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.

Suspension Trapping (S-Trap) Is Compatible with Typical Protein Extraction Buffers and Detergents for Bottom-Up Proteomics.

The analysis of cells and tissue by bottom-up proteomics starts with lysis, followed by in-solution digestion. Lysis buffers commonly used include detergents and other reagents for achieving efficient protein solubility. However, these reagents are, for the most part, incompatible with downstream analytical instrumentation. One method for in-solution digestion and cleanup, termed suspension trapping (S-Trap), has been recently introduced. We present an evaluation of the compatibility of commonly used lysis buffers with S-Trap: SDS, urea, NP-40, RIPA, and SDS with DTT (SDT). We show that S-Trap is compatible with all of the tested buffers, with SDS and SDT performing the best. On the basis of these data, we anticipate that the method will transform experimental planning for mass-spectrometry-based proteomics, making it far more flexible and tolerable of various lysis buffers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD011665.

Database-independent Protein Sequencing (DiPS) Enables Full-length de Novo Protein and Antibody Sequence Determination.

Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence.

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.

CheY's acetylation sites responsible for generating clockwise flagellar rotation in Escherichia coli.

Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K91 repression is manifested in the conformational dynamics of the ß4α4 loop, shifted toward an active state upon mutation. Removal of ß4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination, we further suggest that K109 acetylation is actively involved in generating clockwise rotation.

MS1-based label-free proteomics using a quadrupole orbitrap mass spectrometer.

Presented is a data set for benchmarking MS1-based label-free quantitative proteomics using a quadrupole orbitrap mass spectrometer. Escherichia coli digest was spiked into a HeLa digest in four different concentrations, simulating protein expression differences in a background of an unchanged complex proteome. The data set provides a unique opportunity to evaluate the proteomic platform (instrumentation and software) in its ability to perform MS1-intensity-based label-free quantification. We show that the presented combination of informatics and instrumentation produces high precision and quantification accuracy. The data were also used to compare different quantitative protein inference methods such as iBAQ and Hi-N. The data can also be used as a resource for development and optimization of proteomics informatics tools, thus the raw data have been deposited to ProteomeXchange with identifier PXD001385.

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.

Large-scale proteomics analysis of five brain regions from Parkinson's disease patients with a GBA1 mutation.

Despite being the second most common neurodegenerative disorder, little is known about Parkinson's disease (PD) pathogenesis. A number of genetic factors predispose towards PD, among them mutations in GBA1, which encodes the lysosomal enzyme acid-ß-glucosidase. We now perform non-targeted, mass spectrometry based quantitative proteomics on five brain regions from PD patients with a GBA1 mutation (PD-GBA) and compare to age- and sex-matched idiopathic PD patients (IPD) and controls. Two proteins were differentially-expressed in all five brain regions whereas significant differences were detected between the brain regions, with changes consistent with loss of dopaminergic signaling in the substantia nigra, and activation of a number of pathways in the cingulate gyrus, including ceramide synthesis. Mitochondrial oxidative phosphorylation was inactivated in PD samples in most brain regions and to a larger extent in PD-GBA. This study provides a comprehensive large-scale proteomics dataset for the study of PD-GBA.

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.

A microwave route for the synthesis of nanoflakes and dendrites-type beta-ln2S3 and their characterization.

In this article, a simple microwave route was applied for the synthesis of nanoflakes and dendrite-type beta-indium sulfide (In2S3) in high yield (> 97%), using a homogeneous mixture of indium(lll)chloride and thiourea in an ethylene glycol (EG)/polyethylene glycol (PEG400) solvent. The reaction was conducted in a simple domestic microwave oven (DMO). Powder X-ray diffraction (XRD), low resolution and high resolution transmission electron microscopy (LRTEM and HRTEM), selected area electron diffraction (SAED), and energy dispersive X-ray spectroscopy (EDS), were applied to investigate the crystallinity, structure, morphology, and composition of the In2S3 nano-materials. Both the as-synthesized and calcined In2S3 products were a body-centered tetragonal (bct) phase, observed by XRD and HRTEM. The length and width of the resulting nanoflakes were in the range of 70-600 nm and 4-10 nm, respectively. The optical band gap of the powder was determined by diffuse reflectance spectroscopy (DRS) and was found to be 2.44 eV. The electronic properties of the products were studied by measuring the optical absorption spectra using photoacoustic spectroscopy. The band gap calculated by this method was found to be 2.52 eV. A possible mechanism for the formation of nanoflakes/dendrites-type In2S3 was also discussed.

CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome.

The highly conserved COP9 signalosome (CSN) complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein). We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

The sonochemical synthesis and characterization of mesoporous chiral titania using a chiral inorganic precursor.

The paper presents a successful sonochemical attempt to synthesize mesoporous chiral titania using a chiral inorganic precursor and dodecylamine, as the surfactant template. The resulting porous structure was characterized by nitrogen sorption experiments, transmission electron microscopy, and small-angle XRD. The enantioselectivity of this mesoporous titania after the extraction of the amine was examined by selective adsorption of enantiomers and racemic aqueous solution of camphor. The selective adsorption was measured by circular dichroism (CD) spectroscopy.