The interactome of EBV LMP1 evaluated by proximity-based BioID approach.

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.

Proteomic profiling of NCI-60 extracellular vesicles uncovers common protein cargo and cancer type-specific biomarkers.

Packed with biological information, extracellular vesicles (EVs) offer exciting promise for biomarker discovery and applications in therapeutics and non-invasive diagnostics. Currently, our understanding of EV contents is confined by the limited cells from which vesicles have been characterized utilizing the same enrichment method. Using sixty cell lines from the National Cancer Institute (NCI-60), here we provide the largest proteomic profile of EVs in a single study, identifying 6,071 proteins with 213 common to all isolates. Proteins included established EV markers, and vesicular trafficking proteins such as Rab GTPases and tetraspanins. Differentially-expressed proteins offer potential for cancer diagnosis and prognosis. Network analysis of vesicle quantity and proteomes identified EV components associated with vesicle secretion, including CD81, CD63, syntenin-1, VAMP3, Rab GTPases, and integrins. Integration of vesicle proteomes with whole-cell molecular profiles revealed similarities, suggesting EVs provide a reliable reflection of their progenitor cell content, and are therefore excellent indicators of disease.

Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis.

BACKGROUND: Extracellular vesicles (EVs) are important mediators of cell-to-cell communication in healthy and pathological environments. Because EVs are present in a variety of biological fluids and contain molecular signatures of their cell or tissue of origin, they have great diagnostic and prognostic value. The ability of EVs to deliver biologically active proteins, RNAs and lipids to cells has generated interest in developing novel therapeutics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis and secretion remain unknown. METHODS: Here, we characterized vesicle secretion across the NCI-60 panel of human cancer cells by nanoparticle tracking analysis. Using CellMiner, the quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. RESULTS: Gene products positively associated with the quantity of exosomal-sized vesicles included vesicular trafficking classes of proteins with Rab GTPase function and sphingolipid metabolism. Positive correlates of larger microvesicle-sized vesicle secretion included gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. CONCLUSION: These observations reveal new insights into genes involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. This study offers a foundation for further exploration of targets involved in EV biogenesis and secretion.

ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles.

Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes.

Quantitative proteomic analysis of the Anopheles gambiae (Diptera: Culicidae) midgut infected with o'nyong-nyong virus.

Alphaviruses are arthropod-borne pathogens that infect a range of hosts. In humans and other mammals, alphavirus infection can cause severe disease. In mosquito hosts, however, there are generally few symptoms. Little is known about the cellular responses of mosquitoes that allow them to cope with infection. In this investigation, a six-plex tandem mass tagging proteomic approach was used to study protein accumulation changes in the midgut of Anopheles gambiae (Giles) (Diptera: Culicidae) mosquitoes infected with o'nyong-nyong virus (Togaviridae, Alphavirus). Five hundred thirty-six nonredundant proteins were identified. Twenty-two were found in significantly different quantities in infected midguts compared with controls. Of interest, analysis revealed molecular pathways possibly targeted by virus proteins, such as those involving TAF4 and DNA polymerase phi proteins. Also identified was an FK506-binding protein. FK506-binding protein orthologs have been described as conserved host resistance factors, which suppress dengue and West Nile virus infection in human HeLa cells. This investigation constitutes the first study of the midgut-specific proteome of An. gambiae in relation to alphavirus infection. Our findings offer insight into mosquito immunity, including factors that possibly contribute to the different pathological outcomes observed in vertebrate and insect hosts.

PCR detection of malaria parasites in desiccated Anopheles mosquitoes is uninhibited by storage time and temperature.

BACKGROUND: Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR). METHODS: Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein. RESULTS: Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification. CONCLUSIONS: Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.