Proteome Analysis of Cytoplasmatic and Plastidic β-Carotene Lipid Droplets in Dunaliella bardawil

The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and β-carotene-rich (βC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in βC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The βC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, β-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of βC-plastoglobuli and the biosynthesis of β-carotene within βC-plastoglobuli and hypothesize that βC-plastoglobuli evolved from eyespot lipid droplets.Lipid droplets are the least characterized organelles in both mammalian and plant cells, and they were considered until a few years ago as passive storage compartments for triglycerides (TAG), sterol esters, and some pigments. However, recent studies have shown that they have diverse metabolic functions (Goodman, 2008; Farese and Walther, 2009; Murphy, 2012). Proteomic analyses in plants and some microalgae have shown that lipid droplets in the cytoplasm and in the chloroplast contain a large diversity of proteins including both structural proteins and many enzymes, indicating that they take an active metabolic role in the synthesis, degradation, and mobilization of glycerolipids, sterols, and pigments as well as in regulatory functions that have not yet been clarified (Schmidt et al., 2006; Ytterberg et al., 2006; Nguyen et al., 2011; Lundquist et al., 2012b; Eugeni Piller et al., 2014). A major limitation for determining the proteomes of lipid droplets, particularly in microalgae, is the purity and the homogeneity of the preparation. Green microalgae, for example, may contain three distinct pools of lipid droplets in one cell: the cytoplasmatic lipid droplets (CLD), the major neutral lipid pool, which are induced under stress conditions such as nitrogen limitation or at the stationary growth phase (Wang et al., 2009); plastoglobules, which are smaller lipid droplets within the chloroplast that have been shown to change in size and number under stress conditions and seem to be involved in stress resistance, metabolite transport, and the regulation of photosynthetic electron transport (Bréhélin et al., 2007; Besagni and Kessler, 2013); and the eyespot structure, part of the visual system in green algae, composed of one or several layers of lipid droplets, characterized by their orange color resulting from a high content of β-carotene (Kreimer, 2009). Disruption of microalgal cells, which is required for the isolation of the lipid droplets, usually involves harsh treatments such as sonication, mixing with glass beads, or use of a French press that breaks not only the cell membrane but also the chloroplast. Therefore, it is almost impossible to separate the different lipid droplet classes by the subsequent density gradient centrifugation, making it difficult to assign the origin of identified proteins. The other major difficulty is contamination by proteins released during cell lysis and fractionation, which associate and copurify with lipid droplets. These include cytoplasmic, chloroplastic, and mitochondrial proteins (Moellering and Benning, 2010; James et al., 2011; Nguyen et al., 2011; Nojima et al., 2013). Purification of isolated lipid droplets from loosely associated proteins is possible by treatments with detergents, high salt, and chaotropic agents (Jolivet et al., 2004; Nguyen et al., 2011); however, the danger in such treatments is that they also remove native loosely associated proteins from the lipid droplets.In this work, we tried to circumvent these problems by choosing a special algal species that is suitable for controlled cell lysis and fractionation and by utilizing two different contamination filters.The alga we selected, Dunaliella bardawil, is unique in that it accumulates large amounts of two different types of lipid droplets, CLD and β-carotene-rich (βC) plastoglobuli, under stress conditions (Davidi et al., 2014). The lack of a rigid cell wall in this alga allows lysis of the plasma membrane by a gentle osmotic shock, releasing CLD but leaving the chloroplast intact (Katz et al., 1995). This enables the recovery of large quantities of the two types of highly purified lipid droplets by differential lysis. In a recent study, we described the isolation and lipid compositions of these two lipid pools and showed that they have similar TAG compositions but different lipid-associated major proteins (Davidi et al., 2014).The high nutritional and pharmacological value of β-carotene for humans has promoted intensive research aimed to clarify its biosynthesis and regulation in plants and also led to attempts to increase β-carotene levels by genetic manipulations in crop plants such as tomato (Solanum lycopersicum; Rosati et al., 2000; Giorio et al., 2007) or by the creation of Golden rice (Oryza sativa; Ye et al., 2000). However, the capacity of plants to store β-carotene is limited, and in this respect, D. bardawil is an exceptional example of an organism that can accumulate large amounts of this pigment, up to 10% of its dry weight. This is enabled by the compartmentation and storage of this lipophilic pigment in specialized plastoglobules. Also, the unusual isomeric composition, consisting of around 50% 9-cis- and 50% all-trans-isomers (Ben-Amotz et al., 1982, 1988), is probably of major importance in this respect, due to the better solubility of the cis-isomer in lipids, which enables the storage of high concentrations exceeding 50% of the lipid droplets. The localization of carotenoid biosynthesis in plants appears to be tissue specific: in green tissues, it takes place in chloroplast membranes, probably within the inner chloroplast envelope membrane (Joyard et al., 2009), whereas in carotenoid-accumulating fruits, such as tomato or bell pepper (Capsicum annuum), it takes place in specialized organelles derived from chromoplasts (Siddique et al., 2006; Barsan et al., 2010). In green microalgae, there are at least two types of carotenoid-accumulating organelles: CLD and eyespot. Algae such as Haematococcus pluvialis and Chlorella zofigiensis accumulate carotenoids within CLD. In H. pluvialis, the major pigment, astaxanthin, is synthesized initially in the chloroplast as β-carotene and then transferred to CLD, where it is oxidized and hydroxylated to astaxanthin (Grünewald et al., 2001). The eyespot, which is composed of one or several layers of small β-carotene-containing lipid droplets, has been shown by proteomic analysis to include part of the β-carotene biosynthesis enzymes, indicating that β-carotene is probably synthesized within these lipid droplets (Schmidt et al., 2006). Similarly, plant chromoplasts also contain carotenoid biosynthesis enzymes (Schmidt et al., 2006; Ytterberg et al., 2006; Schapire et al., 2009). D. bardawil and Dunaliella salina are unique in that they accumulate large amounts of β-carotene within βC-plastoglobuli. A special focus in this work was the identification of the β-carotene biosynthesis machinery in D. bardawil. It is not known if the synthesis takes place inside the lipid βC-plastoglobuli or in chloroplast envelope membranes. Since D. bardawil also contains β-carotene and xanthophylls at the photosynthetic system, it is interesting to know whether the β-carotene that accumulates under stress in βC-plastoglobuli is produced by the constitutive carotenoid biosynthetic pathway or by a different stress-induced enzymatic system.     

Man the fat hunter: the demise of Homo erectus and the emergence of a new hominin lineage in the Middle Pleistocene (ca. 400 kyr) Levant

The worldwide association of H. erectus with elephants is well documented and so is the preference of humans for fat as a source of energy. We show that rather than a matter of preference, H. erectus in the Levant was dependent on both elephants and fat for his survival. The disappearance of elephants from the Levant some 400 kyr ago coincides with the appearance of a new and innovative local cultural complex--the Levantine Acheulo-Yabrudian and, as is evident from teeth recently found in the Acheulo-Yabrudian 400-200 kyr site of Qesem Cave, the replacement of H. erectus by a new hominin. We employ a bio-energetic model to present a hypothesis that the disappearance of the elephants, which created a need to hunt an increased number of smaller and faster animals while maintaining an adequate fat content in the diet, was the evolutionary drive behind the emergence of the lighter, more agile, and cognitively capable hominins. Qesem Cave thus provides a rare opportunity to study the mechanisms that underlie the emergence of our post-erectus ancestors, the fat hunters.     

LRP5 sequence and polymorphisms in the baboon

Background  LRP5 is known to have an important relationship with bone density and a variety of other biological processes. Mapping to human chromosome 11q13.2, LRP5 shows considerable evolutionary conservation. Orthologs of this gene exist in many species, although comparison of human LRP5 with other non-human primates has not been performed until now.
Methods  We reported the complementary DNA (cDNA) sequence and deduced amino acid sequence for baboon LRP5 , and compared the baboon and human sequences. cDNA sequences for 21 baboons were examined to identify single-nucleotide polymorphisms (SNPs).
Results  Sequences of coding regions in human and baboon LRP5 showed 97– 99% homology. Twenty-five SNPs were identified in the coding region of baboon LRP5 .
Conclusion  The observed degree of coding sequence homology in LRP5 led us to expect that the baboon may serve as a useful model for future research into the role(s) of this gene in primate metabolic diseases.     

Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures

Endothelial dysfunction/activation underlies the development of long-term cardiovascular complications and atherosclerosis. The aim of this study was to examine a direct role for exogenous sublethal flux of superoxide on endothelial cell dysfunction. Human umbilical vein endothelial cells (HUVEC) were exposed to superoxide generated by 0.1 mM xanthine and 4 mU/ml xanthine oxidase for 15 min and essential endothelial functions were examined. Superoxide dismutase and/or catalase was used as scavenger for O(2)(-)/H(2)O(2) to determine the key culprit. HUVEC detachment was determined by neutral red uptake and apoptosis by annexin V binding. Inflammation was estimated by IL-8 mRNA expression and cellular adhesion molecules (CAM). eNOS and iNOS message and eNOS protein served as an indirect measure for NO. Procoagulable state was evaluated by estimating the intracellular tissue factor. Activation of endothelial NADPH oxidase was determined by lucigenin chemiluminescence. Sublethal superoxide dose evoked: (1) proinflammatory state manifested by increased IL-8 mRNA expression and CAM on the endothelial surface, (2) HUVEC apoptosis and activated endothelial NADPH oxidase, (3) increase in intracellular tissue factor, and (4) decrease in eNOS mRNA and protein and up-regulation of iNOS mRNA. We conclude that extracellular low flux of superoxide exhibits pleiotropic characteristics, triggering activation/dysfunction of endothelial cells.     

A novel ovary-specific and ovulation-associated variant of epoxide hydrolase 2

Ovulation is a complex process initiated by the surge of the pituitary luteinizing hormone (LH) that provokes the expression of specific genes. We report herein the isolation and characterization of an ovulation-associated, ovary-specific novel isoform of epoxide hydrolase 2 (Ephx2), Ephx2C. This variant is exclusively expressed in the granulosa cells of preovulatory mouse ovarian follicles. The LH-induced expression of Ephx2C is mediated by the protein kinase A and partially by the protein kinase C signaling pathways. The involvement of p38 kinase has also been demonstrated.     

Discovering local structure in gene expression data: the order-preserving submatrix problem.

This paper concerns the discovery of patterns in gene expression matrices, in which each element gives the expression level of a given gene in a given experiment. Most existing methods for pattern discovery in such matrices are based on clustering genes by comparing their expression levels in all experiments, or clustering experiments by comparing their expression levels for all genes. Our work goes beyond such global approaches by looking for local patterns that manifest themselves when we focus simultaneously on a subset G of the genes and a subset T of the experiments. Specifically, we look for order-preserving submatrices (OPSMs), in which the expression levels of all genes induce the same linear ordering of the experiments (we show that the OPSM search problem is NP-hard in the worst case). Such a pattern might arise, for example, if the experiments in T represent distinct stages in the progress of a disease or in a cellular process and the expression levels of all genes in G vary across the stages in the same way. We define a probabilistic model in which an OPSM is hidden within an otherwise random matrix. Guided by this model, we develop an efficient algorithm for finding the hidden OPSM in the random matrix. In data generated according to the model, the algorithm recovers the hidden OPSM with a very high success rate. Application of the methods to breast cancer data seem to reveal significant local patterns.     

p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age‐related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage‐induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)‐κB kinase, leading to decreased cell survival. NF‐κB activation induced TNF‐α secretion and JNK activation to mediate death of senescent cells in a caspase‐ and JNK‐dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.     

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin β-ODAP in grass pea

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme—β-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for β-ODAP production in vivo. The identification of BOS may pave the way toward engineering β-ODAP–free grass pea cultivars, which are safe for human and animal consumption.