Plastid terminal oxidase (PTOX) protects photosystem I and not photosystem II against photoinhibition in Arabidopsis thaliana and Marchantia polymorpha.

The plastid terminal oxidase PTOX controls the oxidation level of the plastoquinone pool in the thylakoid membrane and acts as a safety valve upon abiotic stress, but detailed characterization of its role in protecting the photosynthetic apparatus is limited. Here we used PTOX mutants in two model plants Arabidopsis thaliana and Marchantia polymorpha. In Arabidopsis, lack of PTOX leads to a severe defect in pigmentation, a so-called variegated phenotype, when plants are grown at standard light intensities. We created a green Arabidopsis PTOX mutant expressing the bacterial carotenoid desaturase CRTI and a double mutant in Marchantia lacking both PTOX isoforms, the plant-type and the alga-type PTOX. In both species, lack of PTOX affected the redox state of the plastoquinone pool. Exposure of plants to high light intensity showed in the absence of PTOX higher susceptibility of photosystem I to light-induced damage while photosystem II was more stable compared with the wild type demonstrating that PTOX plays both, a pro-oxidant and an anti-oxidant role in vivo. Our results shed new light on the function of PTOX in the protection of photosystem I and II.

Host prion protein expression levels impact prion tropism for the spleen.

Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism.

Multifaceted Study on a Cytochalasin Scaffold: Lessons on Reactivity, Multidentate Catalysis, and Anticancer Properties.

An intramolecular Diels-Alder (IMDA) reaction efficiently accelerated by Schreiner's thiourea is reported, to build a functionalized cytochalasin scaffold (periconiasin series) for biological purposes. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines while actin polymerization is preserved.

Crystal structure and redox properties of a novel cyanobacterial heme protein with a His/Cys heme axial ligation and a Per-Arnt-Sim (PAS)-like domain.

Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E½, of Tll0287 was -255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E½ increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies.

Divergent prion strain evolution driven by PrPC expression level in transgenic mice.

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.

Crystal structure at 1.5Å resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongatus.

PsbV2 is a c-type cytochrome present in a very low abundance in the thermophilic cyanobacterium Thermosynechococcus elongatus. We purified this cytochrome and solved its crystal structure at a resolution of 1.5Å. The protein existed as a dimer in the crystal, and has an overall structure similar to other c-type cytochromes like Cytc6 and Cytc550, for example. However, the 5th and 6th heme iron axial ligands were found to be His51 and Cys101, respectively, in contrast to the more common bis-His or His/Met ligands found in most cytochromes. Although a few other c-type cytochromes were suggested to have this axial coordination, this is the first crystal structure reported for a c-type heme with this unusual His/Cys axial coordination. Previous spectroscopic characterizations of PsbV2 are discussed in relation to its structural properties.

High and low potential forms of the QA quinone electron acceptor in Photosystem II of Thermosynechococcus elongatus and spinach.

The redox potential of Q(A) in Photosystem II (PSII) from Thermosynechococcus elongatus was titrated monitoring chlorophyll fluorescence. A high potential form (E(m)=+60 ± 25 mV) was found in the absence of Mn(4)Ca, the active site for water oxidation. The low potential form (E(m)=-60 ± 48 mV), which is difficult to measure in conventional titration experiments, could be "locked in" by cross-linking the active enzyme. This indicates that the presence of Mn(4)Ca is relayed to the quinone site by significant structural changes in the protein. The presence of high and low potential forms agrees with what has been seen in plants, algae from our lab and in T. elongatus (Shibamoto et al., Biochemistry 48 (2009) 10682-10684). In the latter work, the potentials of Q(A) were shifted to lower potentials compared to other measurements. The redox potential of Q(A) in Mn-depleted PSII from spinach was titrated in the presence of redox mediators and the midpoint potential was shifted by 80 mV towards a more negative value compared to titrations without mediators. The lower values of the midpoint potential of the (Q(A)/Q(A)(-)) redox couple in the literature could be due to a perturbation due to a specific mediator.

D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy.

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA(3) gene over the psbA(1) gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at <10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q(x) band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo(D1) Q(x) band induced by Q(A)(-) (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t(1/2)>50 ms) Q(A)(-) yield was approximately 95% in D1:3, and approximately 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q(x) band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13(1)-keto of Pheo(D1), which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P(D1), in particular Ser153Ala. Photo-induced Q(A)(-) formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+.

Biosynthetic exchange of bromide for chloride and strontium for calcium in the photosystem II oxygen-evolving enzymes.

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light.

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.

Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy.

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrP(res)) isolated from brains of affected animals or humans. However, alternate PrP(res) signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrP(res) of higher apparent molecular mass--called H-type--into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.

Recovery of a recombinant salmonid alphavirus fully attenuated and protective for rainbow trout.

Sleeping disease virus (SDV) is a member of the new Salmonid alphavirus genus within the Togaviridae family. The single-stranded RNA genome of SDV is 11,894 nucleotides long, excluding the 3' poly(A) tail. A full-length cDNA has been generated; the cDNA was fused to a hammerhead ribozyme sequence at the 5' end and inserted into a transcription plasmid (pcDNA3) backbone, yielding pSDV. By transfection of pSDV into fish cells, recombinant SDV (rSDV) was successfully recovered. Demonstration of the recovery of rSDV was provided by immunofluorescence assay on rSDV-infected cells and by the presence of a genetic tag, a BlpI restriction enzyme site, introduced into the rSDV RNA genome. SDV infectious cDNA was used for two kinds of experiments (i) to evaluate the impact of various targeted mutations in nsP2 on viral replication and (ii) to study the virulence of rSDV in trout. For the latter aspect, when juvenile trout were infected by immersion in a water bath with the wild-type virus-like rSDV, no deaths or signs of disease appeared in fish, although they were readily infected. In contrast, cumulative mortality reached 80% in fish infected with the wild-type SDV (wtSDV). When rSDV-infected fish were challenged with wtSDV 3 and 5 months postinfection, a long-lasting protection was demonstrated. Interestingly, a variant rSDV (rSDV14) adapted to grow at a higher temperature, 14 degrees C instead of 10 degrees C, was shown to become pathogenic for trout. Comparison of the nucleotide sequences of wtSDV, rSDV, and rSDV14 genomes evidenced several amino acid changes, and some changes may be linked to the pathogenicity of SDV in trout.

Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features.

It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.

A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes.

Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrP(c)), into a misfolded form, abnormal PrP (PrP(Sc)), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrP(Sc) detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrP(Sc) molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrP(ARR) allele (A(136)R(154)R(171)), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.