Computer-aided design of a PDZ domain to recognize new target sequences

PDZ domains are small globular domains that recognize the last 4-7 amino acids at the C-terminus of target proteins. The specificity of the PDZ-ligand recognition is due to side chain-side chain interactions, as well as the positioning of an alpha-helix involved in ligand binding. We have used computer-aided protein design to produce mutant versions of a Class I PDZ domain that bind to novel Class I and Class II target sequences both in vitro and in vivo, thus providing an alternative to primary antibodies in western blotting, affinity chromatography and pull-down experiments. Our results suggest that by combining different backbone templates with computer-aided protein design, PDZ domains could be engineered to specifically recognize a large number of proteins.     

Effect of drought and high solar radiation on 1-aminocyclopropane-1-carboxylic acid and abscisic acid concentrations in Rosmarinus officinalis plants

The endogenous concentrations of ACC and ABA were measured, at predawn and at maximum solar radiation, during a summer drought, and recovery after autumn rainfalls, in rosemary (Rosmarinus officinalis L.), a drought-tolerant species, growing under Mediterranean field conditions. During the summer, plants were subjected to both water deficit and high solar radiation. Plants showed severe reductions in shoot water potential to -3 MPa, which were associated with drastic stomatal closure (73%), a decrease in net photosynthesis, reaching almost zero, and a severe chlorophyll loss (74%). Despite the severity of the stress, plants recovered after the autumn rainfalls. The concentration of ACC was not enhanced by drought, and at predawn these concentrations remained constant at approximately 600 pmol ACC-1 DW throughout the experiment. Thus, ethylene did not regulate the response of rosemary to drought. However, a sharp increase in ACC levels between predawn and midday was observed. This increase was positively correlated to the intensity of the incident solar radiation. ACC levels recorded in June at midday reached 16 000 pmol g DW and in October values of 1000 pmol g-1 DW were observed. In contrast, in drought-stressed plants predawn concentrations of ABA were up to 130-fold those of recovered plants, and the levels of ABA scored at midday were double of those scored at predawn. In conclusion, although drought-stressed rosemary plants showed a relatively moderate ABA accumulation (approximately 500 pmol g-1 DW#, at predawn), it seems to be an essential factor for the regulation of the plant response to stress, thereby enabling a rapid recovery after stress release, although other mechanisms can not be excluded. As drought stress did not induce ACC accumulation, it was concluded that ethylene production was not a major factor in the drought stress resistance of rosemary plants. The increased ACC and ABA concentrations at midday were correlated with day length and light intensity and not with the water status of the plant.     

The Function of Tocopherols and Tocotrienols in Plants

Referee: Dr. Kozi Asada, Department of Biotechnology, Faculty of Engineering, Fukuyama University, Gakuencho 1, Fukuyama 729-0292, Japan Tocopherols and tocotrienols, which differ only in the degree of saturation of their hydrophobic prenyl side chains, are lipid-soluble molecules that have a number of functions in plants. Synthesized from homogentisic acid and isopentenyl diphosphate in the plastid envelope, tocopherols and tocotrienols are essential to maintain membrane integrity. α-Tocopherol is the major form found in green parts of plants, while tocotrienols are mostly found in seeds. These compounds are antioxidants, thus they protect the plant from oxygen toxicity. Tocopherols and tocotrienols scavenge lipid peroxy radicals, thereby preventing the propagation of lipid peroxidation in membranes, and the ensuing products tocopheroxyl and tocotrienoxyl radicals, respectively, are recycled back to tocopherols and tocotrienols by the concerted action of other antioxidants. Furthermore, tocopherols and tocotrienols protect lipids and other membrane components by physically quenching and reacting chemically with singlet oxygen. The scavenging of singlet oxygen by α-tocopherol in chloroplasts results in the formation of, among other products, α -tocopherol quinone, a known contributor to cyclic electron transport in thylakoid membranes, therefore providing photoprotection for chloroplasts. Moreover, given that α-tocopherol increases membrane rigidity, its concentration, together with that of the other membrane components, might be regulated to afford adequate fluidity for membrane function. Furthermore, α-tocopherol may affect intracellular signaling in plant cells. The effects of this compound in intracellular signaling may be either direct, by interacting with key components of the signaling cascade, or indirect, through the prevention of lipid peroxidation or the scavenging of singlet oxygen. In the latter case, α-tocopherol may regulate the concentration of reactive oxygen species and plant hormones, such as jasmonic acid, within the cell, which control both the growth and development of plants, and also plant response to stress.     

C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.     

Werner syndrome protein directly binds to the AAA ATPase p97/VCP in an ATP-dependent fashion

We have previously shown that the Werner syndrome helicase, WRNp, a member of the RecQ helicase family, forms a tight molecular complex with the p97/Valosin containing protein (VCP), a member of the AAA (ATPases associated with diverse cellular activities) family of proteins. This interaction is disrupted by chemical agents that confer DNA damage, suggesting that VCP plays an important role in the signal-dependent release of WRNp from its nucleolar sequestration site. Here, we characterized the structural requirements for interactions between WRNp and VCP and for the nuclear localization of VCP. We discovered that VCP directly binds to the RQC (RecQ conserved) domain of WRNp, which is a highly conserved motif common to the RecQ helicase family. This interaction is ATP-dependent, suggesting that VCP plays a mechanistic role in releasing WRNp from the nucleolus. Immunohistochemical analysis of various VCP domains and mutated proteins expressed in vitro demonstrated that VCP may contain several hierarchical cellular localization motifs within its domain structure.     

Transgenic expression of CTLA-4 controls lymphoproliferation in IL-2-deficient mice

IL-2-deficient mice develop a lymphoproliferative and autoimmune disease characterized by autoimmune hemolytic anemia (AHA) and inflammatory bowel disease. We have previously reported that IL-2 is necessary for optimal up-regulation of CTLA-4, an inducible negative regulator of T cell activation. In this study, we have tested the hypothesis that reduced expression of CTLA-4 in IL-2-deficient T cells contributes to the pathogenesis of disease in IL-2-deficient mice. Expression of CTLA-4 as a transgene completely prevented lymphoaccumulation and AHA in IL-2-deficient mice. The normalization of T cell numbers was due to inhibition of expansion of conventional CD4+CD25- T cells rather than to rescue of the numbers or function of CD4+CD25+ regulatory T cells, suggesting that CTLA-4 expression on conventional T cells plays a role in maintaining normal T cell homeostasis. In addition, the inhibitory effect of the CTLA-4 transgene on T cell expansion was at least in part independent of CD28 expression. Our results suggest that deficient CTLA-4 expression on conventional T cells contributes to the pathophysiology of the lymphoproliferative disease and AHA in IL-2-deficient mice. Thus, restoring CTLA-4 expression in T cells may be an attractive strategy to control clinical autoimmune diseases in which CTLA-4 expression is reduced.     

In vitro characterization of five humanized OKT3 effector function variant antibodies

Orthoclone OKT 3 (mOKT3) is a highly effective agent for the reversal of steroid-resistant renal allograft rejection. However, its wider use has been limited by the development of a human anti-mouse antibody response (HAMA) and by the "cytokine release syndrome" (CRS). CRS has been associated with T cell/monocyte activation and, secondarily, with activation of the complement cascade. These processes are mediated through Abs' Fc regions by their abilities to cross-link T cells and mononuclear cells and to activate complements. To alleviate these problems, a group of five huIgG1- and huIgG4-based OKT3 wild-type antibodies and their corresponding Fc mutants with altered residues at amino acids 234, 235, and 318, reported to be required for FcgammaRI and FcgammaRII binding and complement fixation, were constructed. Characterization of these humanized OKT3 Abs, denoted huOKT3gamma1, huOKT3gamma4, huOKT3gamma1(A(234), A(235)), huOKT3gamma4(A(234), A(235)), and huOKT3gamma1(A(318)), has demonstrated that huOKT3gamma1(A(234), A(235)) and huOKT3gamma4(A(234), A(235)), and have at least a 100-fold reduced binding to FcgammaRI and FcgammaRII. As expected, they are much less potent in the induction of T cell activation and cytokine release, yet retain in vitro immunosuppressive effects as potent as those of mOKT3. Unexpectedly, while huOKT3gamma1(A(318)) did not show any reduction in its ability to bind C1q and to fix a complement, huOKT3gamma1(A(234), A(235)) was completely inactive. The in vitro characteristics of huOKT3gamma1(A(234), A(235)) are consistent with recent in vivo studies, in which this Ab showed greatly reduced HAMA and CRS with the retention of its ability to reverse ongoing graft rejection in man.     

The xanthophyll cycle is induced by light irrespective of water status in field-grown lavender (Lavandula stoechas) plants

The water relations, the photosynthetic capacity and the pigment content of leaves, i.e. chlorophylls, carotenes and xanthophylls, were analysed during the summer drought and recovery after autumn rainfalls in lavender ( Lavandula stoechas L.) plants grown in Mediterranean field conditions. Summer drought caused photoinhibition of photosynthesis and significant decreases in chlorophylls (by ca 75%), β -carotene (by ca 65%), and lutein and neoxanthin (by ca 50%), although their contents remained unaltered between predawn and midday, suggesting a progressive decrease in response to drought. In contrast, the levels of violaxanthin decreased from predawn to midday, giving rise to enhanced formation of zeaxanthin and antheraxanthin in high light. Zeaxanthin and antheraxanthin formation was not induced by water deficit. Although the levels of photosynthetic pigments were severely affected by water deficit, carotenoids decreased less than chlorophylls, which resulted in increased levels of carotenoids per unit of chlorophyll. We conclude that the enhanced formation of zeaxanthin in high light and the increased levels of carotenoids per unit of chlorophyll observed in water-stressed plants may help to avoid photoinhibitory damage to the photosynthetic apparatus.     

Climate change-driven body size shrinking in a social wasp

1. Climate change is expected to produce shifts in species distributions as well as behavioural, life-history, and/or morphological adaptations to find suitable conditions or cope with the altered environment. Most of our knowledge on this issue comes from studies on vertebrates, mainly endotherm species. However, it remains uncertain how small ectotherms, such as insects, respond to increased temperature. 2. This study tested whether climate change over the last 100 years (1904–2013) has affected morphological and functional traits in workers of the social wasp Dolichovespula sylvestris in the Iberian Peninsula. 3. Head width and forewing length, as well as body mass and wing area (assuming no change in shape), decreased over time and with increased mean annual temperature, even when controlling for geographical location and altitude. Interestingly, wing size decreased with a steeper slope compared with body size. If there is no change in wing shape, this would lead to an invariable wing loading (body mass:wing area ratio) over time, with potential consequences on flying ability of more recent (and thus smaller) wasp individuals. 4. These results suggest that recent climate change is leaving morphological signatures in social wasps, increasing the evidence for this phenomenon in insects. The data furthermore suggest that the known efficient thermoregulatory ability of social insect colonies may not successfully buffer the effect of global warming.