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The exosome membrane localization of histones is independent of DNA and upregulated in response to stress
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.

Keywords [en]
Extracellular histones, extracellular vesicles, exosomes, exoDNA, cellular stress, ESCRT complex, proteomics
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-147419OAI: oai:DiVA.org:umu-147419DiVA, id: diva2:1203614
Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-06-09
In thesis
1. The release of histone proteins from cells via extracellular vesicles
Open this publication in new window or tab >>The release of histone proteins from cells via extracellular vesicles
2018 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes.

In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting.

In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway.

Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent.

The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.

In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.

 

In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 74
Keywords
Histone, extracellular vesicles, exosomes, extracellular histones extracellular DNA, cellular stress, proteomics, ESCRT complex, Lrrn1, mid-hindbrain organizer, ovarian cancer, ascites
National Category
Cell and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:umu:diva-147409 (URN)978-91-7601-888-0 (ISBN)
Presentation
2018-05-28, E04_ R-1, Umeå, 13:00 (English)
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Supervisors
Available from: 2018-05-04 Created: 2018-05-03 Last updated: 2018-06-09Bibliographically approved

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Muthukrishnan, UmaNatarajan, BalasubramanianLevén May, HannaJones, IwanSandblad, LindaNagaev, IvanBaranov, VladimirMincheva-Nilsson, LuciaGilthorpe, Jonathan D.

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Muthukrishnan, UmaNatarajan, BalasubramanianLevén May, HannaJones, IwanSandblad, LindaNagaev, IvanBaranov, VladimirMincheva-Nilsson, LuciaGilthorpe, Jonathan D.
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Umeå Centre for Molecular Medicine (UCMM)Department of Pharmacology and Clinical NeuroscienceDepartment of Molecular Biology (Faculty of Medicine)Department of Clinical Microbiology
Cell and Molecular Biology

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