Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii.
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ABSTRACT:
SUBMITTER: Xue H
PROVIDER: S-EPMC4854336 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.
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Project description:Selenium (Se) deficiency is associated with the occurrence of many diseases. However, excessive Se supplementation, especially with inorganic Se, can result in toxicity. Selenoproteins are the major forms of Se in vivo to exert its biological function. Expression of those selenoproteins, especially with the application of a newly developed system, is thus very important for studying the mechanism of Se in nutrition. The use of Chlamydomonas reinhardtii (C. reinhardtii) as a biological vector to express an heterogeneous protein is still at the initial stages of development. In order to investigate the possibility of using this system to express selenoproteins, human 15-KDa selenoprotein (Sep15), a small but widely distributed selenoprotein in mammals, was chosen for the expression platform test. Apart from the wild-type human Sep15 gene fragment, two Sep15 recombinants were constructed containing Sep15 open reading frame (ORF) and the selenocysteine insertion sequence (SECIS) element from either human Sep15 or C. reinhardtii selenoprotein W1, a highly expressed selenoprotein in this alga. Those Sep15-containing plasmids were transformed into C. reinhardtii CC-849 cells. Results showed that Sep15 fragments were successfully inserted into the nuclear genome and expressed Sep15 protein in the cells. The transgenic and wild-type algae demonstrated similar growth curves in low Se culture medium. To our knowledge, this is the first report on expressing human selenoprotein in green alga.
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Project description:Post-translational modifications (PTMs) are covalent modifications to protein residues which may alter both conformation and activity, thereby modulating signaling and metabolic processes. While PTMs have been largely investigated independently, examination into how different modification interact, or crosstalk, will reveal a more complete understanding of the reciprocity of signaling cascades across numerous pathways. Combinatorial reversible thiol oxidation and phosphorylation in eukaryotes is largely recognized, but rigorous approaches for experimental discovery are underdeveloped. To begin meaningful interrogation of PTM crosstalk in systems biology research, knowledge of targeted proteins must be advanced. Herein, we demonstrate protein-level enrichment of reversibly oxidized proteoforms in Chlamydomonas reinhardtii with subsequent phosphopeptide analysis to determine the extent of phosphorylation in the redox thiol proteome. Label-free quantification was used to quantify 3353 oxidized Cys-sites on 1457 enriched proteins, where sequential phosphopeptide enrichment measured 1094 sites of phosphorylation on 720 proteins with 23% (172 proteins) also identified as reversibly oxidized. Proteins identified with both reversible oxidation and phosphorylation were involved in signaling transduction, ribosome and translation-related machinery, and metabolic pathways. Several redox-modified Calvin-Benson cycle proteins were found phosphorylated and many kinases/phosphatases involved in phosphorylation-dependent photosynthetic state transition and stress-response pathways had sites of reversible oxidation. Identification of redox proteins serves as a crucial element in understanding stress response in photosynthetic organisms and beyond, whereby knowing the ensemble of modifications co-occurring with oxidation highlights novel mechanisms for cellular control.
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2014-04-05 | GSE56505 | GEO