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Article: The Secret Life of Tethers: The Role of Tethering Factors in SNARE Complex Regulation.

Dubuke, Michelle L / Munson, Mary

Frontiers in cell and developmental biology

2016 Volume 4, Page(s) 42

Abstract: Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the ... ...

Abstract	Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC) with the SNAREs and Sec1/Munc18 (SM) proteins. Although these factors are used in different stages of membrane trafficking, e.g., Golgi to plasma membrane transport vs. vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems.
Language	English
Publishing date	2016-05-09
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2016.00042
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Article ; Online: Longitudinal proneuroactive and neuroactive steroid profiles in medication-free women with, without and at-risk for perinatal depression: A liquid chromatography-tandem mass spectrometry analysis.

Deligiannidis, Kristina M / Kroll-Desrosiers, Aimee R / Tan, Yanglan / Dubuke, Michelle L / Shaffer, Scott A

Psychoneuroendocrinology

2020 Volume 121, Page(s) 104827

Abstract: Background: Neuroactive steroids (NAS) are derivatives of cholesterol or steroidal precursors made in the gonads, adrenal gland, placenta and brain. We characterized longitudinal plasma proneuroactive and NAS in healthy perinatal comparison women (HPCW), ...

Abstract	Background: Neuroactive steroids (NAS) are derivatives of cholesterol or steroidal precursors made in the gonads, adrenal gland, placenta and brain. We characterized longitudinal plasma proneuroactive and NAS in healthy perinatal comparison women (HPCW), women at-risk for perinatal depression (AR-PND), and women with PND with/without comorbid anxiety. We hypothesized that AR-PND women who either did or did not go on to develop PND would have elevated NAS concentrations as compared to HPCW and that NAS would be correlated to depressive and anxiety symptoms. Methods: A prospective cohort study evaluated 75 medication-free perinatal women (HPCW, n = 30; AR-PND, n = 19; PND, n = 26). Standardized depression and anxiety assessments and blood samples were completed across 5 visits. Structured Clinical Interviews for DSM-IV TR Disorders were administered at study entry and exit. Plasma pregnenolone, progesterone, 5α- and 5β-dihydroprogesterone, pregnanolone, allopregnanolone, deoxycorticosterone and tetrahydrodeoxycorticosterone were quantified by liquid chromatography-tandem mass spectrometry. Longitudinal relationships between risk-group, depression and anxiety symptoms, and NAS concentrations were analyzed using generalized estimating equations to control for repeated measures correlations. Results: Perinatal 5α-dihydroprogesterone, 5β-dihydroprogesterone, allopregnanolone, deoxycorticosterone, and tetrahydrodeoxycorticosterone concentrations were higher in AR-PND and PND women compared to HPCW (β = 3.57 ± 1.40 and β = 2.11 ± 1.12, p = 0.03; β = 0.18 ± 0.06 and β = 0.03 ± 0.05, p = 0.02; β = 1.06 ± 0.42 and β = 1.19 ± 0.47, p = 0.01; β = 0.17 ± 0.07 and β = 0.11 ± 0.06, p = 0.05; β = 0.03 ± 0.01 and β = 0.03 ± 0.01, p = 0.05, respectively). Perinatal allopregnanolone, 5α-dihydroprogesterone and tetrahydrodeoxycorticosterone were positively associated with HAM-D Conclusion: To our knowledge, this study represents the largest prospective study of 5-α and 5-β reductase products of progesterone and deoxycorticosterone in HPCW and women AR-PND. Data suggest that PND is associated with both a reduction of progesterone to 5β-dihydroprogesterone, 5α-dihydroprogesterone, and allopregnanolone, and the 21-hydroxylation to deoxycorticosterone and tetrahydrodeoxycorticosterone. The shift towards 5α-dihydroprogesterone, deoxycorticosterone and tetrahydrodeoxycorticosterone was associated with a history of depression, a significant risk factor for PND.
MeSH term(s)	20-alpha-Dihydroprogesterone/analysis ; 20-alpha-Dihydroprogesterone/blood ; Adult ; Anxiety/metabolism ; Anxiety/physiopathology ; Chromatography, Liquid/methods ; Depression/metabolism ; Depression/physiopathology ; Depression, Postpartum ; Depressive Disorder/metabolism ; Depressive Disorder/physiopathology ; Desoxycorticosterone/analogs & derivatives ; Desoxycorticosterone/analysis ; Desoxycorticosterone/blood ; Female ; Humans ; Longitudinal Studies ; Neurosteroids/analysis ; Neurosteroids/blood ; Parturition/psychology ; Pregnancy ; Pregnanolone/analysis ; Pregnanolone/blood ; Pregnenolone/analysis ; Pregnenolone/blood ; Prenatal Care/methods ; Prenatal Care/psychology ; Progesterone/analysis ; Progesterone/blood ; Prospective Studies ; Risk Factors ; Tandem Mass Spectrometry/methods
Chemical Substances	Neurosteroids ; 20-alpha-Dihydroprogesterone (145-14-2) ; Desoxycorticosterone (40GP35YQ49) ; tetrahydrodeoxycorticosterone (4AB717DP4A) ; Progesterone (4G7DS2Q64Y) ; Pregnenolone (73R90F7MQ8) ; Pregnanolone (BXO86P3XXW)
Language	English
Publishing date	2020-08-13
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	197636-9
ISSN	1873-3360 ; 0306-4530
ISSN (online)	1873-3360
ISSN	0306-4530
DOI	10.1016/j.psyneuen.2020.104827
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Article ; Online: The secret life of tethers

Michelle L Dubuke / Mary eMunson

Frontiers in Cell and Developmental Biology, Vol

the role of tethering factors in SNARE complex regulation

2016 Volume 4

Abstract	Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC) with the SNAREs and Sec1/Munc18 (SM) proteins. Although these factors are used in different stages of membrane trafficking, e.g. Golgi to plasma membrane transport vs vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems.
Keywords	intracellular trafficking ; exocyst ; GARP ; Hops ; COG ; TRAPP ; Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2016-05-01T00:00:00Z
Publisher	Frontiers Media S.A.
Document type	Article ; Online
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Article ; Online: The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes.

Dubuke, Michelle L / Maniatis, Stephanie / Shaffer, Scott A / Munson, Mary

The Journal of biological chemistry

2015 Volume 290, Issue 47, Page(s) 28245–28256

Abstract: In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, ... ...

Abstract	In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.
MeSH term(s)	Binding Sites ; Chromatography, Gel ; Protein Binding ; SNARE Proteins/metabolism ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; Vesicular Transport Proteins/metabolism
Chemical Substances	SEC6 protein, S cerevisiae ; SNARE Proteins ; Saccharomyces cerevisiae Proteins ; Vesicular Transport Proteins
Language	English
Publishing date	2015-10-07
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M115.673806
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Article ; Online: Rab-E and its interaction with myosin XI are essential for polarised cell growth.

Orr, Robert G / Furt, Fabienne / Warner, Erin L / Agar, Erin M / Garbarino, Jennifer M / Cabral, Sarah E / Dubuke, Michelle L / Butt, Allison M / Munson, Mary / Vidali, Luis

The New phytologist

2020 Volume 229, Issue 4, Page(s) 1924–1936

Abstract: The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as ... ...

Abstract	The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P. patens and A. thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P. patens and the rab-E and myosin XI phenotypes are rescued by A. thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.
MeSH term(s)	Bryopsida/growth & development ; Cell Division ; Cell Proliferation ; Exocytosis ; Myosins ; Plant Proteins ; Two-Hybrid System Techniques
Chemical Substances	Plant Proteins ; Myosins (EC 3.6.4.1)
Language	English
Publishing date	2020-11-28
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	208885-x
ISSN	1469-8137 ; 0028-646X
ISSN (online)	1469-8137
ISSN	0028-646X
DOI	10.1111/nph.17023
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Article ; Online: Pseudouridinylation of mRNA coding sequences alters translation.

Eyler, Daniel E / Franco, Monika K / Batool, Zahra / Wu, Monica Z / Dubuke, Michelle L / Dobosz-Bartoszek, Malgorzata / Jones, Joshua D / Polikanov, Yury S / Roy, Bijoyita / Koutmou, Kristin S

Proceedings of the National Academy of Sciences of the United States of America

2019 Volume 116, Issue 46, Page(s) 23068–23074

Abstract: Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also ...

Abstract	Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the
MeSH term(s)	Codon/genetics ; Codon/metabolism ; Open Reading Frames ; Peptide Chain Elongation, Translational ; Protein Biosynthesis ; Pseudouridine/genetics ; Pseudouridine/metabolism ; RNA Processing, Post-Transcriptional ; RNA, Bacterial/genetics ; RNA, Bacterial/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Transfer/genetics ; RNA, Transfer/metabolism ; Ribosomes/genetics ; Ribosomes/metabolism ; Thermus thermophilus/genetics ; Thermus thermophilus/metabolism ; Uridine/metabolism
Chemical Substances	Codon ; RNA, Bacterial ; RNA, Messenger ; Pseudouridine (1445-07-4) ; RNA, Transfer (9014-25-9) ; Uridine (WHI7HQ7H85)
Language	English
Publishing date	2019-10-31
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1821754116
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Article: Rab‐E and its interaction with myosin XI are essential for polarised cell growth

Orr, Robert G / Furt, Fabienne / Warner, Erin L / Agar, Erin M / Garbarino, Jennifer M / Cabral, Sarah E / Dubuke, Michelle L / Butt, Allison M / Munson, Mary / Vidali, Luis

new phytologist. 2021 Feb., v. 229, no. 4

2021

Abstract	The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live‐cell imaging coupled with targeted gene knockouts and a high‐throughput RNAi assay enabled the first characterisation of the loss of Rab‐E function. Yeast two‐hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab‐E and myosin XI that is conserved between P. patens and A. thaliana. Rab‐E co‐localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab‐E is required for normal plant growth in P. patens and the rab‐E and myosin XI phenotypes are rescued by A. thaliana’s Rab‐E1c and myosin XI‐K/E, respectively. Both PpMyoXI and AtMyoXI‐K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab‐E and myosin XI is a conserved feature of polarised growth in plants.
Keywords	assays ; cell division ; cell growth ; computer simulation ; cytoskeleton ; exocytosis ; image analysis ; kinesin ; myosin ; phenotype ; plant growth ; prediction ; two hybrid system techniques
Language	English
Dates of publication	2021-02
Size	p. 1924-1936.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	NAL-light ; JOURNAL ARTICLE
ZDB-ID	208885-x
ISSN	1469-8137 ; 0028-646X
ISSN (online)	1469-8137
ISSN	0028-646X
DOI	10.1111/nph.17023
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Menthol enhances nicotine-induced locomotor sensitization and in vivo functional connectivity in adolescence.

Thompson, Matthew F / Poirier, Guillaume L / Dávila-García, Martha I / Huang, Wei / Tam, Kelly / Robidoux, Maxwell / Dubuke, Michelle L / Shaffer, Scott A / Colon-Perez, Luis / Febo, Marcelo / DiFranza, Joseph R / King, Jean A

Journal of psychopharmacology (Oxford, England)

2017 Volume 32, Issue 3, Page(s) 332–343

Abstract: Mentholated cigarettes capture a quarter of the US market, and are disproportionately smoked by adolescents. Menthol allosterically modulates nicotinic acetylcholine receptor function, but its effects on the brain and nicotine addiction are unclear. To ... ...

Abstract	Mentholated cigarettes capture a quarter of the US market, and are disproportionately smoked by adolescents. Menthol allosterically modulates nicotinic acetylcholine receptor function, but its effects on the brain and nicotine addiction are unclear. To determine if menthol is psychoactive, we assessed locomotor sensitization and brain functional connectivity. Adolescent male Sprague Dawley rats were administered nicotine (0.4 mg/kg) daily with or without menthol (0.05 mg/kg or 5.38 mg/kg) for nine days. Following each injection, distance traveled in an open field was recorded. One day after the sensitization experiment, functional connectivity was assessed in awake animals before and after drug administration using magnetic resonance imaging. Menthol (5.38 mg/kg) augmented nicotine-induced locomotor sensitization. Functional connectivity was compared in animals that had received nicotine with or without the 5.38 mg/kg dosage of menthol. Twenty-four hours into withdrawal after the last drug administration, increased functional connectivity was observed for ventral tegmental area and retrosplenial cortex with nicotine+menthol compared to nicotine-only exposure. Upon drug re-administration, the nicotine-only, but not the menthol groups, exhibited altered functional connectivity of the dorsal striatum with the amygdala. Menthol, when administered with nicotine, showed evidence of psychoactive properties by affecting brain activity and behavior compared to nicotine administration alone.
MeSH term(s)	Animals ; Brain/drug effects ; Humans ; Locomotion/drug effects ; Male ; Menthol/adverse effects ; Nicotine/adverse effects ; Rats ; Rats, Sprague-Dawley ; Reinforcement (Psychology)
Chemical Substances	Menthol (1490-04-6) ; Nicotine (6M3C89ZY6R)
Language	English
Publishing date	2017-07-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	639313-5
ISSN	1461-7285 ; 0269-8811
ISSN (online)	1461-7285
ISSN	0269-8811
DOI	10.1177/0269881117719265
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Article ; Online: Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity.

Haraszti, Reka Agnes / Miller, Rachael / Stoppato, Matteo / Sere, Yves Y / Coles, Andrew / Didiot, Marie-Cecile / Wollacott, Rachel / Sapp, Ellen / Dubuke, Michelle L / Li, Xuni / Shaffer, Scott A / DiFiglia, Marian / Wang, Yang / Aronin, Neil / Khvorova, Anastasia

Molecular therapy : the journal of the American Society of Gene Therapy

2018 Volume 26, Issue 12, Page(s) 2838–2847

Abstract: Exosomes can deliver therapeutic RNAs to neurons. The composition and the safety profile of exosomes depend on the type of the exosome-producing cell. Mesenchymal stem cells are considered to be an attractive cell type for therapeutic exosome production. ...

Abstract	Exosomes can deliver therapeutic RNAs to neurons. The composition and the safety profile of exosomes depend on the type of the exosome-producing cell. Mesenchymal stem cells are considered to be an attractive cell type for therapeutic exosome production. However, scalable methods to isolate and manufacture exosomes from mesenchymal stem cells are lacking, a limitation to the clinical translation of exosome technology. We evaluate mesenchymal stem cells from different sources and find that umbilical cord-derived mesenchymal stem cells produce the highest exosome yield. To optimize exosome production, we cultivate umbilical cord-derived mesenchymal stem cells in scalable microcarrier-based three-dimensional (3D) cultures. In combination with the conventional differential ultracentrifugation, 3D culture yields 20-fold more exosomes (3D-UC-exosomes) than two-dimensional cultures (2D-UC-exosomes). Tangential flow filtration (TFF) in combination with 3D mesenchymal stem cell cultures further improves the yield of exosomes (3D-TFF-exosomes) 7-fold over 3D-UC-exosomes. 3D-TFF-exosomes are seven times more potent in small interfering RNA (siRNA) transfer to neurons compared with 2D-UC-exosomes. Microcarrier-based 3D culture and TFF allow scalable production of biologically active exosomes from mesenchymal stem cells. These findings lift a major roadblock for the clinical utility of mesenchymal stem cell exosomes.
MeSH term(s)	Animals ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Exosomes/metabolism ; Female ; Gene Silencing ; Mesenchymal Stem Cells/cytology ; Mesenchymal Stem Cells/metabolism ; Mice ; Neurons/metabolism ; Proteome ; RNA, Small Interfering/genetics ; Spheroids, Cellular ; Umbilical Cord/cytology
Chemical Substances	Proteome ; RNA, Small Interfering
Language	English
Publishing date	2018-09-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2010592-7
ISSN	1525-0024 ; 1525-0016
ISSN (online)	1525-0024
ISSN	1525-0016
DOI	10.1016/j.ymthe.2018.09.015
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Article ; Online: Serum Deprivation of Mesenchymal Stem Cells Improves Exosome Activity and Alters Lipid and Protein Composition.

iScience

2019 Volume 16, Page(s) 230–241

Abstract: Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome- ... ...

Abstract	Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an "artificial exosome," in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes.
Language	English
Publishing date	2019-05-27
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2019.05.029
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