Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.     

甾体激素受体超家族的基因调控机制

甾体激素受体超家族是一类基因反式作用因子,对RNA聚合酶Ⅱ转录的某些蛋白质基因和RNA聚合酶Ⅰ转录的核糖体RNA基因均有正或负的转录调节作用.超家族对RNA polⅡ转录的基因调控的机理包括受体激活,相关蛋白解离,磷酸化,同源/异源二聚化,核转位,与正/负激素应答元件及相应转录蛋白作用,最终激活或抑制特异靶基因的转录.甾体激素对RNA polⅠ转录的基因的调节作用以及超家族中的经典受体和孤儿受体非配合的激活机制是目前研究的热点.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

Analysis of inphase interaction pattern in EEG

Inphase interactions among EEG signals recorded using eight electrodes were investigated. The inphase interaction parameters are presented in two ways: (1) matrix form in which the number of inphase interactions are tabulated; and (2) histogram in which the number of inphase interactions are plotted pair-wise between two sites as a function of phase delays in milliseconds. The highest number of interactions occurs between 0 and 8 ms in normal brains. The values of interaction parameters are enhanced by various activities. For example, inphase interaction parameters increase in the motor area in the right hemisphere if the EEG is recorded during repeated left fist clenching. Inphase interactions are drastically altered by the presence of a tumor. We studied the inphase interactions of the EEG of a patient having an occipital tumor. The interaction parameters are greatly diminished in this area, indicating a severe impairment of neuronal communications between both hemispheres in the occipital region. The confidence limits of the changes in inphase interaction parameters during fist clenching are tested statistically using the Student's t test. The test shows that the interaction parameters increase, in general, with 1–5% confidence limits in respective cortex areas as a result of fist clenching.     

A structural model for the chromophore-binding domain of ovine rhodopsin

A putative model for the structure of the relatively independent carboxyl-terminal domain of (rhod)opsin has been developed by use of a combination of several secondary structure prediction methods. The validity of this approach was confirmed by comparing the secondary structure for bacteriorhodopsin as predicted by these methods with its known low resolution structure. The resulting predicted structure agreed well with the experimental data. The model obtained for opsin incorporates two transmembrane α-helical rods linked by an intradiscal loop. Each of the helical sections is interrupted by a short irregular region. One of these includes the lysyl residue to which the chromophore 11-cis retinal is attached. The second non-regular segment, almost opposite the first, contains a cysteinyl and a tryptophanyl residue which may be involved in protein—chromophore interaction. The proposed structure of this whole domain could prove instructive in the elucidation of the primary events of visual transduction.     

Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

Crystal Structures of α-Crystallin Domain Dimers of αB-Crystallin and Hsp20

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised α-crystallin domain from rat Hsp20 and that from human αB-crystallin show that they form homodimers with a shared groove at the interface by extending a β sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the αB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G α-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.     

Adaptive Evolution of p53 Thermodynamic Stability

The thermodynamic stability of a protein plays an important role during evolution and adaptation in order to maintain a folded and active conformation. p53 is a tumour suppressor involved in the regulation of numerous genes. Human p53 has an unusually low thermodynamic stability and is frequently inactivated by oncogenic missense mutations. Here, we examined the thermodynamic and kinetic stability of p53 DNA binding domains from selected invertebrate and vertebrate species by differential scanning calorimetry and equilibrium urea denaturation. There is a correlation in the apparent melting temperature of p53 with the body temperature of homeotherm vertebrates. We found that p53 from these organisms has a half-life for spontaneous unfolding at organismal body temperature of 10-20 min. We also found that p53 from invertebrates has higher stability, bearing more resemblance towards p63 and p73 from humans. Using structure-guided mutagenesis on the human p53 scaffold, we demonstrated that the amino acid changes on the protein surface and in the protein interior lead to the elevated stability of p53 orthologs. We propose a model in which the p53 DNA binding domain has been shaped by the complex interplay of different selective pressures and underwent adaptive evolution leading to pronounced effects on its stability. p53 from vertebrates has evolved to have a low thermodynamic stability and similarly short spontaneous half-life at organismal body temperature, which is related to function.