Dedicated SNARE s and specialized TRIM cargo receptors mediate secretory autophagy
2016; Springer Nature; Volume: 36; Issue: 1 Linguagem: Inglês
10.15252/embj.201695081
ISSN1460-2075
AutoresTomonori Kimura, Jingyue Jia, Suresh Kumar, Seong Won Choi, Yuexi Gu, Michal Mudd, Nicolas Dupont, Shanya Jiang, Ryan Peters, Farzin Farzam, Ashish Jain, Keith A. Lidke, Christopher M. Adams, Terje Johansen, Vojo Deretić,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle8 December 2016free access Transparent process Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy Tomonori Kimura Tomonori Kimura Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Jingyue Jia Jingyue Jia Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Suresh Kumar Suresh Kumar Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Seong Won Choi Seong Won Choi Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Yuexi Gu Yuexi Gu Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Michal Mudd Michal Mudd Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Nicolas Dupont Nicolas Dupont Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Shanya Jiang Shanya Jiang Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Ryan Peters Ryan Peters Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Farzin Farzam Farzin Farzam Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA Search for more papers by this author Ashish Jain Ashish Jain orcid.org/0000-0001-6549-2788 Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø – The Arctic University of Norway, Tromsø, Norway Search for more papers by this author Keith A Lidke Keith A Lidke Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA Search for more papers by this author Christopher M Adams Christopher M Adams Stanford University Mass Spectrometry, Stanford University, Stanford, CA, USA Search for more papers by this author Terje Johansen Terje Johansen orcid.org/0000-0003-1451-9578 Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø – The Arctic University of Norway, Tromsø, Norway Search for more papers by this author Vojo Deretic Corresponding Author Vojo Deretic [email protected] Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Tomonori Kimura Tomonori Kimura Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Jingyue Jia Jingyue Jia Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Suresh Kumar Suresh Kumar Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Seong Won Choi Seong Won Choi Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Yuexi Gu Yuexi Gu Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Michal Mudd Michal Mudd Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Nicolas Dupont Nicolas Dupont Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Shanya Jiang Shanya Jiang Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Ryan Peters Ryan Peters Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Farzin Farzam Farzin Farzam Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA Search for more papers by this author Ashish Jain Ashish Jain orcid.org/0000-0001-6549-2788 Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø – The Arctic University of Norway, Tromsø, Norway Search for more papers by this author Keith A Lidke Keith A Lidke Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA Search for more papers by this author Christopher M Adams Christopher M Adams Stanford University Mass Spectrometry, Stanford University, Stanford, CA, USA Search for more papers by this author Terje Johansen Terje Johansen orcid.org/0000-0003-1451-9578 Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø – The Arctic University of Norway, Tromsø, Norway Search for more papers by this author Vojo Deretic Corresponding Author Vojo Deretic [email protected] Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Author Information Tomonori Kimura1,5,‡, Jingyue Jia1,‡, Suresh Kumar1, Seong Won Choi1, Yuexi Gu1, Michal Mudd1, Nicolas Dupont1, Shanya Jiang1, Ryan Peters1, Farzin Farzam2, Ashish Jain3,6, Keith A Lidke2, Christopher M Adams4, Terje Johansen3 and Vojo Deretic *,1 1Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA 2Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA 3Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø – The Arctic University of Norway, Tromsø, Norway 4Stanford University Mass Spectrometry, Stanford University, Stanford, CA, USA 5Present address: Department of Nephrology, Osaka University School of Medicine, Osaka, Japan 6Present address: Department of Molecular Cell Biology, Centre for Cancer Biomedicine, University of Oslo and Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway ‡These authors contributed equally to this work *Corresponding author. Tel: +1 505 771 2022; E-mail: [email protected] The EMBO Journal (2017)36:42-60https://doi.org/10.15252/embj.201695081 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL-1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R-SNARE Sec22b to recruit cargo to the LC3-II+ sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP-23 and SNAP-29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway. Synopsis Secretory autophagy, an unconventional secretion pathway for cytosolic proteins such as IL-1β and ferritin, diverges from degradative autophagy by utilization of specialized SNAREs and cargo receptors. Secretory autophagy delivers unconventionally secreted cytosolic cargo including IL-1β and ferritin in response to lysosomal damage and signaling. TRIM16 acts as a specialized secretory autophagy receptor for IL-1β. Galectin-8, TRIM16, and R-SNARE Sec22b act coordinately in secretory autophagy. Secretory autophagy requires different SNAREs than degradative autophagy. Introduction Autophagy is a key intracellular quality control process in eukaryotes with multiple roles in development, normal physiology, and disease (Mizushima et al, 2008; Mizushima & Komatsu, 2011). The autophagy pathway governed by ATG factors is primarily known for its degradative cytoplasmic functions (Mizushima et al, 2011). It maintains cellular energy and nutrient supplies during starvation or absence of growth factors (Galluzzi et al, 2014), controls organellar quality and quantity in the cell, prevents accumulation of large protein aggregates, and possesses antimicrobial and other immune functions (Randow & Youle, 2014; Rubinsztein et al, 2015; Sica et al, 2015; Khaminets et al, 2016). Autophagy's potential for secretion has also been considered (Ponpuak et al, 2015), of particular interest in the context of extracellular immune signaling (Deretic et al, 2013; Ma et al, 2013). The core autophagy machinery in mammalian cells has several subsystems interconnected via specific molecular interactions into a unifying apparatus (Mizushima et al, 2011). During autophagy, the cytoplasmic cargo is sequestered by specialized organelles termed autophagosomes, which are characterized by the presence of LC3B, one of the mammalian paralogues of yeast Atg8 (mAtg8s). The conversion of LC3 to LC3-II via its C-terminal lipidation with phosphatidylethanolamine, catalyzed by the E3 ligase complex (ATG5-ATG12/ATG16L1), represents a hallmark of nascent autophagic membranes (Kabeya et al, 2000). The initiation of autophagosome formation depends on upstream Ser/Thr protein kinases, including AMPK (Kim et al, 2011, 2013), mTOR (Egan et al, 2011; Kim et al, 2011; Settembre et al, 2012), and ULK1 (Russell et al, 2013), and lipid kinases centered upon phosphatidylinositol 3-kinase VPS34 complex containing Beclin 1 (Liang et al, 1999) and ATG14L (Sun et al, 2008), to generate phosphatidylinositol 3-phosphate (PI3P) on autophagic membranes. These membranes likely originate from the endoplasmic reticulum (ER) (Axe et al, 2008), with ER-to-Golgi intermediate compartment (ERGIC) playing a key role in LC3 lipidation and formation of autophagosomal membranes (Ge et al, 2014), whereas contributions of the ER–mitochondrial contact sites (Hamasaki et al, 2013), endocytic membranes (Puri et al, 2013; Lamb et al, 2016), and potentially other intracellular compartments (Joachim et al, 2015) play additional roles in redirecting net membrane flow to autophagosomes. The production of PI3P by VPS34 is recognized by WIPI2, which in turn binds to ATG16L (Dooley et al, 2014) of the ATG5-ATG12/ATG16L1 complex, thus localizing LC3 lipidation (Fujita et al, 2008). ATG16L1 is also a binding partner for FIP200, a component of the ULK1 complex (Fujita et al, 2013; Gammoh et al, 2013; Nishimura et al, 2013; Dooley et al, 2014), ensuring that all core subsystems are coming together. Nascent autophagosomes eventually acquire a Qa-SNARE, syntaxin 17 (Itakura et al, 2012; Tsuboyama et al, 2016), which binds to the CCD domain of ATG14L to form a stable binary complex with Qbc-SNARE SNAP-29 (Diao et al, 2015). The binary SNARE complex stabilization and tethering of PI3P-containing membranes by ATG14L permit pairing with the lysosomal R-SNARE VAMP8 to drive a 4-helix bundle SNARE-catalyzed fusion with lysosomes and generate autolysosomes where the captured material is degraded (Mizushima et al, 2011; Itakura et al, 2012; Hamasaki et al, 2013; Takats et al, 2014; Diao et al, 2015). Autophagy can be non-selective or selective with various degrees of precision (Kimura et al, 2016). During selective autophagy, the cargo to be sequestered by autophagosomes is either labeled with tags, including ubiquitin (Khaminets et al, 2016), phosphorylated ubiquitin (Koyano et al, 2014), or galectins (Thurston et al, 2012; Randow & Youle, 2014). These tags can then be recognized by sequestosome 1-like receptors (SLRs) (Birgisdottir et al, 2013; Deretic et al, 2013), including sequestosome 1/p62, NDP52, TAXBP1, NBR1, and optineurin (Bjorkoy et al, 2005; Kirkin et al, 2009; Wild et al, 2011; Newman et al, 2012; Thurston et al, 2012; Lazarou et al, 2015). SLRs recognize ubiquitin on autophagic targets via a variety of ubiquitin binding domains (e.g., UBAN, UBA, UBZ) (Khaminets et al, 2016), whereas some of them can bind galectins, which in turn recognize carbohydrates exposed on exofacial leaflets of damaged endomembranes (Thurston et al, 2012; Randow & Youle, 2014). In addition to SLRs (Lazarou et al, 2015), unique autophagy receptors potentially directly recognizing the cargo have been reported (Sandoval et al, 2008; Zhang et al, 2008; Orvedahl et al, 2011; Liu et al, 2012; Khaminets et al, 2015; Murakawa et al, 2015). Among these, NCOA4 plays the role of a receptor for autophagic degradation of ferritin (Dowdle et al, 2014; Mancias et al, 2014). Recently, a new class of autophagic cargo receptors have been described, which not only recognize targets but also assemble the necessary regulators and execution autophagy factors thus acting dually as receptor regulators of autophagy (Kimura et al, 2016). These factors belong to the TRIM protein family (Reymond et al, 2001), whose members in principle contain N-terminal RING domain, B-box domains, a coiled-coil domain, and a C-terminal domain such as SPRY. TRIMs assemble ULK1 and Beclin 1 complexes and bind to mAtg8s via their LIR (LC3 interaction region) motifs, while recognizing cargo via their SPRY, PYRIN, and potentially other domains (Mandell et al, 2014; Kimura et al, 2015, 2016). Although autophagy is primarily known as a tributary to the degradative lysosomal pathway (Mizushima et al, 2011), functionally different terminations have been considered for autophagocytosed cytosolic material, leading to secretion of cytokines (Ponpuak et al, 2015) or small molecular weight immune mediators (Ma et al, 2013). In particular, autophagy has been examined for its potential in unconventional secretion of leaderless cytosolic proteins that cannot enter the conventional secretory pathway but play extracellular functions (Duran et al, 2010; Manjithaya et al, 2010; Dupont et al, 2011; Zhang & Schekman, 2013; Ponpuak et al, 2015). In mammalian cells, autophagy has been implicated in secretion of IL-1β, as a prototypical cargo for unconventional secretion of cytosolic proteins (Zhang & Schekman, 2013), through its dependence on ATG factors including ATG5 (Dupont et al, 2011). Furthermore, carriers positive for the autophagy marker LC3-II have been identified as IL-1β-sequestration membranes en route for secretion (Zhang et al, 2015). However, how IL-1β is secreted instead of being degraded in autophagic organelles remains unknown. Here we report the key elements of the pathway for unconventional protein secretion through autophagy that separates it from degradative autophagy. This pathway, termed secretory autophagy, requires a specialized receptor recognizing the cytosolic cargo. It also relies on cooperation of this receptor with the R-SNARE present on LC3-II+ ERGIC-derived membranes whereupon the cargo is sequestered. Finally, secretion of the cargo depends on plasma membrane syntaxins instead of delivery to lysosomes via syntaxin 17. The secretory autophagy pathway utilizes a specialized cytosolic cargo receptor regulator from the TRIM family responsive to inducers and agonists of secretion, and a dedicated ERGIC R-SNARE Sec22b that both interacts with the receptor regulator and engages the plasma membrane Qa-SNAREs. This system is utilized by a subset of unconventionally secreted leaderless cytosolic proteins. Results Screen for TRIMs affecting secretory autophagy of IL-1β Diverse lysosome-damaging and inflammasome-activating agents can trigger IL-1β secretion (Schroder & Tschopp, 2010) including silica (Hornung et al, 2008), alum (Hornung et al, 2008), monosodium urate (Martinon et al, 2006), and Leu-Leu-O-Me (LLOMe) (Ito et al, 2015). Lysosomal damage or stress induce autophagy (Maejima et al, 2013; Napolitano & Ballabio, 2016), whereas starvation sensors for induction of autophagy are located on lysosomes (Roczniak-Ferguson et al, 2012; Settembre et al, 2012; Medina et al, 2015; Napolitano & Ballabio, 2016). We used LLOMe as a standard treatment and confirmed that autophagy factors, represented by LC3B and ATG16L1, are required for efficient IL-1β secretion in response to lysosomal damage (Figs 1A and EV1A and B) as well as in response to starvation, a common inducer of autophagy both in general and in association with IL-1β secretion (Dupont et al, 2011; Zhang et al, 2015) (Fig EV1C and D; LDH release was used as a measure of a non-specific leak from cells). A different mAtg8 tested, GABARAP, known to interact strongly with a number of TRIM proteins (Mandell et al, 2014; Kimura et al, 2015, 2016), was also required for IL-1β secretion elicited by either LLOMe or starvation (Fig 1B and Appendix Fig S1A–D). Figure 1. TRIM16 recruits IL-1β to LC3-positive carrier membranes for secretion THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/ml LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants. THP-1 cells were subjected to GABARAP knockdown and processed as in (A). IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe. Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm. IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe. Levels of IL-1β were determined in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/ml LPS overnight and then with 0.25 mM of Silica, Alum, or monosodium urate (MSU). IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data information: Means ± SEM; n ≥ 5, except for (B) where a representative data set from three repeats. *P < 0.05 (t-test for B, C, D, G; ANOVA for A, E, F). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. TRIM16 mediates IL-1β secretion A. Knockdown efficacies as determined by immunoblots. B. LDH release data for samples in Fig 1A. C, D. Levels of (C) IL-1β and (D) LDH release were determined in supernatants from THP-1 cells subjected to knockdowns as indicated, treated with 100 ng/ml LPS overnight, then starved in EBSS for 3 h. E. LDH release results for samples in Fig 1C. F. Co-IP analysis of interactions between flag-TRIM16 with myc-pro-IL-1β in HEK293T cells. G. Immunoblot analyses of knockdown efficacies in THP-1 cells. H. LDH release data for samples in Fig 1E. I. Immunoblot analyses of the levels of mIL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe. J. LDH release data from samples in Fig 1F. K, L. Levels of (K) IL-1β and (L) LDH release was determined in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/ml LPS overnight, then starved in EBSS for 3 h. M. Immunoblot analyses of knockdown efficacies in primary human MDM cells. N. LDH release results for samples in Fig 1G. O. Intracellular localization analysis of TRIM16, pro-IL-1β, and LAMP2 by confocal microscopy. Cells, THP-1. Line tracings correspond to arrows. Scale bars, 5 μm. Data information: Means ± SEM; n ≥ 5, except for immunoblot quantifications and TRIM screen where n ≥ 3. *P < 0.05, †P ≥ 0.05 (t-test for N; ANOVA for B, C, D, H, J, K, L). Download figure Download PowerPoint Members of the TRIM family of proteins have been shown to control autophagy acting as selective autophagy receptors and autophagy regulators (Kimura et al, 2016), whereas several of them, including TRIM16 (Munding et al, 2006) and TRIM20 (Kimura et al, 2015), have been implicated in control of inflammasome activation. We thus screened human TRIMs and found that there were a number of TRIMs influencing IL-1β secretion in response to LLOMe challenge (Figs 1C and EV1E). TRIM16 acts as a receptor for IL-1β in secretion TRIM16 directly interacts with IL-1β as previously reported (Munding et al, 2006), and this association was verified in our experiments (Fig EV1F). TRIM16 colocalized with LC3B upon LLOMe treatment (Fig 1D and Appendix Fig S1E and F), in keeping with its recently described role in autophagic response to and repair of endomembrane and lysosomal damage (Chauhan et al, 2016). Thus, we hypothesized that TRIM16 might connect lysosomal damage with autophagy-sponsored unconventional secretion of IL-1β. We found that TRIM16 was needed for optimal secretion of IL-1β triggered by LLOMe (Figs 1E and EV1G–I), silica (Hornung et al, 2008), alum (Hornung et al, 2008), monosodium urate (Martinon et al, 2006) (Figs 1F and EV1J), and starvation (Fig EV1K and L). TRIM16 was also required for optimal secretion of IL-1β from primary macrophages (human peripheral blood monocyte-derived macrophages, MDM; Figs 1G and EV1M and N). In keeping with these observations, TRIM16 and pro-IL-1β colocalized in cells treated with LLOMe (Fig EV1O). A subset of TRIM16+ profiles overlapped with the lysosomal marker LAMP2 (Fig EV1O), in keeping with the recently described role of TRIM16 in autophagic homeostasis of damaged lysosomes (Chauhan et al, 2016). Another TRIM subjected to follow-up analyses, TRIM10, was confirmed for effects on IL-1β secretion (Figs 1E and EV1G and H) and showed capacity to interact with TRIM16 (Appendix Fig S1G). Thus, TRIM16, a protein that binds IL-1β, as well as additional TRIMs affect IL-1β secretion. Galectin-8 cooperates with TRIM16 in IL-1β secretion in response to lysosomal damage Galectin-3 and galectin-8, the cytosolic lectins which recognize β(1–4)-linked galactosides normally restricted to organellar lumenal membrane leaflets (Nabi et al, 2015), have been associated with autophagic response to endomembrane damage (Thurston et al, 2012; Fujita et al, 2013; Maejima et al, 2013). Hence, we tested whether these galectins contributed to IL-1β secretion. Knockdowns of galectin-8, but not that of galectin-3, suppressed the secretion of IL-1β in THP-1 cells and in primary MDMs upon stimulation (Figs 2A–C and EV2A–E). These relationships were confirmed using bone marrow derived macrophages from galectin-3 and galectin-8 knockout mice (Appendix Fig 2A and B). Zhang et al (2015) have recently shown that HSP90 participates in unconventional secretion of IL-1β. Galectin-8 showed similar overall intracellular distribution and colocalization with HSP90 in cells treated with LLOMe (Fig EV2F). Figure 2. Galectin-8 participates in IL-1β secretion and interacts with TRIM16 A, B. Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS. C. IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. D. Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells. E. Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells. F. Confocal microscopy of THP-1 cells treated with LPS and LLOMe and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Scale bars, 5 μm. G. In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (−) of flag-ULK1 and cold ATP, GST pull downs were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by SDS-PAGE and autoradiography. Amounts of GST fusion proteins are shown in Coomassie brilliant blue (CBB)-stained gels. Data information: Means ± SEM; n ≥ 5, except for immunoblot quantifications where n ≥ 3. *P < 0.05, †P ≥ 0.05 (t-test for F; ANOVA for A, B, C). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Galectin-8 participates in IL-1β secretion and interacts with TRIM16 A. Immunoblot analyses of knockdown efficacies in THP-1 cells. B, C. LDH release data for samples in (B) Fig 2A and (B) Fig 2B. D. Immunoblot analyses of knockdown efficacies in primary human MDMs. E. LDH release data for samples in Fig 2C. F. Confocal microscopy of HeLa cells treated with LLOMe and stained for galectin-8 and HSP90. Arrowheads in enlarged insets indicate colocalization. Scale bars, 5 μm. G. Galectin-8 domains and deletion constructs used. H. In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with full-length- and deletions of GST-galectin-8 in the presence of flag-ULK1 and cold ATP, and GST pull downs were performed and [35S] radiolabeled Myc-HA-TRIM16 detected by SDS-PAGE and autoradiography. Amounts of GST fusion proteins are shown in Coomassie brilliant blue (CBB)-stained gels. Data information: Means ± SEM; n ≥ 5, except for immunoblot quantifications where n ≥ 3. †P ≥ 0.05 (ANOVA for B, C, E). Download figure Download PowerPoint A question arose whether galectin-3 and galectin-8 could influence the TRIM16-LC3 colocalization detected in response to LLOMe illustrated in Fig 1D. Neither galectin-8 knockdown alone (Appendix Fig S2C and D) nor galectin-3 knockdown alone (Appendix Fig S2E and F) affected colocalization between TRIM16 and LC3B elicited in cells by LLOMe. However, a combined knockdown of galectin-3 and galectin-8 reduced the percentage of TRIM16 profiles that were also positive for LC3B (Appendix Fig S2E and F). Thus, galectin-3 and galectin-8 showed redundant effects on bulk (i.e., not differentiated for function) TRIM16-LC3B profiles formed in response to lysosomal damage. Galectin-8 and TRIM16 co-immunoprecipitated in cell extracts (Fig 2D and E) and colocalized in cells treated with LLOMe (Fig 2F). TRIM16 associated with galectin-8 in GST pull-down assays (Figs 2G and EV2G and H), and as described for other galectins, that is, galectin-3 (Chauhan et al, 2016), this association was somewhat enhanced in the presence of ULK1 (Fig 2G). Thus, both the receptor TRIM16 and its interacting partner galectin-8 recognizing membrane damage are required for efficient secretion of IL-1β in response to lysosomal damage. TRIM16 is required for IL-1β delivery to LC3+ carriers Autophagy-dependent IL-1β secretion can be reconstituted in model cell lines whereby IL-1β is sequestered by LC3-II+ carriers en route for secretion (Zhang et al, 2015). These carriers, herein referred to as IL-1β-sequestration membranes, have been shown to fractionate as a 25 k pellet during differential centrifugation of membranes isolated from model cells overexpressing pro-IL-1β and pro-caspase-1 (Zhang et al, 2015). We wondered whether TRIM16, acting as an IL-1β receptor, might play a role in delivering its cargo to IL-1β-sequestration membranes. Endogenous TRIM16 was present along with LC3-II and IL-1β in the 25 k membranes prepared from wild-type HeLa cells reconstituted with flag-pro-IL-1β and myc-pro-caspase-1, as previously described (Fig 3A; Zhang et al, 2015). HeLa cells reconstituted as above were responsive to LLOMe and starvation treatment as evidenced by IL-1β secretion (Fig EV3A and B). Colocalization between LC3 puncta and IL-1β profiles was responsive to LLOMe as determined by high content microscopy in cells reconstituted as above (Appendix Fig S3A). This system permitted us to compare wild-type TRIM16+ HeLa cells and their CRISPR TRIM16 knockout derivative (TRIM16KO cells; Fig EV3C; Chauhan et al, 2016) for their capacity to colocalize LC3 and IL-1β and transfer IL-1β to 25 k membranes. Absence of TRIM16 diminished colocalization between IL-1β and LC3 (Appendix Fig S3A) and abrogated delivery of mature, that is, caspase-1-processed, IL-1β (mIL-1β) to IL-1β-sequestration membranes in cells treated with LLOMe (Fig 3B–D). This was not due to indirect effects inhibiting processing of pro-IL-1β (Fig EV3D). Furthermore, when membrane was subjected to further separation by density gradient centrifugation using previously described methods and fractionation schemes (Zhang et al, 2015), despite equal expression of pro-IL-1β (Fig 3E), mIL-1β co-fractionated with the Sec22b+ LC3-II+ membranes in TRIM16wt cells, but was no longer associated/focused on these membranes in TRIM16KO cells (Fig 3F and G). Of further note was separation of the membrane peak containing mIL-1β, TRIM16, Sec22b, and LC3-II+ from lysosomes (LAMP2) (Fig 3F and G). Thus, TRIM16 is required for IL-1β delivery to IL-1β-sequestration membranes. Figure 3. TRIM16 recruits IL-1β to LC3-positive carrier membranes for secretion and inter
Referência(s)