Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood-Brain Barrier In Vitro System.

The mechanisms involved in the interaction of PrP 106-126, a peptide corresponding to the prion protein amyloidogenic region, with the blood-brain barrier (BBB) were studied. PrP 106-126 treatment that was previously shown to impair BBB function, reduced cAMP levels in cultured brain endothelial cells, increased nitric oxide (NO) levels, and changed the activation mode of the small GTPases Rac1 (inactivation) and RhoA (activation). The latter are well established regulators of endothelial barrier properties that act via cytoskeletal elements. Indeed, liquid chromatography-mass spectrometry (LC-MS)-based proteomic profiling study revealed extensive changes in expression of cytoskeleton-related proteins. These results shed light on the nature of the interaction between the prion peptide PrP 106-126 and the BBB and emphasize the importance of the cytoskeleton in endothelium response to prion- induced stress.

SPEAR: A proteomics approach for simultaneous protein expression and redox analysis.

Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows global identification of redox-modified cysteine (Cys) sites and quantification of their reversible oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of reversibly oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys reversible oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo reversible oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplastic proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of the reversible oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.

Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment.

Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of (15)N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organelle-specific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.

Chimeras taking shape: potential functions of proteins encoded by chimeric RNA transcripts.

Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans.