Análisis de proteínas implicadas en autofagia: Estudios biofísicos de complejos lípido-proteína

En este trabajo nos planteamos avanzar en el conocimiento acerca de los mecanismos de autofagia. Para ello, purificamos GATE G116C, GABARAP G116C y LC3 G120C, las tres proteínas homólogas de Atg8 a las que se les ha modificado la glicina C-terminal por una cisteína terminal, que es capaz de unirse al lípido comercial DPPE-MBP. Así, mediante ensayos de agregación y de fusión en LUVs, y de agregación en GUVs, concluimos que estas tres proteínas son capaces de inducir por sí mismas agregación vesicular in vitro.

Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Features of autophagic cell death in Plasmodium liver-stage parasites

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function.

Functional analysis of Arabidopsis thaliana lesion mimic mutant cfs1 in ESCRT complex-related protein trafficking

The Endosomal Sorting Complex Required for Transport (ESCRT)-complex is composed of four complexes, ESCRT-0-III. They sequentially act on a late endosome to sort mono-ubiquitinated transmembrane proteins into the intralumenal vesicle, forming of a multivesicular body(MVB) that is delivered to vacuole for degradation. In Arabidopsis thaliana, the loss of an ESCRT-I component, elch displays a cytokinesis defect; while a dominant negative expression of an ESCRT-III component results in cell death due to vacuolar loss. In this work, the function of a plant-specific ELCH-interactor, CELL DEATH RELATED FYVE/SYLF DOMAIN CONTAINING 1 (CFS1) and its influences on the ESCRT-complex function are investigated. CFS1 is a phosphatidylinositol-3-phosphate- and actin-binding protein. The cfs1 mutants mimic lesions in the first eldest leaf that propagate to the next eldest one. Genetic analyses have demonstrated that cell death in cfs1 does not require a functional ESCRT-I component; nevertheless, the loss of CFS1 alleviates elchcytokinesis defect, suggesting its influence on the ESCRT-I function. Further analyses reveal that cfs1 accumulates autophagosomes throughout its lifespan due to a decrease in autophagosome degradation, suggesting that as the plant ages, the cumulated autophagosomes falsely trigger effectors-triggered immunity that executes cell death in cfs1. As the ESCRT-complex has been demonstrated to be involved in the delivery of autophagosomes to vacuole and CFS1 homolog, CFS2 reportedly interacts with ATG8, it can be postulated from the results of this work that CFS1 alone or together with CFS2 function in sequestering mature autophagosomes onto MVBs. At the MVBs, the ESCRT-complex then mediates the fusion of autophagosome and MVB for subsequent delivery to vacuole.