Extracellular vesicles (EVs) act as key mediators of intercellular communication and are secreted and taken up by all cell types in the central nervous system (CNS). While detailed study of EV-based signaling is likely to significantly advance our understanding of human neurobiology, the technical challenges of isolating EVs from CNS tissues have limited their characterization using 'omics' technologies. We therefore developed a new Protein Organic Solvent Precipitation (PROSPR) method that can efficiently isolate the EV repertoire from human biological samples.

Aggregation of malformed proteins is a key feature of many neurodegenerative diseases, but the mechanisms that drive proteinopathy in the brain are poorly understood. We aimed to characterize aggregated proteins in human brain tissues affected by dementia.

Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.

Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves 'fuzzy' processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.

Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.

Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown.

Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated.

Neutrophil extracellular traps (NETs) consist of a decondensed DNA scaffold decorated with neutrophil-derived proteins. The proteome of NETs, or "NETome," has been largely elucidated in vitro. However, components such as plasma and extracellular matrix proteins may affect the NETome under physiological conditions. Here, using a reductionistic approach, we explored the effects of two proteases active during injury and wounding, human thrombin and plasmin, on the NETome. Using high-resolution mass spectrometry, we identified a total of 164 proteins, including those previously not described in NETs. The serine proteases, particularly thrombin, were also found to interact with DNA and bound to NETs in vitro. Among the most abundant proteins were those identified previously, including histones, neutrophil elastase, and antimicrobial proteins. We observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels upon the addition of the two proteases. Analyses of NET-derived tryptic peptides identified subtle changes upon protease treatments. Our results provide evidence that exogenous proteases, present during wounding and inflammation, influence the NETome. Taken together, regulation of NETs and their proteins under different physiological conditions may affect their roles in infection, inflammation, and the host response.

Vascular dementia (VaD) is a leading cause of dementia in the elderly together with Alzheimer's disease with limited treatment options. Poor understanding of the pathophysiology underlying VaD is hindering the development of new therapies. Hence, to unravel its underlying molecular pathology, an iTRAQ-2D-LC-MS/MS strategy was used for quantitative analysis of pooled lysates from Brodmann area 21 of pathologically confirmed cases of VaD and matched non-neurological controls. A total of 144 differentially expressed proteins out of 2281 confidently identified proteins (false discovery rate=0.3%) were shortlisted for bioinformatics analysis. Western blot analysis of selected proteins using samples from individual patients (n=10 per group) showed statistically significant increases in the abundance of SOD1 and NCAM and reduced ATP5A in VaD. This suggested a state of hypometabolism and vascular insufficiency along with an inflammatory condition during VaD. Elevation of SOD1 and increasing trend for iron-storage proteins (FTL, FTH1) may be indicative of an oxidative imbalance that is accompanied by an aberrant iron metabolism. The synaptic proteins did not exhibit a generalized decrease in abundance (e.g. syntaxin) in the VaD subjects. This reported proteome offers a reference data set for future basic or translational studies on VaD.

Lewy body dementias are the second most common cause of neurodegenerative dementia in the elderly after Alzheimer's disease (AD). The two clinical subgroups of Lewy body dementias, namely, dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), are differentiated by the chronology of cognitive symptoms relative to parkinsonism. At present, there remains a debate on whether DLB and PDD are separate disease entities, or fall within the same spectrum of Lewy body dementias. In this study, we compared the detergent-soluble proteome via an 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) analysis of pooled lysates from the prefrontal cortex (BA9) of DLB (n = 19) and PDD (n = 21) patients matched a priori for amyloid (total Aβ42) burden, semi-quantitative scores for Lewy bodies and neurofibrillary tangles together with age-matched control (n = 21) subjects. A total of 1914 proteins were confidently identified by iTRAQ (false discovery rate = 0%). None of the proteins showed a significant yet opposite regulation in between DLB and PDD when compared to aged controls in the proteomic data set as well as following immunoblot analysis of the pooled and individual lysates involving all 61 subjects. The postsynaptic protein, synaptopodin (SYNPO) was significantly down-regulated in both DLB and PDD subgroups, suggesting a defective synaptic transmission in the demented patients. In conclusion, the largely similar proteome of DLB and PDD matched for amyloid burden suggests that variations in concomitant AD-related pathology, abnormal post-translational modifications or protein-protein interactions, defective intracellular trafficking or misfolding of proteins could play a part in driving the clinically observed differences between these two subgroups of Lewy body dementias. This further indicates that amyloid-targeting therapeutic strategies may show different efficacies in DLB versus PDD.

Brain degenerative protein modifications (DPMs) are associated with the apparition and progression of dementia, and at the same time, Alzheimer's disease with cerebrovascular disease (AD + CVD) is the most prevalent form of dementia in the elder population. Thus, understanding the role(s) of brain DPMs in this dementia subtype may provide novel insight on the disease pathogenesis and may aid on the development of novel diagnostic and therapeutic tools. Two essential DPMs known to promote inflammation in several human diseases are the ureido DPMs (uDPMs) arginine citrullination and lysine carbamylation, although they have distinct enzymatic and non-enzymatic origins, respectively. Nevertheless, the implication of uDPMs in the neuropathology of dementia remains poorly understood.

Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFβ (transforming growth factor β) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin-proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy-lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.

Malaria causes every year over half-a-million deaths. The emergence of parasites resistant to available treatments makes the identification of new targets and their inhibitors an urgent task for the development of novel anti-malaria drugs. Protein kinase CK2 is an evolutionary-conserved eukaryotic serine/threonine protein kinase that in Plasmodium falciparum (PfCK2) has been characterized as a promising target for chemotherapeutic intervention against malaria. Here we report a crystallographic structure of the catalytic domain of PfCK2α (D179S inactive single mutant) in complex with ATP at a resolution of 3.0 Å. Compared to the human enzyme, the structure reveals a subtly altered ATP binding pocket comprising five substitutions in the vicinity of the adenine base, that together with potential allosteric sites, could be exploited to design novel inhibitors specifically targeting the Plasmodium enzyme. We provide evidence for the dual autophosphorylation of residues Thr63 and Tyr30 of PfCK2. We also show that CX4945, a human CK2 inhibitor in clinical trials against solid tumor cancers, is effective against PfCK2 with an IC50 of 13.2 nM.

Here, we provide data related to the research article entitled "Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes" by Adav et al. (J. Proteomics (2015) [1]). Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). The data presented here represents the detailed comparative abundances of diverse groups of biomass hydrolyzing enzymes including cellulases, hemicellulases, lignin degrading enzymes, and peptidases and proteases; and their post translational modification like deamidation.

Apart from the skin surface, hair represents a significant tissue component with a capacity of bacterial interactions. New information can be obtained about hair function through the characterization of bacterial adherence, colonization, and responses to hair shafts per se. In this proof-of-principle study, we examine the growth kinetics of Gram-positive Staphylococcus aureus and Staphylococcus epidermidis, and Gram-negative Pseudomonas aeruginosa and Escherichia coli in the presence of human hair shafts. We explore the ability of these bacteria to adhere to and colonize hair shaft surfaces, as well as the resulting impact on the hair's surface morphology. We show that hair shafts inhibit the growth of Gram-positive S. aureus and S. epidermidis, while the growth kinetics of P. aeruginosa and E. coli remain unaffected. Scanning electron microscope analysis and steeping studies show that P. aeruginosa and E. coli to adhere to and colonize on human hair shafts without significantly affecting the hair shaft's surface morphology. P. aeruginosa produced a substantial amount of biofilm on the hair shaft surfaces, while E. coli specifically inhabited the edges of the cuticle scales. Taken together, our results demonstrate differences in bacterial responses to human hair shafts, which may provide novel insights into hair and scalp health.

The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies.

Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire 'gain-of-function' isoDGR motifs that might play a role in atherosclerotic pathology.

Metabolic disorders in T2DM generate multiple sources of free radicals and oxidative stress that accelerate nonenzymatic degenerative protein modifications (DPMs) such as protein oxidation, disrupt redox signaling and physiological function, and remain a major risk factor for clinical diabetic vascular complications. In order to identify potential oxidative biomarkers in the blood plasma of patients with T2DM, we used LC-MS/MS-based proteomics to profile plasma samples from patients with T2DM and healthy controls. The results showed that human serum albumin (HSA) is damaged by irreversible cysteine trioxidation, which can be a potential oxidative stress biomarker for the early diagnosis of T2DM. The quantitative detection of site-specific thiol trioxidation is technically challenging; thus, we developed a sensitive and selective LC-MS/MS workflow that has been used to discover and quantify three unique thiol-trioxidized HSA peptides, ALVLIAFAQYLQQC(SO3H)PFEDHVK (m/z 1241.13), YIC(SO3H)ENQDSISSK (m/z 717.80) and RPC(SO3H)FSALEVDETYVPK (m/z 951.45), in 16 individual samples of healthy controls (n = 8) and individuals with diabetes (n = 8). Targeted quantitative analysis using multiple reaction monitoring mass spectrometry revealed impairment of the peptides with m/z 1241.13, m/z 717.80 and m/z 951.45, with significance (P < 0.02, P < 0.002 and P < 0.03), in individuals with diabetes. The results demonstrated that a set of three HSA thiol-trioxidized peptides, which are irreversibly oxidatively damaged in HSA in the plasma of patients with T2DM, can be important indicators and potential biomarkers of oxidative stress in T2DM.