Defining the proteolytic landscape during enterovirus infection.

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Author Correction: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins.

A range of cell types, including embryonic stem cells, neurons and astrocytes have been shown to release extracellular vesicles (EVs) containing molecular cargo. Across cell types, EVs facilitate transfer of mRNA, microRNA and proteins between cells. Here we describe the release kinetics and content of EVs from mouse retinal progenitor cells (mRPCs). Interestingly, mRPC derived EVs contain mRNA, miRNA and proteins associated with multipotency and retinal development. Transcripts enclosed in mRPC EVs, include the transcription factors Pax6, Hes1, and Sox2, a mitotic chromosome stabilizer Ki67, and the neural intermediate filaments Nestin and GFAP. Proteomic analysis of EV content revealed retinogenic growth factors and morphogen proteins. mRPC EVs were shown to transfer GFP mRNA between cell populations. Finally, analysis of EV mediated functional cargo delivery, using the Cre-loxP recombination system, revealed transfer and uptake of Cre+ EVs, which were then internalized by target mRPCs activating responder loxP GFP expression. In summary, the data supports a paradigm of EV genetic material encapsulation and transfer within RPC populations. RPC EV transfer may influence recipient RPC transcriptional and post-transcriptional regulation, representing a novel mechanism of differentiation and fate determination during retinal development.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Identification of ginkgolide targets in brain by photoaffinity labeling.

Ginkgolides are terpene trilactones in Ginkgo biloba, a popular medicinal herb for memory disorders. Although ginkgolides are known for various neurobiological effects, their macromolecular target in brain is unknown. In this work, we employed benzophenone derivatives of ginkgolides to identify their binding target in brain. Photolabeling of bovine hippocampus homogenates identified a series of α-tubulin isotypes. Selective photolabeling of α-tubulin over ß-tubulin, which is equally abundant in brain, suggested that ginkgolides might modulate microtubule biology differently than typical microtubule-binding agents, such as taxol. In fact, ginkgolide A did not affect microtubule polymerization or cell proliferation; instead, it inhibited detyrosination of α-tubulin and reorientation of microtubule-organizing centers. Taken together, the current findings indicate that ginkgolides constitute a new class of microtubule-binding agents with distinct effects on α-tubulin biology.

Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor ß secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.