Cellular Fatty Acid and Ester Formation by Brewers′ Yeast

The activity of alcohol acetyltransferase, bound to the cell membrane and responsible for the formation of acetate esters, was affected by the fatty acid composition of the cell membrane. When saturated fatty acids, which only slightly inhibit alcohol acetyltransferase activity, were in-corporated into the cell membrane, the enzyme activity and ester formation were only slightly affected. On. the other hand, when unsaturated fatty acids, which strongly inhibit the enzyme activity, accumulated in the cell membrane, ester formation was suppressed with inhibition of the enzyme activity. The mechanism of formation of acetate esters by brewers′ yeast was explained by the alcohol acetyltransferase activity under the influence of the fatty acid composition of the cell membrane.     

Spectroscopic studies on free radical coalescing antioxidants and brain protein cystatin

Cystatins are the inhibitors of thiol proteinases and are ubiquitously present in mammalian system. In brain, they put off unwanted proteolysis and are also involved in several neurodegenerative diseases. In the present study, it was demonstrated that photo-activated HOCl-induced modifications in brain cystatin leading to its inactivation and degradation due to hydroxyl radicals. It has been shown that oxidation of cystatin by ROS in vivo leads to oxidative modification which may direct the damage of this significant protein, as it is so well pronounced in vitro. The interplay between free radicals, antioxidants and co-factors is important in maintaining health, aging and age-related diseases. Body’s endogenous antioxidant systems stabilize free radical-induced oxidative stress by the ingestion of exogenous antioxidants. If the generation of free radicals goes beyond the protective effect of antioxidants, this can cause oxidative damage which accumulates during the life cycle and has been implicated in aging and age-related diseases such as cardiovascular disease, cancer, neurodegenerative disorders and other chronic conditions. Activation of neutrophils in certain diseases (e.g., inflammatory conditions and atherosclerosis) results in the production of highly reactive species, such as OH^? and the release of the enzyme myeloperoxidase. Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. Hypochlorous acid (HOCl) is a potent oxidant formed by myeloperoxidase that causes aggregation of many proteins and damage of proteins by reaction with amino-acid side-chains or backbone cleavage.

Communicated by Ramaswamy H. Sarma     

重组人胱抑素C的原核表达及鉴定

目的构建重组人胱抑素C（cystatinC,CysC）的原核高效表达质粒,诱导表达并纯化获得CysC重组蛋白。方法根据大肠埃希菌编码蛋白的特性设计CysC编码基因序列,人工合成目的基因克隆至pET-22b（＋）表达载体中,测序及酶切鉴定正确后诱导其在大肠埃希菌BL21中表达,所获得的包涵体蛋白经亲和层析纯化后采用SDS—PAGE及Western印迹鉴定。结果酶切结果证实构建的表达质粒结构正确;测序结果显示克隆的基因序列所编码的蛋白与GenBank中的CysC氨基酸序列相符;SDS-PAGE及Western印迹结果证实获得的重组CysC融合蛋白分子量约为16kD,经NP亲和层析纯化获得纯度大于90％的目的蛋白。结论建立了重组人CysC的原核高效表达系统并获得了CysC重组蛋白。     

Val55点突变对鸡胱抑素I66Q结构稳定性影响的分子动力学研究

Val55是鸡胱抑素(Chicken cystatin,cC)铰链环状区的重要位点。本文采用分子动力学模拟的方法研究了V55位点突变对cC典型的淀粉样突变体I66Q结构稳定性的影响情况,并深入探讨了其分子机制。研究表明V55N和V55D对I66Q突变体都有稳定其结构的作用,但V55N的稳定作用更显著。进一步研究发现V55N和V55D对I66Q的这种稳定作用是由于突变后的55位残基与邻近残基形成了较多稳定的氢键,从而增加了自身位点及Loop1、β2-β3的稳定性,并进一步稳定了I66Q的α-螺旋和疏水核心结构。这可能最终阻碍胱抑素淀粉样突变体I66Q结构域交换的发生。     

Inhibitory effects of human cystatin C on plum pox potyvirus proteases

The effect of different protease inhibitors on the proteolytic processing of the plum pox potyvirus (PPV) polyprotein has been analyzed. Human cystatin C, an inhibitor of cysteine proteases, interfered with the outoprocessing of the viral papain-like cysteine protease HCPro. Unexpectedly, it also had an inhibitory effect on the autocatalytic cleavage of the Nla protease which, although it has a Cys residue in its active center, has been described as structurally related to serine proteases. Other protease inhibitors tested had no effect on any of the cleavage events analyzed.     

Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.     

Domain swapping in N-truncated human cystatin C

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.     

Structural comparison between wild-type and P25S human cystatin A by NMR spectroscopy. Does this mutation affect the α-helix conformation ?

The effect of substituting Pro²⁵, located in the α-helical region of the cystatin A structure, with Ser has been studied. The structures of wild type and P25S cystatin A were determined by multidimensional NMR spectroscopy under comparable conditions. These two structures were virtually identical, and the α-helix between Glu¹⁵-Lys³⁰ exists with uninterrupted continuity, with a slight bend at residue 25. In order to characterize the possible substitution effects of Pro²⁵ with Ser on the α-helix, the chemical shifts of the amide nitrogens and protons, the generalized order parameters obtained by the analyses of the ¹⁵N-¹H relaxation data, the amide proton exchange rates, and the NOE networks among the α-helical and surrounding residues were carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the α-helical regions of the wild-type and P25S cystatin A proteins. We therefore conclude that our previous structure of the wild-type cystatin A, in which the α-helix exhibited a sharp kink at Pro²⁵, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the α-helix and the rolled β-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the α-helix in both variants and for the destabilized hydrogen bonding of the α-helical residues that follow Pro²⁵/Ser²⁵, as evidenced by increased amide exchange rates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy‐2 and Icy‐4 cystatin genes. Artificial microRNA lines independently silencing the two drought‐induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay‐green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy‐2 and Icy‐4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap‐1, HvPap‐12, and HvPap‐16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.