Role of proteolytic enzymes in the development of amino acid imbalance in wheat endosperm proteins

Three proteases with caseinolytic activity have been isolated from the developing wheat endosperm. Two have been purified. The activity of protease A, the one that appears early in endosperm development, is inhibited by - SH inhibitors. Protease C, the one that appears late in endosperm development, is not affected. Protease A cleaves polyaspartic acid and polyglutamic acid but not polylysine. Protease C, on the other hand, cleaves polylysine but not polyaspartic acid and polyglutamic acid. Protease C degrades lysine-rich proteins isolated from wheat endosperm more efficiently than protease A.     

Hyperacetylated histones facilitate chromatin assembly in vitro.

We have examined the effect of histone acetylation on the in vitro assembly of nucleosomes with DNA and purified histones at physiological ionic strength in the presence of polyglutamic acid. We have found that hyperacetylated histones assemble nucleosomes with greater efficiency, and to a greater extent, than either control or hypoacetylated histones. Assembly reactions were performed over a range of histone to DNA ratios (0.25 to 3.0, w/w) and polyglutamic acid to histone ratios (0 to 1.6, w/w). Although polyglutamic acid may act as a sink to prevent nonspecific histone-DNA interactions, our data suggest that the polyanion primarily facilitates the assembly of nucleosomes by organizing histones into a form that is amenable to deposition.     

Potentiometric titration behavior of a copolymer of glutamic acid and alanine prepared by thermal polycondensation

The dissociation behavior of a copolymer of glutamic acid and alanine was investigated by means of potentiometric titration at various ionic strengths. From the curves of the apparent dissociation constants versus the degree of dissociation, the intrinsic dissociation constant of the carboxyl groups in the copolymer was estimated to be 3.92. This value is different from that of -linked polyglutamic acid and is similar to those of γ-linked polyglutamic acids such as bacterial poly-γ-D-glutamic acid. An analytical method of titration was used to determine the ratio of - and γ-glutamyl residues in the copolymer. From the results of this method and from the intrinsic dissociation constant, it was concluded that the glutamyl residues in this copolymer are mostly or entirely γ-linked.     

Real-time observation of nucleoplasmin-mediated DNA decondensation and condensation reveals its specific functions as a chaperone

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro. We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein–protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level.     

Decondensation of mouse sperm chromatin and reassembly into nucleosomes mediated by polyglutamic acid in vitro

The organization of the mammalian sperm nucleus was probed with staphylococcal nuclease. Although isolated nuclei are resistant to cleavage, following reduction and alkylation, 30% of the sperm DNA could be digested and the remaining DNA had a heterodisperse size distribution. By morphological criteria, a model acidic protein, polyglutamic acid was capable of decondensing purified sperm nuclei that had been reduced and alkylated. The maximal extent of nuclease digestion increased to 85–90%. The subsequent addition of purified, exogenous core histones in 0.1 M NaCl partially reversed this vulnerability to nuclease cleavage such that only 55% of the DNA was digested. Furthermore, analysis of the remaining DNA revealed a nucleosome ladder pattern with unit length repeat of 150 bp. These results strongly suggest that polyglutamic acid can mediate not only decondensation of sperm nuclei but also the assembly of sperm chromatin into nucleosomes.     

The inhibition by strongly acidic polypeptides of the repair of double-stranded DNA breaks induced by the gamma irradiation of Chinese hamster fibroblast cells]

The ability of polypeptides consisted of aspartic and glutamic acids to inhibit the repair and to promote the formation of unrepaired double-strand DNA breaks and chromosomal aberrations in gamma-ray induced Chinese hamster cells was shown. A complete inhibition of the double-strand DNA breaks repair was observed at the concentrations of 20 mu M/l (polyglutamic acid with molecular weight 2000-15,000 daltons) and 100 mu M/l (aspartylglutamic acid with molecular weight 1500-4500 daltons). Both polypeptides were low toxic at the given concentrations.     

Synthesis and anti-influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives

We synthesized polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en analogues on the polyglutamic acid back bone, via a spacer of alkyl ether at the C-7 position. These multivalent conjugates 9 and 10 showed enhancement of antiviral activity against infuenza A virus and more potent efficacy in vivo relative to a monomeric sialidase inhibitor.     

Acidic phospholipids stimulate the autophosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase

The autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase was stimulated by the acidic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol. Other phospholipids (phosphatidylethanolamine, phosphatidylcholine, sphingomyelin), acidic compounds (dextran sulfate, polyglutamic acid, chondroitin sulfate, hyaluronic acid) and calciumcalmodulin were essentially inactive. Sodium dodecyl sulfate also stimulated the catalytic subunit autophosphorylation, but other detergents (Triton X-100 and deoxycholic acid) did not. The combination of phosphatidic acid and sodium dodecyl sulfate was as effective as each agent alone, suggesting similar stimulation mechanisms. The data suggest that acidic membrane phospholipids might have a role in regulating the autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.     

A model chromatin assembly system. Factors affecting nucleosome spacing

Poly[d(A-T)].poly[d(A-T)], when reconstituted with chicken erythrocyte core histones and subsequently incubated with sufficient histone H5 in a solution containing polyglutamic acid, forms structures resembling chromatin. H5 induces nucleosome alignment in about two hours at physiological ionic strength and 37 degrees C. The nucleosome spacing and apparent linker heterogeneity in the assembled nucleoprotein are very similar to those in chicken erythrocyte chromatin. Also, condensed chromatin-like fibers on the polynucleotide can be visualized. The binding of one mole of H5 per mole of core octamer is necessary to generate the physiological nucleosome spacing, which remains constant with the addition of more H5. The nucleosome repeat length is not a function of the core histone to poly[d(A-T)] ratio for values lower than the physiological ratio. With increasing ratios, in excess of the physiological value, nucleosome spacing first becomes non-uniform, and then takes on the close packing limit of approximately 165 base-pairs. In addition to eliminating possible base sequence effects on nucleosome positioning, poly[d(A-T)] allows nucleosomes to slide more readily than does DNA, thereby facilitating alignment. Evidence is presented that polyglutamic acid facilitates the nucleosome spacing activity of histone H5, primarily by keeping the nucleoprotein soluble. This model system should be useful for understanding how different repeat lengths arise in chromatin.     

Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract

Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of 1), Xenopus egg extracts and 2), purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone Nucleosome Assembly Protein (NAP-1) or negatively charged macromolecules such as polyglutamic acid and RNA. The comparison shows that the compaction rates can differ by a factor of up to 1000 for the same amount of histones, depending on the system used and on the presence of histone tails, which can be subjected to post-translational modifications. Reactions with purified reconstitution systems follow a slow and sequential mechanism, compatible with the deposition of one (H3-H4)(2) tetramer followed by two (H2A-H2B) dimers. Addition of the histone chaperone NAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects cannot be obtained with polyglutamic acid or RNA, suggesting that yNAP-1 impact on the reaction cannot simply be explained in terms of charge screening. Faster compaction kinetics and higher packing ratios are reproducibly reached with extracts, indicating a role of additional components present in this system. Data are discussed and models proposed to account for the kinetics obtained in our single-molecule assay.     

Electrophoretic behavior of poly-L-glutamic acid and poly-L-lysine

Two electrophoretic components have been detected on the ascending boundary in unbuffered solutions of polyglutamic acid and polylysine under certain experimental conditions. If the conditions are favorable to aggregation, another maximum, the third in order, is formed on this boundary. We attribute this maximum to the formation of aggregates. The behavior of both boundaries and all components at various pH, concentrations, and temperature of solutions and at different molecular weights of polymers was described.     

Conformational studies of anthrax polypeptide, subtilis polypeptide, and synthetic poly- -L-glutamic acid

Poly-γ-L -glutamic acid has been synthesized by the activated pentachlorophenyl ester polymerization method and the molecule weight of the polymer was found to be 16,000. Comparative conformational studies on the synthetic and on the native polyglutamic acid mbtained from B. anthracis and B. subtilis were carried out using optical rotatory dispersion, circular dichroism, peptide absorption spectrum, and titration data. These results show that poly-γ-glutamic acid does not exhibit any conformational order under the conditions of investigation. At low degrees of ionization, restriction of conformational freedom via random “hypercoiling” of the chain appears likely.     

Conformational analysis of an ionizable polydipeptide

A semi-empirical conformational energy calculation has been performed on the ionizable polydipeptide poly(Glu-Ala). The results indicate that; (1)the ionized polymer assumes the lefthanded extended helix conformation is aqueous solution; (2) the poly(Glu-Ala) extended helix is less stable than that of polyglutamic acid; (3) the unionized polydipeptide will preferentially assume the “β-helical” conformation (isolated 2₁ degenerate helix) in aqueous solution. These conclusions are supported by experiment.     

Are density functional theory predictions of the Raman spectra accurate enough to distinguish conformational transitions during amyloid formation?

We report density functional theory (DFT) calculations of the Raman spectra for hexapepetides of glutamic acid and lysine in three different conformations (α, β and PPII). The wave numbers of amide I, amide II and amide III bands of all three conformations predicted at B3LYP/6-31G and B3LYP/6-31G* are in good agreement with previously reported experimental values of polyglutamic acid and polylysine. Agreement with experiment improves when polarization functions are included in the basis set. Explicit water molecules, H-bonded to the backbone amide groups were found to be absolutely necessary to obtain this agreement. Our results indicate that DFT is a promising tool for assignment of the spectral data on kinetics of conformational changes for peptides during amyloid formation.     

Remodeling of the conformational ensemble of the repeat domain of tau by an aggregation enhancer

Misfolding of the microtubule‐associated protein Tau is a hallmark of Alzheimer disease and several other neurodegenerative disorders. Because of the dynamic nature of the Tau protein, little is known about the changes in Tau structure that occur during misfolding. Here we studied the structural consequences upon binding of the repeat domain of Tau, which plays a key role in pathogenic aggregation, to an aggregation enhancer. By combining NMR experiments with molecular simulations we show that binding of the aggregation enhancer polyglutamic acid remodels the conformational ensemble of Tau. Our study thus provides insight into an early event during misfolding of Tau.     

Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment

Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca(2+) from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca(2+) to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca(2+) binding was investigated by means of a Ca(2+)-sensitive electrode. In all cases, Ca(2+) binds with low affinity; the half-maximum binding constant K(1/2) ranges from 6 to 16 mM. The binding stoichiometry between Ca(2+) ions and carboxylic groups is approximately 1:1; an exception is GARP2, where a binding stoichiometry of approximately 1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca(2+) binding.     

Binding of polyanions to carrier ampholytes in isoelectric focusing.

The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate.     

Primary structure of rat chromogranin A and distribution of its mRNA

The primary structure of rat chromogranin A has been deduced from a rat adrenal cDNA clone. A comparison of rat and bovine chromogranin A reveals similar features: clusters of polyglutamic acid, similar amino acid composition, position of seven of 10 pairs of basic amino acids, identical placement of the only two cysteine residues, a highly conserved N- and C-terminus, and a sequence homologous to porcine pancreastatin 1-49 [(1986) Nature 324, 476-478]. Unique features of rat chromogranin A are an eicosaglutamine sequence and two potential N-linked glycosylation sites. Chromogranin A mRNA is detectable in adrenal medulla, anterior pituitary, cerebral cortex, and hippocampus, as well as tumor cell lines derived from pancreas, pituitary, and adrenal medulla.     

Glutamic acid removal and PHB storage in the activated sludge process under dynamic conditions

Glutamic acid removal in the activated sludge process is studied herein, primarily the formation of storage polymers under dynamic conditions. The activated sludge process was operated by using a sequencing batch reactor (sludge age of 6 d) fed with a synthetic mixture of readily available carbon sources, including glutamic acid. Removal of glutamic acid as the only carbon sources was studied in batch tests, along with oxygen consumption, ammonia uptake-release, and formation of storage polymers. It was found that poly-3-hydroxybutyrate (PHB) was stored and that the storage also occurred simultaneously to biomass growth. PHB storage accounted for 16% of the overall solids that were formed from glutamic acid, as the average value of nine batch tests. Neither other Polyhydroxyalkanoates nor polyglutamic acid were detected. Nuclear magnetic resonance analysis, performed on biomass extracts, allowed us to clarify the main metabolic pathways involved in glutamic acid removal and, in particular, the pathways involved in PHB storage. It was found that glutamic acid enters the Krebs cycle as alpha-ketoglutaric acid and exits to form pyruvic acid and then acetyl-CoA, which is the starting point of PHB production pathway.