Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc --> Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.     

Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome

Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.     

Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab

The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy.     

Oxidized proteins: intracellular distribution and recognition by the proteasome

The formation of oxidized proteins is one of the highlights of oxidative stress. In order not to accumulate such proteins have to be degraded. The major proteolytic system responsible for the removal of oxidized proteins is the proteasome. The proteasome is distributed throughout the cytosolic and nuclear compartment of mammalian cells, with high concentrations in the nucleus. On the other hand a major part of protein oxidation is taking place in the cytosol. The present review highlights the current knowledge on the intracellular distribution of oxidized proteins and put it into contrast with the concentration and distribution of the proteasome.     

Degradation of HNE-modified proteins--possible role of ubiquitin

4-Hydroxynonenal (HNE) is a lipid peroxidation product that is able to modify proteins. HNE-modified proteins are degraded to a considerable extend by the proteasomal system. It is unclear whether the recognition of HNE-modified proteins is mediated by ubiquitin, or whether the ubiquitin-independent proteasomal pathway is involved. In this study we demonstrate that HNE-modified GAPDH is preferentially ubiquitinated in vitro. In an attempt to demonstrate the formation of poly-ubiquitinated HNE-modified proteins in living cells we explored E36 fibroblasts. A clear rise in HNE-protein modification could be demonstrated after HNE treatment of the cells. Using inhibitors, we could show that the ubiquitin-dependent, ubiquitin-independent, and the lysosomal pathways affect the presence of HNE-modified proteins. We conclude that, although several proteolytic pathways exist for the degradation of HNE-modified proteins, there is the possibility of involvement of ubiquitin-dependent degradation.     

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population

Dinoflagellates are a major cause of harmful algal blooms (HABs), with consequences for coastal marine ecosystem functioning and services. Alexandrium fundyense (previously Alexandrium tamarense) is one of the most abundant and widespread toxigenic species in the temperate Northern and Southern Hemisphere and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. fundyense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. fundyense strains and a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4 + Alex5 and Alex2 + Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious HAB species.     

Characterizing a hybrid zone between a cryptic species pair of freshwater snails

Characterizing hybrid zones and their dynamics is a central goal in evolutionary biology, but this is particularly challenging for morphologically cryptic species. The lack of conspicuous divergence between parental types means intermediate hybrid forms often go undetected. We aimed to detect and characterize a suspected hybrid zone between a pair of morphologically cryptic lineages of the freshwater snail, Radix. We sampled Radix from across a contact zone between two mitochondrial lineages (Radix balthica and an undescribed lineage termed ‘MOTU3’) and detected admixture between two nuclear genotype clusters, which were significantly but not categorically associated with the mitochondrial lineages. Using a model selection approach, we show that the admixture cline is best explained by an interaction between precipitation and temperature gradients over the area, rather than geographic distance. We thus hypothesize that the correlation with climatic gradients suggests environmental selection has played a role in maintaining the hybrid zone. In a 2050 climate change scenario, we furthermore predict an expansion of one of the nuclear clusters and a widening of the hybrid zone as the climate warms and dries.     

The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l^−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

The biodiversity‐dependent ecosystem service debt

Habitat destruction is driving biodiversity loss in remaining ecosystems, and ecosystem functioning and services often directly depend on biodiversity. Thus, biodiversity loss is likely creating an ecosystem service debt: a gradual loss of biodiversity‐dependent benefits that people obtain from remaining fragments of natural ecosystems. Here, we develop an approach for quantifying ecosystem service debts, and illustrate its use to estimate how one anthropogenic driver, habitat destruction, could indirectly diminish one ecosystem service, carbon storage, by creating an extinction debt. We estimate that c. 2–21 Pg C could be gradually emitted globally in remaining ecosystem fragments because of plant species loss caused by nearby habitat destruction. The wide range for this estimate reflects substantial uncertainties in how many plant species will be lost, how much species loss will impact ecosystem functioning and whether plant species loss will decrease soil carbon. Our exploratory analysis suggests that biodiversity‐dependent ecosystem service debts can be globally substantial, even when locally small, if they occur diffusely across vast areas of remaining ecosystems. There is substantial value in conserving not only the quantity (area), but also the quality (biodiversity) of natural ecosystems for the sustainable provision of ecosystem services.