A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 muM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.

Background:Courtship behavior plays a critical role in attracting females and reproduction success. However, the effects of exposure to a ubiquitous contaminant di(2-ethylhexyl) phthalate (DEHP) on these behaviors and, in particular, on courtship vocalizations have not been examined.Objective:The effects of adult exposure to DEHP on courtship and mating behaviors and gonadotropic axis and neural mechanisms involved in DEHP-induced effects were analyzed in male mice.Methods:Adult C57BL/6J males were orally exposed to DEHP (0, 0.5, 5, and 50μg/kg/d) for 4 wk. Olfactory preference, ultrasonic vocalizations (USVs), partner preference and mating, as well as locomotor activity and motor coordination, were measured. The kisspeptin system and testosterone levels were analyzed. Proteomic and molecular studies were conducted on the hypothalamic preoptic nucleus, the key region involved in sexual motivation to vocalize and mate.Results:DEHP at 50μg/kg/d reduced the emission of USVs, whereas lower doses changed the ratio of syllable categories. This was associated with diminished sexual interest of female partners toward males exposed to 5 or 50μg/kg/d and increased latency to mate, despite normal olfactory preference. The kisspeptin system and circulating testosterone levels were unaffected. In DEHP-exposed males, proteomic analysis of the preoptic nucleus identified differentially expressed proteins connected to the androgen receptor (AR). Indeed, exposure to 5 or 50μg/kg/d of DEHP induced selective AR downregulation in this nucleus and upstream chemosensory regions. The involvement of AR changes in the observed alterations was further supported by the reduced emission of courtship vocalizations in males with disrupted neural AR expression.Conclusions:These data demonstrate the critical role of neural AR in courtship vocalizations and raises the possibility that the vulnerability of this signaling pathway to exposure to endocrine disrupters may be detrimental for courtship communication and mating in several species. https://doi.org/10.1289/EHP1443

Glutathionylation, the reversible post-translational formation of a mixed disulfide between a cysteine residue and glutathione (GSH), is a crucial mechanism for signal transduction and regulation of protein function. Until now this reversible redox modification was studied mainly in eukaryotic cells. Here we report a large-scale proteomic analysis of glutathionylation in a photosynthetic prokaryote, the model cyanobacterium Synechocystis sp. PCC6803. Treatment of acellular extracts with N,N-biotinyl glutathione disulfide (BioGSSG) induced glutathionylation of numerous proteins, which were subsequently isolated by affinity chromatography on streptavidin columns and identified by nano LC-MS/MS analysis. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and mass spectrometry analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H2 production. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acids metabolism, was confirmed by biochemical studies on the purified recombinant proteins. These results suggest that glutathionylation constitutes a major mechanism of global regulation of the cyanobacterial metabolism under oxidative stress conditions.