Добірка наукової літератури з теми "Trimers or higher oligomers of RNase A"

Bovine pancreatic RNase A (ribonuclease A) aggregates to form various types of catalytically active oligomers during lyophilization from aqueous acetic acid solutions. Each oligomeric species is present in at least two conformational isomers. The structures of two dimers and one of the two trimers have been solved, while plausible models have been proposed for the structures of a second trimer and two tetrameric conformers. In this review, these structures, as well as the general conditions for RNase A oligomerization, based on the well known 3D (three-dimensional) domain-swapping mechanism, are described and discussed. Attention is also focused on some functional properties of the RNase A oligomers. Their enzymic activities, particularly their ability to degrade double-stranded RNAs and polyadenylate, are summarized and discussed. The same is true for the remarkable antitumour activity of the oligomers, displayed in vitro and in vivo, in contrast with monomeric RNase A, which lacks these activities. The RNase A multimers also show an aspermatogenic action, but lack any detectable embryotoxicity. The fact that both activity against double-stranded RNA and the antitumour action increase with the size of the oligomer suggests that these activities may share a common structural requirement, such as a high number or density of positive charges present on the RNase A oligomers.

ABSTRACT As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.

Analogues of the 2′,5′-linked adenylate trimer 5′-monophosphates, p5′A2′p5′A2′p5′A (pA3) (1a), containing 8-hydroxyadenosine and 8-mercaptoadenosine in the first, second, and third nucleotide positions were tested for their ability to bind to and activate RNase L of mouse L cells. The oligomer, p5′ASH2′p5′ASH2′p5′ASH (pASH3) (1c) had little capacity to bind to RNase L. On the other hand, an analogue of the p5′AOH2′p5′AOH2′p5′AOH (pAOH3) (1b) bound almost as well as the parent 2-5A [pppA(2′p5′A)2] (P3A3) (1d) to RNase L. The 8-substituted analogues of 2-5A were more resistant to degradation by (2′,5′) phosphodiesterase. Finally, the monophosphate, pASH3 (1c) which possessed higher anti-HIV activity than pAg (1a) or pAOH3 (1b).

Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40–50% acetic acid solutions. The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides. Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates. In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer. These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.

An experimental method has been developed which enables the determination of the critical molar mass (Mmc) of ovalbumin oligomers degraded by ultrasound of known frequency. To test the validity of the Mmc postulate, a series of ovalbumin oligomers was prepared by the radiolytic cross-linking of 1% solutions of ovalbumin monomer dissolved in 50mMNa/K-phosphate buffer pH 7.0 saturated withN2O. Under these conditions, irradiation with 5 kGy from a 60 Co source, yielded ovalbumin dimers, trimers, tetramers, and higher order oligomers. On the basis of the results obtained with the ovalbumin oligomers, it was concluded that for ultrasound of 23 kHz frequency and 5mm amplitude, the Mmc was 274000 - 14000 g/mol. Our results confirmed that the two postulates in the chemistry of polymer degradation by ultrasound are valid when ovalbumin oligomers are used as substrates, i.e., (1) that the higher the molar mass of the original macromolecule, the faster is its degradation rate, and (2) that a lower molar mass limit (LMmL) exists below which the macromolecules are resistent to further degradation.

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell–surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell–cell contacts, suggests that such multimeric complexes represent a fusion machine.

The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG–Sepharose and calmodulin–agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.

Human cystatin C (HCC), an amyloidogenic protein, forms dimers and higher oligomers (trimers, tetramers and donut like large oligomers) via a domain-swapping mechanism. The aim of this study was the characterization of the HCC oligomeric states observed within the pH range from 2.2 to 10.0 and also in conditions promoting oligomerization. The HCC oligomeric forms obtained in different conditions were characterized using size exclusion chromatography, dynamic light scattering and small-angle X-ray scattering. The marked ability of HCC to form tetramers at low pH (2.3 or 3.0) and dimers at pH 4.0–5.0 was observed. HCC remains monomeric at pH levels above 6.0. Based on the SAXS data, the structure of the HCC tetramer was proposed. Changes in the environment (from acid to neutral) induced a breakdown of the HCC tetramers to dimers. The tetrameric forms of human cystatin C are formed by the association of the dimers without a domain-swapping mechanism. These observations were confirmed by their dissociation to dimers at pH 7.4.

Abstract VWF is a plasma protein that binds platelets to an injured vascular wall during thrombosis. When exposed to the shear forces found in flowing blood, VWF molecules undergo lateral self-association that results in a meshwork of VWF fibers. Fiber formation has been shown to involve thiol/disulfide exchange between VWF molecules. A C-terminal fragment of VWF was expressed in mammalian cells and examined for unpaired cysteine thiols using tandem mass spectrometry (MS). The VWF C2 domain Cys2431-Cys2453 disulfide bond was shown to be reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers when expressed in mammalian cells. Mutagenesis studies showed that both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulfide bonds were involved in oligomer formation. Our present findings imply that lateral VWF dimers form when a Cys2431 thiolate anion attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond of another VWF molecule, whereas the Cys2451-Cys2468 disulfide/dithiol mediates formation of trimers and higher-order oligomers. These observations provide the basis for exploring defects in lateral VWF association in patients with unexplained hemorrhage or thrombosis.

"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.<br>"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.

Elemental silicon on which the entire technology is based is typically obtained by reduction of the mineral silica with carbon at high temperatures: . . . SiO2 + 2C → Si 2CO (2.1) . . . The silicon is then converted directly to tetrachlorosilane by the reaction . . . Si + 2Cl2 → SiCl4 (2.2) . . Tetrachlorosilane can be used to form an organosilane by the Grignard Reaction . . . SiCl4 + 2 RMgX → R2SiCl2 + 2 MgClX (2.3). . . This relatively complicatreaction has been replaced by the so-called Direct Process or Rochow Process, which starts from elemental silicon as is illustrated by the reaction . . . Si + 2 RCl → R2SiCl2 (2.4) . . . This process also yields RSiCl3 and R3SiCl, which­­ can be removed by distillation. Compounds of formula R2SiCl2 are extremely important as intermediates to a variety of substances having both organic and inorganic character. Hydrolysis gives dihydroxy structures, which can condense to give the basic [–SiR2O–] repeat unit. The nature of the product obtained depends greatly on the reaction conditions. Basic catalysts and higher temperatures favor higher molecular weight linear polymers. Acidic catalysts tend to produce cyclic small molecules or low molecular weight polymers. The hydrolysis approach to polysiloxane synthesis has been largely replaced by ring-opening polymerization of organosilicon cyclic trimers and tetramers, with ionic initiation. These cyclic monomers are produced by the hydrolysis of dimethyldichlorosilane. Under the right conditions, at least 50 wt % of the products are cyclic oligomers. The desired cyclic species are separated from the mixture for use in ring-opening polymerizations such as those described in the following section. In addition, “click” chemistry has been developed for new synthesis techniques in general, and polymerizations in particular. These approaches have been used to prepare polysiloxane elastomers and polydimethylsiloxane (PDMS) copolymers that can function as thermoplastic elastomers. New synthetic strategies for structured silicones, based on B(C6F5)3 have also been developed. Another new approach involves enzymes, such as the lipase enzymatically catalyzed synthesis of silicone aromatic polyesters and silicone aromatic polyamides.