Hagit Zer

Photoinhibition — a historical perspective

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II ‘life cycle.’

UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in Skeletal Muscle?

Background Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. Methodology/Principal Findings We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed α-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and α-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. Conclusions/Significance The interaction of GNE with α-actinin 1 might point to its involvement in α-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of α-actinin, α-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of α-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with α-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of α-actinin 1, and to the muscle-restricted pathology of HIBM.

Light, redox state, thylakoid-protein phosphorylation and signaling gene expression

Two recent publications concerning the chloroplast membrane-protein phosphorylation and state transition might lead to further progress in the elucidation of the mechanism and role of this process. A thylakoid-bound protein TSP9 is released to the chloroplast matrix upon redox-dependent stepwise phosphorylation of three threonine sites and might signal redox-dependent gene transcription. The state-transition process and phosphorylation of the light-harvesting complex II require the activity of a novel protein kinase Stt7.

Deletion of PsbM in Tobacco Alters the QB Site Properties and the Electron Flow within Photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic ΔpsbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in ΔpsbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in ΔpsbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in ΔpsbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher QB/Q –B ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the QA/QB sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the QB site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

A quantitative evaluation of the molecular binding affinity between a monoclonal antibody conjugated to a nanoparticle and an antigen by surface plasmon resonance.

We have designed a site-specific drug colloidal carrier ultimately for improving pancreatic and lung cancer treatment. It is based on a nanoparticulate drug delivery system that targets tumors overexpressing H-ferritin. A monoclonal antibody, AMB8LK, specifically recognizing H-ferritin was thiolated and conjugated to maleimide-activated polylactide nanoparticles (NPs) resulting in the formation of immunonanoparticles (immunoNPs). The AMB8LK immunoNPs exhibited a mean diameter size of 112+/-20nm and a density of 76 antibody molecules per NP. AMB8LK immunoNPs were evaluated for uptake and binding properties on CAPAN-1 and A-549 cell lines, using confocal microscopy. ImmunoNPs demonstrated specific binding and increased uptake of the desired cells by means of monoclonal antibodies (MAbs), compared to nonconjugated NPs. A lipophilic paclitaxel derivative, paclitaxel palmitate (pcpl), was encapsulated within the various NP formulations, and their cytotoxic effect was evaluated on A-549 cells using MTT assay. Pcpl-loaded AMB8LK immunoNPs showed a significantly increased cytotoxic effect when compared to pcpl solution and pcpl NPs. Surface plasmon resonance (SPR) was used to determine quantitatively the affinity constants of native AMB8LK and AMB8LK immunoNPs to gain insight on the affinity of the MAbs following the conjugation process onto NPs. The results of the association/dissociation and affinity kinetics of the interaction between H-ferritin and native AMB8LK or AMB8LK immunoNPs revealed similar constant values, showing that the conjugation process of the MAb to the NPs did not alter the intrinsic specificity and affinity of the MAb to the antigen. In conclusion, at the cellular level, AMB8LK immunoNPs may carry drugs to desired overexpressing antigen cells with adequate affinity properties, potentially leading to improved drug therapy and reduced systemic adverse effects.

Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s)

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31–35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20–100 μmol photons m−2 s−1 as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness.

Inverse correlation between resistance towards copper and towards the redox-cycling compound paraquat: A study in copper-tolerant hepatocytes in tissue culture

The essential mediatory role of copper or iron in the manifestation of paraquat toxicity has been demonstrated (Kohen and Chevion (1985) Free Rad. Res. Commun. 1, 79-88; Korbashi, P. et al. (1986) J. Biol. Chem. 261, 12472-12476). Several liver cell lines, characterized by their resistance to copper, were challenged with paraquat and their cross-resistance to paraquat and copper was studied. Cell growth and survival data showed that copper-resistant cells, containing elevated copper, are more sensitive towards paraquat than wild type cells. Copper-deprived resistant cells did not have this sensitivity. Paraquat was also shown to cause a marked degradation of cellular glutathione in all cell lines. Albeit the fact that the basal glutathione levels are higher in copper-resistant than in wild type cells, there is more paraquat-induced degradation of cellular glutathione (GSH + GSSG) in resistant cells. It is suggested that in copper-resistant cells which contain elevated levels of copper, paraquat-induced cellular injury is potentiated even where glutathione levels are elevated. Additionally, in vitro experiments are presented that support the in vivo findings demonstrating a role for copper in glutathione degradation.

BAF-1 mobility is regulated by environmental stresses.

Barrier to autointegration factor (BAF) is an essential mobile protein that binds lamins, LEM-domain proteins, histones, and DNA. Under environmental stress, BAF becomes immobile. This phenomenon is not shared with other chromatin-binding proteins. The ability of BAF mutants to be immobilized by heat shock in gut cells correlated with normal or increased affinity for emerin.

The hierarchy of transition metal homeostasis: Iron controls manganese accumulation in a unicellular cyanobacterium

Iron and manganese are part of a small group of transition metals required for photosynthetic electron transport. Here, we present evidence for a functional link between iron and manganese homeostasis. In the unicellular cyanobacterium, Synechocystis sp. PCC 6803, Fe and Mn deprivation resulted in distinct modifications of the physiological status. The effect on growth and photosynthetic activity under Fe limitation were more severe than those observed under Mn limitation. Moreover, the intracellular elemental quotas of Fe and Mn were found to be linked. Fe limitation reduced the intracellular Mn quota. Mn limitation did not exert a reciprocal effect on Fe quotas. Microarray analysis comparing Mn and Fe limitation revealed a stark difference in the extent of the transcriptional response to the two limiting conditions, reflective of the physiological responses. The effects of Fe limitation on the transcriptional network are widespread while the effects on Mn limitation are highly specific. Our analysis also revealed an overlap in the transcriptional response of specific Fe and Mn transporters. This overlap provides a framework for explaining Fe limitation induced changes in Mn quotas.

Novel Aspects on the Regulation of Thylakoid Protein Phosphorylation

Thylakoid membrane proteins are phosphorylated by different enzymes, which are subject to different control mechanisms. Activation of the light harvesting complex (LHCII) kinase is signaled by the redox state of plastoquinone and the cytochrome b/f complex and modulated by the thiol reduction state. Phosphorylation of Photosystem II (PS II) proteins may involve kinase(s) associated with the PS II core complex that do not involve the cytochrome b/f complex. Exposure of the phosphoprotein phosphorylation site(s) to protein kinases is regulated by light-induced conformational changes. Thus, thylakoid protein phosphorylation is regulated at both the enzyme and substrate levels. Thylakoid protein dephosphorylation is also under regulatory control, involving interaction between an immunophilin and a membrane-bound phosphatase. The physiological significance of thylakoid protein phosphorylation is not fully understood. Phosphorylation of LHCII is suggested to have a dual role: i) regulation of the LHCII/PSII/PS I interaction, underlying the mechanism of energy transfer balance and ii) prevention of the light-induced aggregation of LHCII or LHCII-PS II complexes. The formation of such macrodomains may affect the dynamics of the thylakoid membrane, which requires unhindered lateral diffusion of integral protein complexes. Phosphorylation of PS II subunits appear to be essential for the repair of photodamage to its reaction center occurring during light stress conditions.