Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b₆f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.Desiccation tolerance, the ability to survive absolute water contents down to approximately 0.1 g water g⁻¹ dry weight, is a trait found in some bacteria, algae, fungi, as well as animals and plants. In the plant kingdom, desiccation tolerance is common in ferns, mosses, and most seeds and pollen of flowering plants (angiosperms). Resurrection plants, a diverse group of approximately 300 angiosperm species, possess this trait also in their vegetative tissues. These plants are able to withstand prolonged periods of dehydration and to recover within hours to a few days once water is available. A major and interesting aspect in the study of desiccation tolerance in resurrection plants is how they protect themselves against oxidative damage during dehydration, which is often accompanied by conditions of high irradiance (for review, see Bartels and Hussain, 2011; Farrant and Moore, 2011; Morse et al., 2011).A decrease in water content quickly results in lowered leaf stomatal conductance and, consequently, decreased uptake of CO₂. This hinders and ultimately blocks the Calvin cycle. The light-driven reactions, however, typically continue well after the onset of water deficiency, with intact chlorophyll-protein complexes absorbing light energy. The imbalance between the light reactions and the downward biochemical pathways results in a lack of electron sinks and in the system becoming overenergized. This, in turn, leads to enhanced generation of reactive oxygen species (ROS), which inflict damage onto photosynthetic components as well as onto other chloroplast and cellular constituents. At times, the damage may be severe and lead to irreversible impairment and finally plant death (Dinakar et al., 2012).Resurrection plants minimize such potential ROS damage by shutting down photosynthesis during early stages of dehydration (Farrant, 2000; Farrant et al., 2007). There are two mechanisms whereby this is achieved. In poikilochlorophyllous resurrection plants, chlorophyll, along with photosynthetic protein complexes, are degraded, and thylakoids, the membranes that host the photosynthetic pigment-protein complexes, are dismantled. This straightforward mechanism prevents the formation of ROS, yet it comes at the cost of resynthesizing photosynthetic components de novo upon rehydration. On the other hand, homoiochlorophyllous species retain most of their photosynthetic complement and so must rely on other means to protect themselves from oxidative damage in the desiccated state. Some of these, such as leaf folding or curling, which minimize the exposure of inner leaves and/or of adaxial (upper) leaf surfaces to the light, and the accumulation of anthocyanins in leaf surfaces, which act as sunscreens, and the presence of reflective hairs and waxy cuticles, reduce the overall absorption of radiation and thus protect against photodamage (Sherwin and Farrant, 1998; Farrant, 2000; Bartels and Hussain, 2011; Morse et al., 2011). ROS that are generated are dealt with by antioxidants, ROS scavengers, and in some cases also by anthocyanins and other polyphenols (Moore et al., 2005; Kytridis and Manetas, 2006; Farrant et al., 2007). Nevertheless, all of these mechanisms are insufficient to completely prevent and/or detoxify all ROS that are formed, necessitating additional means to prevent or deal with possible damage that ROS may inflict during dehydration and while desiccated (Dinakar et al., 2012).The major photoprotective mechanism in plants and algae is nonphotochemical quenching (NPQ), in which excess light energy absorbed at the antennae of PSII is dissipated as heat. NPQ has been shown to be active in desiccation-tolerant bryophytes and pteridiophytes (Eickmeier et al., 1993; Oliver, 1996), in homoiochlorophyllous angiosperms (Alamillo and Bartels, 2001; Georgieva et al., 2009; Dinakar and Bartels, 2012; Huang et al., 2012), and during the initial stages of drying in poikilochlorophyllous angiosperms (Beckett et al., 2012). Photoinhibition, when damage to PSII (mainly to its D1 subunit) exceeds the repair capacity, typically under conditions of light stress, is also observed in homoiochlorophyllous resurrection plants (e.g. Georgieva and Maslenkova, 2006). Other ways to avoid ROS-induced damage include the rerouting of reducing equivalents to alternative electron sinks, such as the water-water cycle and/or photorespiration, as well as structural rearrangements of PSII and light-harvesting antenna (LHCII) complexes into energy-dissipating states (for review, see Dekker and Boekema, 2005; Yamamoto et al., 2014). These latter processes, in particular the ones pertaining to possible changes in PSII-LHCII macrostructure, have not yet been characterized in homoiochlorophyllous resurrection plants.To gain insight into the ways homoiochlorophyllous resurrection plants cope with dehydration while retaining most of their photosynthetic apparatus, we combined microscopic, spectroscopic, and biochemical approaches. Investigation of the supramolecular organization of photosynthetic complexes was carried out using cryoscanning electron microscopy (cryo-SEM) of high-pressure frozen, freeze-fractured leaf samples; to our knowledge, this combination of procedures has not been utilized previously to investigate thylakoid membranes within plant tissues.The studies reveal that during dehydration, the density of PSII in grana membranes gradually decreases. Notably, in the dehydrated state, in which photosynthetic activity is halted, PSII complexes are also observed to be arranged into rows and two-dimensional arrays. These arrangements are proposed to represent quenched PSII complexes that likely minimize the generation of ROS during desiccation. Furthermore, we observe inverted hexagonal (H_II) phases in this dry state, and these two structural rearrangements are correlated with the massive accumulation of Suc. Biochemical studies of thylakoid membrane fractions support the finding that the relative level of PSII proteins decreases during dehydration. These analyses also reveal that the level of the cytochrome f subunit of the cytochrome b₆f complex decreases quite dramatically and early during dehydration. This provides evidence for an additional level of regulation that inhibits/shuts down the photosynthetic light reactions during desiccation.     

Cerebral nitric oxide represses choroid plexus NFκB‐dependent gateway activity for leukocyte trafficking

Chronic neuroinflammation is evident in brain aging and neurodegenerative disorders and is often associated with excessive nitric oxide (NO) production within the central nervous system (CNS). Under such conditions, increased NO levels are observed at the choroid plexus (CP), an epithelial layer that forms the blood–cerebrospinal fluid barrier (BCSFB) and serves as a selective gateway for leukocyte entry to the CNS in homeostasis and following injury. Here, we hypothesized that elevated cerebral NO levels interfere with CP gateway activity. We found that induction of leukocyte trafficking determinants by the CP and sequential leukocyte entry to the CSF are dependent on the CP epithelial NFκB/p65 signaling pathway, which was inhibited upon exposure to NO. Examining the CP in 5XFAD transgenic mouse model of Alzheimer''s disease (AD-Tg) revealed impaired ability to mount an NFκB/p65-dependent response. Systemic administration of an NO scavenger in AD-Tg mice alleviated NFκB/p65 suppression at the CP and augmented its gateway activity. Together, our findings identify cerebral NO as a negative regulator of CP gateway activity for immune cell trafficking to the CNS.     

The COP9 signalosome is vital for timely repair of DNA double-strand breaks

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.     

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.     

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.     

Discriminative motif finding for predicting protein subcellular localization

Many methods have been described to predict the subcellular location of proteins from sequence information. However, most of these methods either rely on global sequence properties or use a set of known protein targeting motifs to predict protein localization. Here, we develop and test a novel method that identifies potential targeting motifs using a discriminative approach based on hidden Markov models (discriminative HMMs). These models search for motifs that are present in a compartment but absent in other, nearby, compartments by utilizing an hierarchical structure that mimics the protein sorting mechanism. We show that both discriminative motif finding and the hierarchical structure improve localization prediction on a benchmark data set of yeast proteins. The motifs identified can be mapped to known targeting motifs and they are more conserved than the average protein sequence. Using our motif-based predictions, we can identify potential annotation errors in public databases for the location of some of the proteins. A software implementation and the data set described in this paper are available from http://murphylab.web.cmu.edu/software/2009_TCBB_motif/.     

Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice

Reduced expression of the Indy (I'm Not Dead, Yet) gene in D.?melanogaster and its homolog in C.?elegans prolongs life span and in D.?melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which Indy does this is unknown. Here, we report on the knockout mouse model of the mammalian Indy (mIndy) homolog, SLC13A5. Deletion of mIndy in mice (mINDY(-/-) mice) reduces hepatocellular ATP/ADP ratio, activates hepatic AMPK, induces PGC-1α, inhibits ACC-2, and reduces SREBP-1c levels. This signaling network promotes hepatic mitochondrial biogenesis, lipid oxidation, and energy expenditure and attenuates hepatic de novo lipogenesis. Together, these traits protect mINDY(-/-) mice from the adiposity and insulin resistance that evolve with high-fat feeding and aging. Our studies demonstrate a profound effect of mIndy on mammalian energy metabolism and suggest that mINDY might be a therapeutic target for the treatment of obesity and type 2 diabetes.     

Learning cellular sorting pathways using protein interactions and sequence motifs

Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/.