at lower cholesterol content, Δπ(X) is practically identical to the value obtained with the pure glycerophospholipid. With mixtures of coprostanol and glycerophospholipids, the break in the Δπ(X) curves occurs when X=0.33±0.03. Cholesterol-sphingomyelin and epicoprostanol-phosphatidylethanolamine mixtures show an increase of Δπ(X) when X>0. The data seem to support earlier claims that cholesterol can form stoichiometric complexes with glycerophospholipids, the stoichiometries revealed by the band 3-monolayer interactions being 2:1 and 1:2. They also show that cholesterol-sphingomyelin complexes, if they should exist, must be structurally different from the cholesterol-glycerophospholipid complexes.     

A basement membrane-like matrix formed by cell-released proteins at the medium/air interface supports growth of keratinocytes

Epithelial cells require adherence to a matrix for regular growth. During standard keratinocyte cell culture in serum-free medium, we observed that cell colonies formed not only on the bottom of the culture vessels but also at the medium/air interface. Coomassie blue staining detected a protein membrane that extended up to several centimeters between the colonies of floating cells. Ultrastructural investigation of this membrane revealed structures closely resembling those of basement membranes, and immunochemical staining confirmed the presence of laminins-1 and -5 as well as collagen IV, representative components of basement membranes. Cells attached to the floating membrane proliferated and could be cultivated for up to six months. When keratinocyte-conditioned medium was filtered and transferred to a culture vessel without cells, the protein membrane at the liquid/air interface formed within one week suggesting self-assembly of cell-released proteins. Our findings provide a basis for the production of epidermal basement membranes for potential medical uses.     

Production and characterization of antibodies against C-terminal peptide of protein F1: A novel phosphorylation at serine 209 of the peptide by protein kinase C

Protein F1 (GAP-43, B-50, neuromodulin, P-57), a neural tissue-specific phosphoprotein enriched in the growth cones of elongating neurites, is suggested to be involved in synaptic plasticity, neuronal development, and neurotransmitter release. In this study, a 21 amino acid polypeptide (AKPKES^* ARQDEGKEDPEADQE) that corresponds to the C-terminus sequence of protein F1 (from position 204–224) was synthesized and used to produce anti-protein F1 antibodies. Immunoblot analysis has demonstrated that the prepared antibodies recognized intact protein F1. Protein F1 and the synthesized F1 peptide were phosphorylated in vitro by PKC. Furthermore, phosphorylated protein F1 was immunoprecipitated by anti-F1 peptide antibodies demonstrating that these antibodies recognized both native, non-phosphorylated and phosphorylated protein. The anti-protein F1 antibodies also stained the plasma membranes of cell bodies and neurities of mouse neuronal cultures obtained from 14-day old spinal embryonic tissue. By contrast, no glial cells were stained. These data suggest that serine 209 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. In addition, antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined all neurites of cultured mouse spinal neurons.Abbreviations used IgG immunoglobulin G - KLH keyhole limpet haemocyanin - OAG L--1-oleoyl-2-acetoyl-sn-3-glycerol - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PKC protein kinase C - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Higher-plant plasma membrane cytochromeb 561: A protein in search of a function

Summary During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducibleb-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochromeb ₅₆₁ (cytb ₅₆₁) according to the wavelength maximum of its -band in the reduced form. More recent evidence suggests that this protein is homologous to ab-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cytb ₅₆₁ is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromesb ₅₆₁ probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromesb ₅₆₁. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cytb ₅₆₁ is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cytb ₅₆₁, compares it to the animal cytb ₅₆₁, and discusses the possible physiological function of these proteins in plants.Abbreviations Asc ascorbate - cyt cytochrome - DHA dehydroascorbate - E₀ standard redox potential - EST expressed sequence tag - His histidine - MDA monodehydroascorbate - Met methionine - PM plasma membrane     

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca²⁺-dependent protien kinase activities as well as a basal (cAMP- and Ca²⁺-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K_m values of cAMP and Ca²⁺ for the membrane-bound protein kinase were 5·10^?8 M and 8.3·10^?4M (in the presence of 1 mM EGTA), respectively. The apparent K_m values of Mg²⁺ were 7·10^?4 M (without cAMP and Ca²⁺, 5·10^?4 M (with cAMP) and 1.3·10^?3 M (with Ca²⁺), and those ATP were 3.5·10^?5 M (with or without cAMP) and 8.5·10^?5 M (with Ca²⁺). The Ca²⁺-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using ³²P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca²⁺, but was sitmulated by a rather broad range (5–25 mM) of Mg²⁺ and Mn²⁺. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca²⁺-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica

Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).     

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk.

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.     

Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca²⁺ (in the absence or presence of calmodulin) or independent of these factors. In these present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.     

The proteinopathy of D169G and K263E mutants at the RNA Recognition Motif (RRM) domain of tar DNA-binding protein (tdp43) causing neurological disorders: A computational study

One of the multitasking proteins, transactive response DNA-binding protein 43 (tdp43) plays a key role in RNA regulation and the two pathogenic mutations such as D169G and K263E, located at the RNA Recognition Motif (RRM) of tdp43, are reported to cause neurological disorders such as Amyotrophic Lateral Sclerosis and FrontoTemporal Lobar Degeneration. As the exploration of the proteinopathy demands both structural and functional characterizations of mutants, a comparative analysis on the wild type and mutant tdp43 (D169G and K263E) and their complexes with RNA has been performed using computational approaches. Molecular dynamics simulations revealed comparatively stable mutant structures compared to wild type tdp43. Both mutants show lesser binding affinity toward RNA molecule when compared to the wild type tdp43. Some of the observed features, including the increased solvent-accessible surface area, conformational flexibility as well as unfolding of tdp43, and the altered RNA conformation in tp43-RNA complex, reveal the susceptibility of these mutants to induce conformational changes in tdp43 for a possible aggregation in the cytoplasm. Particularly, the enhanced aggregation propensity of both mutants also evidences the higher probability of cytoplasmic aggregation of tdp43 mutants. Hence, the present analysis highlighting the structural and functional aspects of wild and mutant tdp43 will form the basis to gain insight into the proteinopathy of tdp43 and the related structure-based drug discovery. Thus, tdp43 can be used as target to develop novel therapeutic approaches or drug designing.     

Yeast protein kinase Ptk2 localizes at the plasma membrane and phosphorylates in vitro the C-terminal peptide of the H-ATPase

Glucose triggers posttranslational modifications that increase the activity of the Saccharomyces cerevisiae plasma membrane H⁺-ATPase (Pma1). Glucose activation of yeast H⁺-ATPase results from the change in two kinetic parameters: an increase in the affinity of the enzyme for ATP, depending on Ser⁸⁹⁹, and an increase in the V_max involving Thr⁹¹². Our previous studies suggested that Ptk2 mediates the Ser⁸⁹⁹-dependent part of the activation. In this study we find that Ptk2 localized to the plasma membrane in a Triton X-100 insoluble fraction. In vitro phosphorylation assays using a recombinant GST-fusion protein comprising 30 C-terminal amino acids of Pma1 suggest that Ser⁸⁹⁹ is phosphorylated by Ptk2. Furthermore, we show that the Ptk2 carboxyl terminus is essential for glucose-dependent Pma1 activation and for the phosphorylation of Ser⁸⁹⁹.     

Role of tyrosine hot‐spot residues at the interface of colicin E9 and immunity protein 9: A comparative free energy simulation study

The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high‐affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot‐spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein–protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr–Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein–protein interactions dominating the free energy change due to the Tyr54Ala mutation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.     

Detection of a Concanavalin A binding protein in the mollicute Spiroplasma citri and purification from the plasma membrane

A protein that binds Concanavalin A (Con A) was detected on Western blots of Spiroplasma citri proteins. Its apparent molecular weight was 84000. It was localized in the plasma membrane. Affinity chromatography on Con A-agarose was used to isolate this protein. The glycosylation inhibitor, tunicamycin, inhibits S. citri growth and seems to block the glycosylation of the Con A-binding protein.     

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

A locally-induced increase in intracellular Ca propagates cell-to-cell in the presence of plasma membrane Ca ATPase inhibitors in non-excitable cells

Intercellular Ca²⁺ waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca²⁺ waves are mediated by gap junctional diffusion of a Ca²⁺ mobilizing messenger such as IP₃. Since Ca²⁺ is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca²⁺ to intercellular Ca²⁺ waves is limited. Here, we report that in the presence of plasma membrane Ca²⁺ ATPase inhibitors, locally-released Ca²⁺ from the flash-photolysis of caged-Ca²⁺ appeared to induce further Ca²⁺ release and were propagated from one cell to another, indicating that Ca²⁺ was self-amplified to mediate intercellular Ca²⁺ waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells.     

A model for the formation, growth, and lysis of clots in quiescent plasma. A comparison between the effects of antithrombin III deficiency and protein C deficiency

A mathematical model comprised of 23 reaction-diffusion equations is used to simulate the biochemical changes and transport of various reactants involved in coagulation and fibrinolysis in quiescent plasma. The growth and lysis of a thrombus, as portrayed by the model equations, is governed by boundary conditions that include the surface concentration of TF-VIIa, the generation of XIa by contact activation (in vitro), and the secretion of tPA due to endothelial activation. We apply the model to two clinically relevant hypercoagulable states, caused by deficiency of either antithrombin III or protein C. These predictions are compared with published experimental data which validate the utility of the developed model under the special case of static conditions. The incorporation of varying hemodynamic conditions in to the current fluid static model remains to be performed.