Association between life-span extension by caloric restriction and thiol redox state in two different strains of mice

The hypothesis that the life-extending effect of caloric restriction (CR) is associated with an attenuation of the age-related pro-oxidant shift in the thiol redox state was tested employing a novel experimental design. Amounts of GSH, GSSG, and protein mixed disulfides (Pr-SSG) in the skeletal muscle and liver were compared between two strains of mice that have similar life spans when fed ad libitum (AL), but different life spans under the standard CR regimen. The life span of one strain, C57BL/6, is extended under CR, whereas it remains unaffected in the other strain, DBA/2. Mice were fed AL or 40% less food starting at 4 months and compared at 6 and 24 months of age. The amounts of GSSG and Pr-SSG increased and the GSH:GSSG ratios decreased with age in both strains of AL-fed mice. CR prevented these age-related changes in the C57BL/6, whose life span is extended by CR, but not in the DBA/2 mice, in which it remains unaffected. CR enhanced the activity of glutamate-cysteine ligase in the C57BL/6, but not in the DBA/2 mice. The results suggest that longevity extension by CR may be associated with the attenuation of age-related pro-oxidizing shifts in the thiol redox state.     

An indirect haemagglutination test to detect serum antibodies toGiardia lamblia

We used an indirect haemagglutination test withGiardia lamblia trophozoites as the antigen to detectanti-Giardia lamblia antibodies in serum, the soluble tritonatedGiardia lamblia antigen being used for detecting anti-giardial antibodies in sera of 60 human subjects. Titers in some of these subjects were 1 : 80-1 : 2560. whereas titers in some subjects were negative button to 1 : 20. The results indicated thatGiardia lamblia, an intestinal parasite, induced a systemic antibody response and the indirect haemagglu tination test foranti-Giardia lamblia antibodies is a simple specific and reproducible system which may be useful in epidemiologic and immunologic studies of giardiasis. The specificity of the anti-bodies was demonstrated by the ability of liveGiardia lamblia trophozoites, but not Entamoeba histolytica to absorb the antibody activity.     

Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

分析超速离心研究多聚酸性氨基酸对SnRK2.6的影响

分析超速离心技术是一种通过检测分子在离心场作用下的沉降行为,分析获得其沉降系数、扩散系数、流体力学半径、摩尔质量、结合常数等水力学和热力学性质的方法,被广泛应用于蛋白质分子溶液性质的研究中.本文利用分析超速离心技术研究了拟南芥Sn RK2.6(sucrose non-fermenting1-related protein kinase 2.6)末端一段多聚酸性氨基酸序列对其溶液性质的影响,并将多聚酸性氨基酸序列Sn RK2.6(333~362)及人源PDI(protein disulfide isomerase)(441~491)连接至拟南芥PYL10(PYR like protein 10)分子末端进行分析,同时结合分子排阻层析和静态光散射技术,研究了上述蛋白质分子的分子质量和聚合状态.结果表明,多聚酸性氨基酸序列可以引起蛋白质分子轴长比增加,在溶液中运动时摩擦系数增加,水合半径明显增大,分子排阻层析洗脱体积明显变小.     

Structural classification of small, disulfide-rich protein domains

Disulfide-rich domains are small protein domains whose global folds are stabilized primarily by the formation of disulfide bonds and, to a much lesser extent, by secondary structure and hydrophobic interactions. Disulfide-rich domains perform a wide variety of roles functioning as growth factors, toxins, enzyme inhibitors, hormones, pheromones, allergens, etc. These domains are commonly found both as independent (single-domain) proteins and as domains within larger polypeptides. Here, we present a comprehensive structural classification of approximately 3000 small, disulfide-rich protein domains. We find that these domains can be arranged into 41 fold groups on the basis of structural similarity. Our fold groups, which describe broader structural relationships than existing groupings of these domains, bring together representatives with previously unacknowledged similarities; 18 of the 41 fold groups include domains from several SCOP folds. Within the fold groups, the domains are assembled into families of homologs. We define 98 families of disulfide-rich domains, some of which include newly detected homologs, particularly among knottin-like domains. On the basis of this classification, we have examined cases of convergent and divergent evolution of functions performed by disulfide-rich proteins. Disulfide bonding patterns in these domains are also evaluated. Reducible disulfide bonding patterns are much less frequent, while symmetric disulfide bonding patterns are more common than expected from random considerations. Examples of variations in disulfide bonding patterns found within families and fold groups are discussed.     

G protein-coupled receptor oligomerization for what?

Although the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last decade, under some premises the existence of a supramolecular organization of these receptors begins now to be widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlie this phenomenon remains still unexplained. Recently, a trans-conformational switching model has been proposed as a mechanism allowing direct inhibition of receptor activation. Thus, heterotropic receptor–receptor allosteric regulations are behind the GPCR oligomeric function. Accordingly, we revise here how GPCR oligomerization impinge in several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. Overall, the rationale of receptor oligomerization might lie in the cellular need of sensing complex extracellular signals and to translate into a simple computational mode.     

Large-Scale, One-Step Purification of Oxidized and Reduced Forms of Bovine Brain S100b Protein by HPLC

A rapid and simple method, using a reverse-phase column in a HPLC system, has been developed to purify high yields of both oxidized and reduced S100b proteins from a bovine brain S100 protein mixture. The final proteins were characterized by amino acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ab-sorbance and fluorescence spectroscopy. Both S100b subtypes appeared highly purified and differed only by their oxidation state: all four cysteinyl sulfhydryl groups were free in reduced S100b protein whereas two of them gave disulfide bridges in oxidized S100b protein. The stability of the oxidation state of the two isolated subtypes suggests that the two forms were not in rapid equilibrium and probably coexisted in vivo.     

Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.     

Protein oligomerization through domain swapping: role of inter-molecular interactions and protein concentration

Domain swapping has been shown to be an important mechanism controlling multiprotein assembly and has been suggested recently as a possible mechanism underlying protein aggregation. Understanding oligomerization via domain swapping is therefore of theoretical and practical importance. By using a symmetrized structure-based (Gō) model, we demonstrate that in the free-energy landscape of domain swapping, a large free-energy barrier separates monomeric and domain-swapped dimeric configurations. We investigate the effect of finite monomer concentration, by implementing a new semi-analytical method, which involves computing the second virial coefficient, a thermodynamic indicator of inter-molecular interactions. This method, together with the symmetrized structure-based (Gō) model, minimizes the need for expensive many-protein simulations, providing a convenient framework to investigate concentration effect. Finally, we perform direct simulations of domain-swapped trimer formation, showing that this modeling approach can be used for higher-order oligomers.     

Kinetic stability and crystal structure of the viral capsid protein SHP

SHP, the capsid-stabilizing protein of lambdoid phage 21, is highly resistant against denaturant-induced unfolding. We demonstrate that this high functional stability of SHP is due to a high kinetic stability with a half-life for unfolding of 25 days at zero denaturant, while the thermodynamic stability is not unusually high. Unfolding experiments demonstrated that the trimeric state (also observed in crystals and present on the phage capsid) of SHP is kinetically stable in solution, while the monomer intermediate unfolds very rapidly. We also determined the crystal structure of trimeric SHP at 1.5A resolution, which was compared to that of its functional homolog gpD. This explains how a tight network of H-bonds rigidifies crucial interpenetrating residues, leading to the observed extremely slow trimer dissociation or denaturation. Taken as a whole, our results provide molecular-level insights into natural strategies to achieve kinetic stability by taking advantage of protein oligomerization. Kinetic stability may be especially needed in phage capsids to allow survival in harsh environments.