Susana Shochat

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.’

Changes in the properties of reaction center II during the initial stages of photoinhibition as revealed by thermoluminescence measurements

Abstract Analysis of the early events occurring during photoinhibition of Chlamydomonas reinhardtii cells by the thermoluminescence technique shows a shift of the B-emission band at 30°C ascribed to charge recombination of S2Q−B to lower temperatures (15–17°C). The appearance of this modified emission band is gradual, affects the whole population of reaction centers and occurs already at relatively low light intensities and short periods of exposure (20–60 min, 300–1000 W · −2). Under these conditions a reduction of only 30–40% occurs in the intensity of the emission band ascribed to charge recombination of S2Q−A. The loss of the S2Q−B response at 30°C is interpreted as a destabilization of this state and seems to correlate with an increase in the value of the intrinsic fluorescence F0 while the reduction in the S2Q−A signal parallels the reduction of the maximal variable fluorescence in the presence of DCMU. Measurements of oxygen flash yield and oscillation under these conditions show that the S-states cycle is not impaired. Following more extensive photoinhibition the B-type signal was completely lost while the S2Q−A band emission persisted and remained at approx. 20% of its initial value. The light intensity, required for the complete shift of the B-emission band at 30°C to 15°–17°C, seems to be sufficient to accelerate the rate of D1 protein synthesis which continues for a while even if the cells are reexposed to low light intensity (recovery). These results indicate that during the initial stage of photoinhibition changes are induced in the reaction center which lead to some alteration of the D1 protein, resulting in a destabilisation of the S2Q−B charge recombination. These events may be connected with the light-dependent turnover of the D1 protein.

Residual Structure in Islet Amyloid Polypeptide Mediates Its Interactions with Soluble Insulin

Islet amyloid polypeptide (IAPP), a 37-amino acid polypeptide hormone of the calcitonin family, is colocalized and cosecreted with insulin in secretory granules of pancreatic islet beta cells. IAPP can assemble into toxic oligomers and amyloid fibrils, a hallmark of type 2 diabetes. Its interactions with insulin in the secretory granules might influence the formation of cytotoxic oligomers and amyloid fibrils. Presented NMR analysis shows that IAPP, free in solution and in complex with insulin, retains elements of residual secondary structure. NMR chemical shifts and (15)N relaxation data as well as 49 ns replica exchange molecular dynamic simulations indicate that the transiently populated helical structure in residues 11-18 is essential for interactions with insulin. These interactions are mediated by salt bridges between positively charged residues Arg11 or Arg18 of rat IAPP and Glu13 of insulin B chain as well as by hydrophobic interactions flanking the salt bridges. The insulin binding region is composed of the same amino acids in amyloidogenic human IAPP and soluble rat IAPP (with the sole exception of His/Arg-18), implying the same binding mode for both hormones. This His/Arg-18 mutation results in reduced affinity binding of human IAPP to insulin in comparison to rat IAPP as it is detected by surface plasmon resonance biosensor analysis. Implications of the described interactions between soluble forms of IAPP and insulin in preventing oligomerization of human IAPP are discussed.

The dichlorophenyldimethylurea-binding site in thylakoids of Chlamydomonas reinhardii. Role of Photosystem II reaction center and phosphorylation of the 32–35 kilodalton polypeptide in the formation of the high-affinity binding site

Abstract Binding of 3H-labeled dichlorophenyldimethylurea (DCMU) to thylakoids from two conditional mutants of Chlamydomonas reinhardii, having normal or altered polypeptide composition, was studied. Membranes could be obtained having polypeptide(s) participating in the formation of Photosystem II (PS II) reaction center (44–54 kDa) and the 32–35 kDa polypeptide involved in herbicide binding, having only one of these polypeptides or none. The 32–35 kDa polypeptide in Chlamydomonas is phosphorylated and its phosphorylation state is affected by membrane-bound kinase and phosphatase. The latter preferentially removes phosphate from the 32–35 kDa polypeptide in vitro. Normal membranes possess approx. 1 DCMU-binding site/600 chlorophyll molecules with a binding constant of 2–6 · 10−8 M (high-affinity site). Alteration of the PS II reaction center, without loss of the 32–35 kDa polypeptide, reduces significantly the number of high-affinity binding sites and increases the value of the binding costant to about 10−7 M (low-affinity site). A similar situation is obtained following in vitro dephosphorylation of the 32 kDa polypeptide. A reduction in the number of high-affinity sites and an increase in the binding constant are also observed in membranes having an active PS II reaction center but depleted of the 32–35 kDa polypeptide. The number of high-affinity sites increases following insertion of the 32–35 kDa polypeptide into such membranes in vivo. It is concluded that the formation of the high-affinity DCMU-binding site requires the presence of a phosphorylated 32–35 kDa polypeptide and a functional organization of the PS II reaction center.

Blood-cell-specific acetylcholinesterase splice variations under changing stimuli

Developmental and trauma‐induced mechanism(s) that modify inflammation and immune responses in blood cells were recently found to be regulated by acetylcholine. Here, we report corresponding blood cell‐specific changes in acetylcholinesterase splice variants. Plasmon resonance and flow cytometry using acetylcholinesterase variant‐specific antibody probes, revealed a progressive increase in myeloid cell fractions expressing the apoptosis‐related acetylcholinesterase‐S variant from newborns to adult controls and post‐delivery mothers. Hematopoietic cell fractions positive for the myeloproliferative acetylcholinesterase‐R variant, were similarly high in post‐partum blood, both intracellular and on the cell surface. Moreover, intracellular acetylcholinesterase‐S protein amounts as reflected by fluorescence intensity measurements remained unchanged in myeloid cells from post‐partum mothers as compared with matched controls. Unlike brain neurons, which over‐express intracellular acetylcholinesterase‐R under stress, lymphocytes from post‐partum mothers presented increased surface acetylcholinesterase‐S and pronounced decreases in both the expression and contents of surface acetylcholinesterase‐R. Peripheral stimuli‐induced modulations in acetylcholine regulation may hence reflect blood cell lineage‐dependent acetylcholinesterase splice variations.

Mechanism of the Light Dependent Turnover of the D1 Protein

The D1 protein is one of the major components of reaction center II (RCII) and contains within one of its membrane intrinsic loops the binding site of Qb (1). In light exposed chloroplasts in vivo, the protein is specifically and continuously degraded and resynthesized (turnover, (2)). The synthesis of D1 appears to be subject to translation control (3). The rate of D1 turnover is light intensity dependent (4), and it has been proposed that the phenomenon of photoinhibition and its recovery are related to the light induced turnover of D1 (5). Thermoluminescence measurements (6) have indicated that the primary light induced damage to RCII is localized at the level of the D1 protein and affects the redox potential of Qb − (7). This phenomenon correlated in time with the light accelerated synthesis of the D1 protein. The D1 protein encoded by the chloroplast psbA gene, is synthesized as a precursor (pD1) by thylakoid bound ribosomes located on the stroma lamellae (8) and appears as a mature protein in grana localized RCII (2). The degradation of D1 can occur in absence of simultaneous pD1 synthesis (9). Experimental results, part of which are presented here, demonstrate that the turnover (7,10) of D1 is a direct result of a light induced conformational change of RCII leading to an irreversible modification of D1 which triggers its degradation. The modified RCII, impaired in its electron flow activity, translocates to the nonappressed stroma domains where it serves as an acceptor for pD1. This shuttle process regulates the synthesis of D1 and accounts for the maintenance of a constant population of functional RCII in light exposed chloroplasts.

Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semi-synthetic phage display library

Current Alzheimers disease therapies suppress acetylcholine hydrolysis by inhibiting acetylcholinesterase (AChE) at cholinergic synapses. However, anticholinesterases promote alternative splicing changing the composition of brain AChE variants. To study this phenomenon we developed monoclonal antibodies to acetylcholinesterase synaptic peptide (ASP), a synthetic peptide with the C-terminal sequence unique to the human synaptic variant AChE-S. Screening of a phage display human antibody library allowed the isolation of single-chain Fv (scFv) antibodies that were highly specific for ASP, and displayed closely related third complementarity determining regions of the variable heavy chain domain (V(H)-CDR3). BIAcore analysis demonstrated dissociation constants at the micromolar range: 1.6 x 10(-6) and 2.0 x 10(-6) M for ASP and the complete AChE-S protein, respectively. The anti-ASP antibodies provide a novel tool for studying the synaptic AChE-S variant, the expression of which is altered in ageing and dementia.

Photoinactivation of photosystem II and degradation of the D 1 protein are reduced in a cytochrome b6/f-less mutant of Chlamydomonas reinhardtii.

Abstract The effect of unoccupancy of the QB site by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QB site are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QB charge recombination, rise in in trinsic fluorescence and loss of variable fluorescence. These parameters relate to functions in volving the reaction center II D1 protein. The light-induced degradation of D1 in the mutant cells was also considerably reduced, with a t 1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D1 protein are related and require occupancy of the QB site by PQ and its light-driven reduction.

Inactivation of Photosystem II and Turnover of the D1-Protein by Light and Heat Stresses

The photosynthetic electron flow activity is sensitive to physiological stress conditions such as light, heat, cold or water stress1,2. Interestingly, photosystem I (PS I) is not as sensitive as photosystem II (PS II), and in most cases, PS I continues to operate normally under conditions in which PS II activity is completely inhibited. As plants are exposed to stress conditions in their natural habitats, the questions arise as to what are the molecular mechanisms of the stress induced inactivation of PS II and whether there are physiological processes in the chloroplasts which may protect the photosynthetic electron flow activity of PS II from the specific damage induced by environmental stresses. Among these, the inactivation of PS II by light and heat stress have been extensively studied using the green alga Chlamydomonas reinhardtii.