Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted Mr 2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.

Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete

Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C—C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity

While the interactions of cells with polymeric substrata are widely studied, the influence of cell–cell cohesivity on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or “tug-of-war” between cell–cell and cell–substratum adhesions. We have generated both a “library” of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesivity. Cell–substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell–cell cohesivity was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell–cell cohesivity on a series of copolymeric substrata having different levels of cell–substratum adhesivity. In these experiments, cell–cell cohesivity was measured by tissue surface tensiometry, and cell–substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell–cell cohesivity dramatically slowed the spreading rate of cell aggregates.

Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I− polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA

In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3′ end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.

Ubiquitin-mediated degradation of active Src tyrosine kinase

Src family tyrosine kinases are involved in modulating various signal transduction pathways leading to the induction of DNA synthesis and cytoskeletal reorganization in response to cell-cell or cell-matrix adhesion. The critical role of these kinases in regulating cellular signaling pathways requires that their activity be tightly controlled. Src family proteins are regulated through reversible phosphorylation and dephosphorylation events that alter the conformation of the kinase. We have found evidence that Src also is regulated by ubiquitination. Activated forms of Src are less stable than either wild-type or kinase-inactive Src mutants and can be stabilized by proteasome inhibitors. In addition, poly-ubiquitinated forms of active Src have been detected in vivo. Taken together, our results establish ubiquitin-mediated proteolysis as a previously unidentified mechanism for irreversibly attenuating the effects of active Src kinase.

Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones

The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.

X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein

The poly(A)-binding protein (PABP) recognizes the 3′ mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein–protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four α-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells1

Glucose (Glc) starvation of suspension-cultured carrot (Daucus carota L.) cells resulted in sequential activation of phospholipid catabolic enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at the early part of starvation, and these activities were followed by β-oxidation and the glyoxylate cycle enzymes in order. The activity of PLD and lipolytic acyl hydrolase was further confirmed by in vivo-labeling experiments. It was demonstrated that Glc added to a medium containing starving cells inhibited the phospholipid catabolic activities, indicating that phospholipid catabolism is negatively regulated by Glc. There was a burst of ethylene production 6 h after starvation. Ethylene added exogeneously to a Glc-sufficient medium activated PLD, indicating that ethylene acts as an element in the signal transduction pathway leading from Glc depletion to PLD activation. Activation of lipid peroxidation, suggestive of cell death, occurred immediately after the decrease of the phospholipid degradation, suggesting that the observed phospholipid catabolic pathway is part of the metabolic strategies by which cells effectively survive under Glc starvation.

Degradation of CYC1 mRNA in the yeast Saccharomyces cerevisiae does not require translation.

Several studies have indicated that degradation of certain mRNAs is tightly coupled to their translation, whereas, in contrast, other observations suggested that translation can be inhibited without changing the stability of the mRNA. We have addressed this question with the use of altered CYC1 alleles, which encode iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. The cyc1-1249 mRNA, which lacks all in-frame and out-of-frame AUG triplets, was as stable as the normal mRNA. This finding established that translation is not required for the degradation of CYC1 mRNAs. Furthermore, poly(G)18 tracks were introduced within the CYC1 mRNA translated regions to block exonuclease degradation. The recovery of 3' fragments revealed that the translatable and the AUG-deficient mRNAs are both degraded 5'-->3'. Also, the increased stability of CYC1 mRNAs in xrn1-delta strains lacking Xrn1p, the major 5'-->3' exonuclease, established that the normal and AUG-deficient mRNAs are degraded by the same pathway. In addition, deadenylylation, which activates the action of Xrn1p, occurred at equivalent rates in both normal and AUG-deficient mRNAs. We conclude that translation is not required for the normal degradation of CYC1 mRNAs, and that translatable and untranslated mRNAs are degraded by the same pathway.