Role of Gate-16 and Gabarap in Prevention of Caspase-11-Dependent Excess Inflammation and Lethal Endotoxic Shock.

Sepsis is a life-threating multi-organ disease induced by host innate immunity to pathogen-derived endotoxins including lipopolysaccharide (LPS). Direct sensing of LPS by caspase-11 activates inflammasomes and causes lethal sepsis in mice. Inhibition of caspase-11 inflammasomes is important for the prevention of LPS-induced septic shock; however, whether a caspase-11 inflammasome-specific suppressive mechanism exists is unclear. Here we show that deficiency of GABARAP autophagy-related proteins results in over-activation of caspase-11 inflammasomes but not of canonical inflammasomes. Gate-16-/-Gabarap-/- macrophages exhibited elevated guanylate binding protein 2 (GBP2)-dependent caspase-11 activation and inflammatory responses. Deficiency of GABARAPs resulted in formation of GBP2-containing aggregates that promote IL-1ß production. High mortality after low dose LPS challenge in Gate-16-/-Gabarap-/- mice primed with poly(I:C) or polymicrobial sepsis was ameliorated by compound GBP2 deficiency. These results reveal a critical function of Gate-16 and Gabarap to suppress GBP2-dependent caspase-11-induced inflammation and septic shock.

Stress-associated endoplasmic reticulum protein 1 (SERP1) and Atg8 synergistically regulate unfolded protein response (UPR) that is independent on autophagy in Candida albicans.

Cellular stresses could activate several response processes, such as the unfolded protein response (UPR), autophagy and oxidative stress response to restore cellular homeostasis or render cell death. Herein, we identified the Candida albicans stress-associated endoplasmic reticulum protein 1 (SERP1), also known as Ysy6, which was involved in endoplasmic reticulum (ER) stress response. We found that deletion of both SERP1/YSY6 and ATG8 led to hypersensitivity to tunicamycin (TN), and resulted in severe mitochondrial dysfunction under this stress. UPR reporting systems illustrated that the double mutation attenuated splicing of HAC1 mRNA, followed by decreased level of UPR activation. In addition, the atg8Δ/Δ ysy6Δ/Δ double mutant had normal autophagic degradation of the ER component Sec63 under ER stress, suggesting that SERP1/Ysy6 and Atg8 synergistically regulated UPR that is independent on autophagy. We also found that deletion of both SERP1/YSY6 and ATG8 caused the loss of virulence. This study reveals the important role of SERP1/Ysy6 and Atg8 in ER stress response and virulence in C. albicans.

Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux.

Selective autophagy and macroautophagy sequester specific organelles/substrates or bulk cytoplasm, respectively, inside autophagosomes as cargo for delivery to lysosomes. The mammalian ATG8 orthologues (MAP1LC3A/B/C and GABARAP/L1/L2) are ubiquitin (UB)-like proteins conjugated to the autophagosome membrane and are thought to facilitate cargo receptor recruitment, vesicle maturation, and lysosomal fusion. To elucidate the molecular functions of the ATG8 proteins, we engineered cells lacking genes for each subfamily as well as all six mammalian ATG8s. Loss of GABARAPs alone attenuates autophagic flux basally and in response to macroautophagic or selective autophagic stimuli, including parkin-dependent mitophagy, and cells lacking all ATG8 proteins accumulate cytoplasmic UB aggregates, which are resolved following ectopic expression of individual GABARAPs. Autophagosomes from cells lacking GABARAPs had reduced lysosomal content by quantitative proteomics, consistent with fusion defects, but accumulated regulators of late endosome (LE)/autophagosome maturation. Through interaction proteomics of proteins accumulating in GABARAP/L1/L2-deficient cells, we identified C18orf8/RMC1 as a new subunit of the CCZ1-MON1 RAB7 guanine exchange factor (GEF) that positively regulates RAB7 recruitment to LE/autophagosomes. This work defines unique roles for GABARAP and LC3 subfamilies in macroautophagy and selective autophagy and demonstrates how analysis of autophagic machinery in the absence of flux can identify new regulatory circuits.

The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome.