Expression of Chlamydomonas reinhardtii chloroplast diacylglycerol acyltransferase 3 is induced by light in concert with triacylglycerol accumulation.

Considerable progress has been made towards the understanding of triacylglycerol (TAG) accumulation in algae. One key aspect is finding conditions that trigger TAG production without reducing cell division. Previously, we identified a soluble diacylglycerol acyltransferase (DGAT), related to plant DGAT3, with heterologous DGAT activity. In this work, we demonstrate that Chlamydomonas reinhardtii DGAT3 localizes to the chloroplast and that its expression is induced by light, in correspondence with TAG accumulation. Dgat3 mRNAs and TAGs increase in both wild-type and starch-deficient cells grown with acetate upon transferring them from dark or low light to higher light levels, albeit affected by the particularities of each strain. The response of dgat3 mRNAs and TAGs to light depends on the pre-existing levels of TAGs, suggesting the existence of a negative regulatory loop in the synthesis pathway, although an effect of TAG turnover cannot be ruled out. Altogether, these results hint towards a possible role of DGAT3 in light-dependent TAG accumulation in C. reinhardtii.

Native Desmodesmus sp. and Chlorella sp. isolated from the Reconquista River display a different binding preference for Cu(II) and Zn(II).

The present work was developed to study the metal removal performance of unicellular algae isolated from the Reconquista River and to evaluate the effect of the presence of more than one metal in the removal process. Thus, native species of unicellular algae were isolated from the highly contaminated Reconquista River. All of the isolates were classified, at genus level, based on their morphological appearance. Nine isolates were screened for their Zn(II) removal capacities. Chlorella sp. RR5 and Desmodesmus sp. RR7 were selected based on their removal performance, and their potential in the remediation of multiple metals was analyzed. Therefore, zinc (Zn(II)), copper (Cu(II)), and chromium (Cr(VI)) removal was evaluated in mono- and multi-metallic solutions. Biosorption capacities were high (0.8-1.8 mmol g-1) for Zn(II) and Cu(II) in mono-metallic solutions. Removal capacities decreased up to 48% in multi-metallic solutions. Interestingly, when multi-metallic systems were considered, each strain showed a metal preference. Chlorella sp. removed better Cu(II) meanwhile Desmodesmus sp. showed a preference for Zn(II). Thus, a metal-binding selectivity in each strain was determined. Chromium (VI) remediation was almost null in the conditions analyzed in this work. Fourier transformation infrared spectroscopy (FT-IR) analysis showed that polysaccharides were the main functional group involved in metal adsorption and, in some cases, also the carboxylates played an important role. Overall, we were able to analyze a new source of algal diversity and perform a metal removal characterization of them, leading to the identification of a metal selectivity based on the characteristics of the tested algal strains.

Reactive Oxygen Species in Emulated Martian Conditions and Their Effect on the Viability of the Unicellular Alga Scenedesmus dimorphus.

Formation of oxygen-based free radicals from photochemical decomposition of hydrogen peroxide (H2O2) on Mars may be a key factor in the potential survival of terrestrial-like organisms on the red planet. Martian conditions that generate reactive oxygen species involve the decomposition of H2O2 at temperatures of around 278 K under relatively high doses of C-band ultraviolet radiation (UVC). This process is further amplified by the presence of iron oxides and perchlorates. Photosynthetic organisms exhibit a number of evolutionary traits that allow them to withstand both oxidative stress and UVC radiation. Here, we examine the effect of free radicals produced by the decomposition of H2O2 under emulated martian conditions on the viability of Scenedesmus dimorphus, a unicellular alga that is resistant to UVC radiation and varying levels of perchlorate and H2O2, both of which are present on Mars. Identification and quantification of free radicals formed under these conditions were performed with Electron Paramagnetic Resonance spectroscopy. These results were correlated with the viability of S. dimorphus, and the formation of oxygen-based free radicals and survival of the alga were found to be strongly dependent on the amount of H2O2 available. For H2O2 amounts close to those present in the rarefied martian environment, the products of these catalytic reactions did not have a significant effect on the algal population growth curve.

Computational Functional Analysis of Lipid Metabolic Enzymes.

The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.

Analysis of triglyceride synthesis unveils a green algal soluble diacylglycerol acyltransferase and provides clues to potential enzymatic components of the chloroplast pathway.

BACKGROUND: Microalgal triglyceride (TAG) synthesis has attracted considerable attention. Particular emphasis has been put towards characterizing the algal homologs of the canonical rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Less work has been done to analyze homologs from a phylogenetic perspective. In this work, we used HMMER iterative profiling and phylogenetic and functional analyses to determine the number and sequence characteristics of algal DGAT and PDAT, as well as related sequences that constitute their corresponding superfamilies. We included most algae with available genomes, as well as representative eukaryotic and prokaryotic species. RESULTS: Amongst our main findings, we identified a novel clade of DGAT1-like proteins exclusive to red algae and glaucophyta and a previously uncharacterized subclade of DGAT2 proteins with an unusual number of transmembrane segments. Our analysis also revealed the existence of a novel DGAT exclusive to green algae with moderate similarity to plant soluble DGAT3. The DGAT3 clade shares a most recent ancestor with a group of uncharacterized proteins from cyanobacteria. Subcellular targeting prediction suggests that most green algal DGAT3 proteins are imported to the chloroplast, evidencing that the green algal chloroplast might have a soluble pathway for the de novo synthesis of TAGs. Heterologous expression of C. reinhardtii DGAT3 produces an increase in the accumulation of TAG, as evidenced by thin layer chromatography. CONCLUSIONS: Our analysis contributes to advance in the knowledge of complex superfamilies involved in lipid metabolism and provides clues to possible enzymatic players of chloroplast TAG synthesis.

The diversity of algal phospholipase D homologs revealed by biocomputational analysis.

Phospholipase D (PLD) participates in the formation of phosphatidic acid, a precursor in glycerolipid biosynthesis and a second messenger. PLDs are part of a superfamily of proteins that hydrolyze phosphodiesters and share a catalytic motif, HxKxxxxD, and hence a mechanism of action. Although HKD-PLDs have been thoroughly characterized in plants, animals and bacteria, very little is known about these enzymes in algae. To fill this gap in knowledge, we performed a biocomputational analysis by means of HMMER iterative profiling, using most eukaryotic algae genomes available. Phylogenetic analysis revealed that algae exhibit very few eukaryotic-type PLDs but possess, instead, many bacteria-like PLDs. Among algae eukaryotic-type PLDs, we identified C2-PLDs and PXPH-like PLDs. In addition, the dinoflagellate Alexandrium tamarense features several proteins phylogenetically related to oomycete PLDs. Our phylogenetic analysis also showed that algae bacteria-like PLDs (proteins with putative PLD activity) fall into five clades, three of which are novel lineages in eukaryotes, composed almost entirely of algae. Specifically, Clade II is almost exclusive to diatoms, whereas Clade I and IV are mainly represented by proteins from prasinophytes. The other two clades are composed of mitochondrial PLDs (Clade V or Mito-PLDs), previously found in mammals, and a subfamily of potentially secreted proteins (Clade III or SP-PLDs), which includes a homolog formerly characterized in rice. In addition, our phylogenetic analysis shows that algae have non-PLD members within the bacteria-like HKD superfamily with putative cardiolipin synthase and phosphatidylserine/phosphatidylglycerophosphate synthase activities. Altogether, our results show that eukaryotic algae possess a moderate number of PLDs that belong to very diverse phylogenetic groups.

Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase.

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.

Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin.

Selected vulnerability of neurons in Huntington's disease suggests that alterations occur in a cellular process that is particularly critical for neuronal function. Supporting this idea, pathogenic Htt (polyQ-Htt) inhibits fast axonal transport (FAT) in various cellular and animal models of Huntington's disease (mouse and squid), but the molecular basis of this effect remains unknown. We found that polyQ-Htt inhibited FAT through a mechanism involving activation of axonal cJun N-terminal kinase (JNK). Accordingly, we observed increased activation of JNK in vivo in cellular and mouse models of Huntington's disease. Additional experiments indicated that the effects of polyQ-Htt on FAT were mediated by neuron-specific JNK3 and not by ubiquitously expressed JNK1, providing a molecular basis for neuron-specific pathology in Huntington's disease. Mass spectrometry identified a residue in the kinesin-1 motor domain that was phosphorylated by JNK3 and this modification reduced kinesin-1 binding to microtubules. These data identify JNK3 as a critical mediator of polyQ-Htt toxicity and provide a molecular basis for polyQ-Htt-induced inhibition of FAT.

Proteomics analysis of human coronary atherosclerotic plaque: a feasibility study of direct tissue proteomics by liquid chromatography and tandem mass spectrometry.

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.

Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts.

Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30-40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties.