28,613 research outputs found
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Proteomic analyses of Urine Exosomes reveal New Biomarkers of Diabetes in Pregnancy.
ObjectiveTo evaluate 24 hour urine exosome protein content changes among pregnant US subjects with diabetes and obesity during early pregnancy.MethodsThe exosome proteome content from 24 hour urine samples of pregnant subjects with gestational diabetes mellitus (GDM, N=8) and pre-gestational Type 2 diabetes (PGD, N = 10) were compared with control samples (CTRL, N = 10) obtained at week 20 of pregnancy. Differences in exosome protein load between groups was identified by liquid chromatography/mass spectrometry, analyzed by linear regression in negative binomial distribution, visualized in MetaboAnalyst (version 3.0), and validated by western immunoblotting.ResultsAt the 20th week of pregnancy, we identified 646, 734 and 856 proteins in exosomes from 24 hour urine samples of patients from the CTRL, GDM and PGD groups, respectively. S100 calcium binding protein A9, damage associated molecular pattern (DAMP) signal, was found to be significantly increased in both GDM and PGD subjects. In GDM subjects the peptide counts for S100A9 protein independently correlated with maternal obesity and macrosomia of the newborn infants. Early to late pregnancy developmental changes in the GDM group were shown to utilize pathways and protein expression levels differently from those in PGD or CTRL groups.ConclusionsUrinary exosome proteomic analysis non-invasively provides insights into maternal changes during diabetic pregnancy. Exosome biomarkers early in pregnancy can be potentially used to better understand pathophysiologic mechanisms of diabetes at a cellular level, and to distinguish between gestational and pre-gestational diabetes at the pathway level. This information can aid intervention efforts to improve pregnancy outcomes in women with diabetes
The Exosome Subunit Rrp44 Plays a Direct Role in RNA Substrate Recognition
The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNAiMet lacking a single m1A58 modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNAiMet, showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNAiMet by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein
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Exosomes regulate neurogenesis and circuit assembly.
Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development
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Mechanisms of nuclear content loading to exosomes.
Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development
Writing a wrong: Coupled RNA polymerase II transcription and RNA quality control
Processing and maturation of precursor RNA species is coupled to RNA polymerase II transcription. Co-transcriptional RNA processing helps to ensure efficient and proper capping, splicing, and 3' end processing of different RNA species to help ensure quality control of the transcriptome. Many improperly processed transcripts are not exported from the nucleus, are restricted to the site of transcription, and are in some cases degraded, which helps to limit any possibility of aberrant RNA causing harm to cellular health. These critical quality control pathways are regulated by the highly dynamic protein-protein interaction network at the site of transcription. Recent work has further revealed the extent to which the processes of transcription and RNA processing and quality control are integrated, and how critically their coupling relies upon the dynamic protein interactions that take place co-transcriptionally. This review focuses specifically on the intricate balance between 3' end processing and RNA decay during transcription termination. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Processing > 3' End Processing RNA Processing > Splicing Mechanisms RNA Processing > Capping and 5' End Modifications
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Pseudotyping exosomes for enhanced protein delivery in mammalian cells.
Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells
The 3' to 5' exoribonuclease DIS3: from structure and mechanisms to biological functions and role in human disease
DIS3 is a conserved exoribonuclease and catalytic subunit of the exosome, a protein complex involved in the 3’ to 5’ degradation and processing of both nuclear and cytoplasmic RNA species. Recently, aberrant expression of DIS3 has been found to be implicated in a range of different cancers. Perhaps most striking is the finding that DIS3 is recurrently mutated in 11% of multiple myeloma patients. Much work has been done to elucidate the structural and biochemical characteristics of DIS3, including the mechanistic details of its role as an effector of RNA decay pathways. Nevertheless, we do not understand how DIS3 mutations can lead to cancer. There are a number of studies that pertain to the function of DIS3 at the organismal level. Mutant phenotypes in S.pombe, S.cerevisae and Drosophila suggest DIS3 homologues have a common role in cell-cycle progression and microtubule assembly. DIS3 has also recently been implicated in antibody diversification of mouse B-cells. This article aims to review current knowledge of the structure, mechanisms and functions of DIS3 as well as highlighting the genetic patterns observed within myeloma patients, in order to yield insight into the putative role of DIS3 mutations in oncogenesis
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Assessing Neuronal and Astrocyte Derived Exosomes From Individuals With Mild Traumatic Brain Injury for Markers of Neurodegeneration and Cytotoxic Activity.
Mild traumatic brain injury (mTBI) disproportionately affects military service members and is very difficult to diagnose. To-date, there is currently no blood-based, diagnostic biomarker for mTBI cases with persistent post concussive symptoms. To examine the potential of neuronally-derived (NDE) and astrocytic-derived (ADE) exosome cargo proteins as biomarkers of chronic mTBI in younger adults, we examined plasma exosomes from a prospective longitudinal study of combat-related risk and resilience, marine resiliency study II (MRSII). After return from a combat-deployment participants were interviewed to assess TBI exposure while on deployment. Plasma exosomes from military service members with mTBI (mean age, 21.7 years, n = 19, avg. days since injury 151), and age-matched, controls (deployed service members who did not endorse a deployment-related TBI or a pre-deployment history of TBI; mean age, 21.95 years, n = 20) were precipitated and enriched against a neuronal adhesion protein, L1-CAM, and an astrocyte marker, glutamine aspartate transporter (GLAST) using magnetic beads to immunocapture the proteins and subsequently selected by fluorescent activated cell sorting (FACS). Extracted protein cargo from NDE and ADE preparations were quantified for protein levels implicated in TBI neuropathology by standard ELISAs and on the ultra-sensitive single molecule assay (Simoa) platform. Plasma NDE and ADE levels of Aβ42 were significantly higher while plasma NDE and ADE levels of the postsynaptic protein, neurogranin (NRGN) were significantly lower in participants endorsing mTBI exposure compared to controls with no TBI history. Plasma NDE and ADE levels of Aβ40, total tau, and neurofilament light (NFL), P-T181-tau, P-S396-tau were either undetectable or not significantly different between the two groups. In an effort to understand the pathogenetic potential of NDE and ADE cargo proteins, neuron-like cultures were treated with NDE and ADE preparations from TBI and non-TBI groups. Lastly, we determined that plasma NDE but not ADE cargo proteins from mTBI samples were found to be toxic to neuron-like recipient cells in vitro. These data support the presence of markers of neurodegeneration in NDEs of mTBI and suggest that these NDEs can be used as tools to identify pathogenic mechanisms of TBI
Exosomes released from breast cancer carcinomas stimulate cell movement
For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis
Lysosomal Delivery of Bioactive Proteins to Living Human Cells via Engineered Exosomes
Exosomes are naturally secreted nanovesicles derived from mammalian cells that are used for intercellular communication in vivo. As a result, they can potentially be used for intracellular delivery of therapeutics for disease treatment. We have developed an exosome pseudotyping approach using vesicular stomatitis virus glycoprotein (VSVG) to produce protein chimeras that optimize production of modified exosomes containing protein therapeutics and facilitate effective delivery to their target cells. To the VSVG transmembrane scaffold, we have fused both fluorescent and luminescent reporters for exosome tracking/visualization and quantification of activity respectively. Through our design, we have shown the biogenesis of VSVG modified exosomes from transfected producer cells through fluorescence imaging and the production of a VSVG-based stable cell line. In addition, we have characterized isolated engineered exosomes and shown that they exhibited the correct size, distribution, and molecular markers, while retaining the bioactivity of their protein cargo. Furthermore, we show that our engineered exosomes and their protein cargo are internalized by multiple cell lines into the endosomal and lysosomal compartments of those cells. Lastly, these modified exosomes can confer their bioactive cargo, either a luminescent reporter or puromycin resistance into these target cells. In summary, this study presents a novel approach to exosome engineering to enhance therapeutic protein loading and delivery, and more importantly, shows the delivery of modified exosomes to intracellular lysosomal compartments. This aspect leads to the assumption that in future studies, these engineered exosomes can be used as a vehicle for delivery of therapeutic proteins for treatment of lysosomal storage diseases
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