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Targeting by AutophaGy proteins (TAG): Targeting of IFNG-inducible GTPases to membranes by the LC3 conjugation system of autophagy.

ABSTRACT: LC3 has been used as a marker to locate autophagosomes. However, it is also well established that LC3 can localize on various membranous structures other than autophagosomes. We recently demonstrated that the LC3 conjugation system (ATG7, ATG3, and ATG12-ATG5-ATG16L1) is required to target LC3 and IFNG (interferon, gamma)-inducible GTPases to the parasitophorus vacuole membrane (PVM) of a protist parasite Toxoplasma gondii and consequently for IFNG to control T. gondii infection. Here we show that not only LC3, but also its homologs (GABARAP, GABARAPL1, and GABARAPL2) localize on the PVM of T. gondii in a conjugation-dependent manner. Knockout/knockdown of all LC3 homologs led to a significant reduction in targeting of the IFNG-inducible GTPases to the PVM of T. gondii and the IFNG-mediated control of T. gondii infection. Furthermore, when we relocated the ATG12-ATG5-ATG16L1 complex, which specifies the conjugation site of LC3 homologs, to alternative target membranes, the IFNG-inducible GTPases were targeted to the new target membranes rather than the PVM of T. gondii. These data suggest that the localization of LC3 homologs onto a membrane by the LC3 conjugation system is necessary and sufficient for targeting of the IFNG-inducible GTPases to the membrane, implying Targeting by AutophaGy proteins (TAG). Our data further suggest that the conjugation of ubiquitin-like LC3 homologs to the phospholipids of membranes may change the destiny of the membranes beyond degradation through lysosomal fusion, as the conjugation of ubiquitin to proteins changes the destiny of the proteins beyond proteasomal degradation.

SUBMITTER: Park S

PROVIDER: S-EPMC4990996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins.

Project description:Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance.

| S-EPMC3542218 | biostudies-literature

Viral Replication Complexes Are Targeted by LC3-Guided Interferon-Inducible GTPases.

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| S-EPMC5591033 | biostudies-literature

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| S-EPMC10485044 | biostudies-literature

CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy.

Project description:Rationale: Sustained cardiac hypertrophy often leads to heart failure (HF). Understanding the regulation of cardiomyocyte growth is crucial for the treatment of adverse ventricular remodeling and HF. Cell division cycle 20 (CDC20) is an anaphase-promoting complex activator that is essential for cell division and tumorigenesis, but the role of CDC20 in cardiac hypertrophy is unknown. We aimed to test whether CDC20 participates in the regulation of pathological cardiac hypertrophy and investigate the underlying mechanism in vitro and in vivo. Methods: Male C57BL/6 mice were administered a recombinant adeno-associated virus serotype 9 (rAAV9) vector expressing CDC20 or a siRNA targeting CDC20 and their respective controls by tail intravenous injection. Results: Microarray analysis showed that CDC20 was significantly upregulated in the heart after angiotensin II infusion. Knockdown of CDC20 in cardiomyocytes and in the heart reduced cardiac hypertrophy upon agonist stimulation or transverse aortic constriction (TAC). Conversely, enforced expression of CDC20 in cardiomyocytes and in the heart aggravated the hypertrophic response. Furthermore, we found that CDC20 directly targeted LC3, a key regulator of autophagy, and promoted LC3 ubiquitination and degradation by the proteasome, which inhibited autophagy leading to hypertrophy. Moreover, knockdown of LC3 or inhibition of autophagy attenuated Ang II-induced cardiomyocyte hypertrophy after deletion of CDC20 in vitro. Conclusions: Our study reveals a novel cardiac hypertrophy regulatory mechanism that involves CDC20, LC3 and autophagy, and suggests that CDC20 could be a new therapeutic target for patients with hypertrophic heart diseases.

| S-EPMC6299438 | biostudies-literature

The LC3 conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles

Project description:This SuperSeries is composed of the SubSeries listed below.

2019-12-22 | GSE137618 | GEO

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Project description:Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

| S-EPMC7007875 | biostudies-literature

MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins.

Project description:Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.

| S-EPMC3541284 | biostudies-literature

The LC3 conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles (smRNA-seq dataset)

Project description:We employed small non-coding RNA sequecing to profile the small RNAs in extracellular vesicles (EVs) from WT cells and cells deficient for the LC3 conjugation machinery

2019-12-26 | GSE137617 | GEO

The LC3 conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles (lrgRNA-seq dataset)

Project description:We employed RNA sequecing to profile large RNAs (mRNAs and lincRNAs) packaged into extracellular vesicles (EVs) from WT cells and cells deficient for the LC3 conjugation machinery

2019-12-26 | GSE137616 | GEO

Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins.

Project description:Microtubule-associated protein 1 light chain 3 α (LC3)/GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved in (macro)autophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane vesicles called autophagosomes. The only currently known cellular molecules covalently modified by LC3/GABARAP are membrane phospholipids such as phosphatidylethanolamine in the autophagosome membrane. Autophagy-related 4 cysteine peptidase (ATG4) proteases process inactive pro-LC3/GABARAP before lipidation, and the same proteases can also deconjugate LC3/GABARAP from lipids. To determine whether LC3/GABARAP has other molecular targets, here we generated a pre-processed LC3B mutant (Q116P) that is resistant to ATG4-mediated deconjugation. Upon expression in human cells and when assessed by immunoblotting under reducing and denaturing conditions, deconjugation-resistant LC3B accumulated in multiple forms and at much higher molecular weights than free LC3B. We observed a similar accumulation when pre-processed versions of all mammalian LC3/GABARAP isoforms were expressed in ATG4-deficient cell lines, suggesting that LC3/GABARAP can attach also to other larger molecules. We identified ATG3, the E2-like enzyme involved in LC3/GABARAP lipidation, as one target of conjugation with multiple copies of LC3/GABARAP. We show that LC3B-ATG3 conjugates are distinct from the LC3B-ATG3 thioester intermediate formed before lipidation, and we biochemically demonstrate that ATG4B can cleave LC3B-ATG3 conjugates. Finally, we determined ATG3 residue Lys-243 as an LC3B modification site. Overall, we provide the first cellular evidence that mammalian LC3/GABARAP post-translationally modifies proteins akin to ubiquitination ("LC3ylation"), with ATG4 proteases acting like deubiquitinating enzymes to counteract this modification ("deLC3ylation").

| S-EPMC6709618 | biostudies-literature

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