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Proteins to Exosomes Soluble Mycobacterial and Eukaryotic Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes This information is current as Victoria L. Smith, Liam Jackson and Jeffrey S. Schorey of September 28, 2021. J Immunol published online 5 August 2015 http://www.jimmunol.org/content/early/2015/08/05/jimmun ol.1403186 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/08/05/jimmunol.140318 Material 6.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 5, 2015, doi:10.4049/jimmunol.1403186 The Journal of Immunology Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes Victoria L. Smith, Liam Jackson, and Jeffrey S. Schorey Exosomes are extracellular vesicles of endocytic origin that function in intercellular communication. Our previous studies indicate that exosomes released from Mycobacterium tuberculosis-infected macrophages contain soluble mycobacterial proteins. However, it was unclear how these secreted proteins were targeted to exosomes. In this study, we determined that exosome production by the murine macrophage cell line RAW264.7 requires the endosomal sorting complexes required for transport and that trafficking of mycobacterial proteins from phagocytosed bacilli to exosomes was dependent on protein ubiquitination. Moreover, soluble mycobacterial proteins, when added exogenously to RAW264.7 or human HEK293 cells, were endocytosed, ubiquitinated, and released via exosomes. This suggested that endocytosed proteins could be recycled from cells through exosomes. This hypothesis was supported using the tumor-associated protein He4, which, when endocytosed by RAW264.7 or HEK293 cells, was transported Downloaded from to exosomes in a ubiquitin-dependent manner. Our data suggest that ubiquitination is a modification sufficient for trafficking soluble proteins within the phagocytic/endocytic network to exosomes. The Journal of Immunology, 2015, 195: 000–000. uberculosis (TB) is a leading cause of death globally, with for degradation, some traffic to the plasma membrane, which, upon an estimated 1.4 million deaths annually. Mycobacterium fusion with the membrane, releases the intraluminal vesicles as tuberculosis, the causative agent of TB, is an intracellular exosomes. Despite their discovery nearly 30 years ago, the T http://www.jimmunol.org/ pathogen that primarily infects macrophages. Despite its intra- mechanism for biogenesis is still not well understood. Evidence cellular lifestyle, M. tuberculosis, and other members of the my- suggests that biogenesis may vary within a cell and between cell cobacterial species, shed and release bacterial components that types (7–9). To date, several models have been suggested in me- gain access to the immune system (1). Evidence suggests that one diating the formation of MVBs and sorting of protein cargo into mechanism that facilitates the release of mycobacterial proteins the intraluminal vesicles, including the endosomal sorting com- into the extracellular milieu is through exosomes (2, 3). Exosomes plex required for transport (ESCRT) machinery (10–13). Two such are membrane-bound vesicles of endocytic origin that are 30–100 nm protein complexes, ESCRT-0 and ESCRT-1, have been shown to in sizes that have been shown to function in intercellular com- be involved in MVB protein sorting and loading in dendritic and munication and immune cell activation and serve as a source of neuroglia cells as well as other cell types. Protein sorting by by guest on September 28, 2021 disease biomarkers. In the context of a M. tuberculosis infection, ESCRT-0 and ESCRT-1 is mediated by ubiquitin-interacting exosomes have been shown to carry mycobacterial components motifs that are found on the Hrs and Tsg101 subunits, which including .40 bacterial proteins, many of which are known constitute part of ESCRT-0 and ESCRT-1 complexes, respectively. immune-dominate Ags (4). Moreover, these exosomes when used Through its ubiquitin-interacting domains, ESCRT-0 clusters as a vaccine can protect mice against an aerosolized infection (5). ubiquitinated proteins for delivery into MVBs (14). ESCRT-0 Exosomes carrying various pathogen- or cancer-derived Ags were subsequently recruits ESCRT-1 to the endosomal membrane, also found to elicit a protective immune response (6). Although which in turn recruits the remaining members of the ESCRT much of the published work has characterized the host T cell machinery: ESCRT-II and ESCRT-III (15, 16). It is believed that response to antigenic proteins present within exosomes, we filaments formed by ESCRT-III ultimately promote invagination know little about the mechanism by which these proteins are of the membrane and intraluminal vesicles formation. Although targeted to exosomes. a general model for exosome biogenesis through the ESCRT Exosome biogenesis begins with the invagination of endosomal proteins has been elucidated, identifying specific proteins traf- membranes to form intraluminal vesicles. Whereas most late ficked to exosomes through this mechanism has been difficult, endosomes, or multivesicular bodies (MVB), fuse with lysosomes particularly for nonmembrane proteins. Previously, we reported that, of the mycobacterial proteins Department of Biological Sciences, Eck Institute for Global Health, University of identified on exosomes, the vast majority were experimentally Notre Dame, Notre Dame, IN 46556 shown to be secreted, soluble proteins (4). Interestingly, macro- Received for publication December 22, 2014. Accepted for publication July 8, 2015. phages treated with culture filtrate protein (CFP), which contains This work was supported by National Institute of Allergy and Infectious Diseases mycobacterial proteins secreted or shed in culture, release exo- Grant AI052439. somes containing many of the same mycobacterial proteins that Address correspondence and reprint requests to Dr. Jeffrey S. Schorey, Department of are on exosomes following a M. tuberculosis infection. This Biological Sciences, University of Notre Dame, 129 Galvin Life Science Center, Notre Dame, IN 46556. E-mail address: [email protected] finding suggests that these soluble mycobacterial proteins have the The online version of this article contains supplemental material. necessary signal to be trafficked to the MVB during exosome Abbreviations used in this article: BCA, bicinchoninic acid assay; CFP, culture biogenesis independent of their entry mechanism into macro- filtrate protein; ESCRT, endosomal sorting complex required for transport; MVB, phages. The data presented in this study suggest that ubiq- multivesicular body; siRNA, small interfering RNA; TB, tuberculosis; WT, uitination of both mycobacterial- as well as host-derived soluble wild-type. proteins within the phagocytic/endocytic network is an important Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 mechanism for their trafficking to MVBs and onto exosomes. It www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403186 2 SORTING SOLUBLE UBIQUITINATED PROTEINS TO EXOSOMES also suggests that endocytosed proteins can be recycled back to lysates were prepared in radioimmunoprecipitation assay buffer. Suspen- the extracellular environment by incorporation into exosomes. sion was mixed with Laemmli buffer and heated at 95˚C for 5 min before loading onto SDS gel. Immunoblots probed with Abs for proteins: ubiq- uitin (P4D1, 1:1,000; Santa Cruz), Tsg101 (C-2, 1:1,000; Santa Cruz), Hrs Materials and Methods (V-20, 1:500; Santa Cruz), tubulin (T5293; Sigma-Aldrich), CFP (C192, Bacterial strains and media 1:1,000; American Type Culture Collection), Kat-G (IT-42, 1:20; Ameri- can Type Culture Collection), His (1:500; Santa Cruz), GroES (SA-12, The mouse macrophage cell line RAW264.7 and human HEK293 cells were 1:20; American Type Culture Collection), HspX (IT-20, 1:15; American maintained in DMEM supplemented with 10% FBS, 10 mM sodium py- Type Culture Collection), and Park2 (ARP43038, 1:500; Aviva). Primary ruvate, and 25 mM HEPES. M. tuberculosis H37Rv was grown in Mid- Ab incubation was followed with HRP-conjugated secondary Abs (1:25,000; dlebrook 7H9 broth supplemented with oleic albumin dextrose catalase Pierce) and detected using ECL kit (Pierce). until midlogarithmic growth phase and frozen down as stocks in growth media plus 15% glycerol. Prior to use, the bacterial stocks were thawed Immunoprecipitation of ubiquitinated proteins and the mycobacteria were declumped by a brief sonication and passed through a syringe fitted with a 27-gauge needle at least 10 times. In brief, ubiquitin Ab (P4D1) was conjugated to Sepharose A/G beads. Conjugation mixture containing 2 mg Ab, 40 mL
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