Atg8 Conjugation Research Articles

Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.

Potential role of autophagy in proteolysis in Trichomonas vaginalis.

Autophagy has been shown to be involved in the pathogenesis of several protists, offering prospects for the developments of new drugs targeting autophagy. However, there is no evidence illustrating functional autophagy in the deep-branching trichomonads. The human parasitic protist Trichomonas vaginalis has been predicted to possess reduced autophagic machinery, with only autophagy-related protein 8 (Atg8) conjugation system required for autophagosome formation. The recombinant protein of TvAtg8 (rTvAtg8) and the polyclonal antibody against rTvAtg8 were generated. The expression and localization of TvAtg8 was monitored upon autophagy induction by glucose restriction (GR) compared with glucose-rich cultivation. The role of TvAtg8 in proteolysis was clarified. Here, we report that T. vaginalis Atg8 (TvAtg8) is upregulated and conjugated to autophagosome-like vesicles upon autophagy induction by GR. Moreover, we investigate, for the first time, the role of autophagy in T. vaginalis. Proteasome inhibition (PI)-induced autophagy compensates for the removal of polyubiquitinated proteins under glucose-rich condition. GR-induced autophagy is a major proteolytic system in T. vaginalis. These results suggest that autophagy is vital for proteolysis in T. vaginalis with an impaired ubiquitin-proteasome system or under glucose-limited environment. Our findings unveiled previously unidentified functions of autophagy in proteostasis in trichomonads, advancing our understanding of this highly conserved process in the ancient eukaryote.

Abstract 354: Accelerated Autophagy is Required for Doxorubicin-induced Oxidative Damage and Cardiomyopathy

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is limited by the risk of severe cardiotoxicity. In patients, the acute onset of cardiomyopathy often precludes chemotherapy treatment, resulting in reduced quality of life and increased morbidity. While the mechanisms by which DOX promotes cardiac dysfunction are not well understood, our previous findings show that intracellular metabolism of DOX elicits mitochondrial reactive oxygen species (ROS) generation, and subsequent activation of the autophagy/lysosomal system. Equally, evidence suggests that elimination of vital cellular organelles and proteins within the cell as a result of pathologically elevated autophagy may in turn stimulate an increase in ROS generation. Therefore, we hypothesize that DOX-induced autophagy promotes cardiac dysfunction as a result of an autophagy-induced increase in oxidative stress in the heart. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV-dnATG5) to female Sprague-Dawley rats via tail vein administration four weeks prior to either DOX (20 mg/kg ip) or saline treatment. Two days following treatment, assessment of cardiac function showed significant alterations to the myocardial performance index and fractional shortening percentage in control, DOX treated animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and increased appearance of autophagic vacuoles within the heart. In contrast, rAAV-dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 to ATG5 thereby reducing autophagosome formation and attenuating cardiac dysfunction. Interestingly, protein expression of the antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. These findings indicate that accelerated autophagy promotes DOX-induced cardiac dysfunction and is an underlying cause of chemotherapy-induced oxidative damage to the heart.

Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes

ABSTRACTPost-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12–ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.

Autophagy promotes cancer chemotherapy‐induced oxidative stress and skeletal muscle dysfunction

Chemotherapy‐induced cachexia is a debilitating condition characterized by fatigue and muscle wasting. The onset of muscle weakness in patients often limits treatment, resulting in reduced quality of life and increased morbidity. Our previous findings show that the chemotherapeutic agent doxorubicin (DOX) causes myotoxicity as a result of reactive oxygen species (ROS) generation during intracellular metabolism of DOX by the mitochondria, and subsequent activation of the autophagy/lysosomal system. We hypothesize that DOX‐induced accelerated autophagy results in the elimination of vital cellular organelles and proteins, resulting in functional deficits to the muscle tissue. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV‐dnATG5) via intramuscular injection of the rat soleus muscle four weeks prior to treatment. Animals received either DOX (20 mg/kg ip) or saline. Two days after chemotherapy, soleus muscle specific force production and fiber cross‐sectional area were significantly reduced in control animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and the increased appearance of autophagic vacuoles within the muscle. In contrast, rAAV‐dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 and ATG5 thereby reducing autophagosome formation within the soleus muscle. Interestingly, the protein expression of catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. Finally, DOX‐induced muscle fiber atrophy and contractile dysfunction were prevented in rAAV‐dnATG5 animals. These findings indicate that accelerated autophagy is an underlying cause of chemotherapy‐induced oxidative damage and skeletal muscle weakness.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii.

Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.

Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy.

Ribosomes are abundant cellular machines1,2 regulated by assembly, supernumerary subunit turnover, and nascent chain quality control mechanisms1–5. Moreover, nitrogen starvation in yeast has been reported to promote selective ribosome delivery to the vacuole in an autophagy conjugation system-dependent manner, a process called “ribophagy”6,7. However, whether ribophagy in mammals is selective or regulated is unclear. Using Ribo-Keima flux reporters, we find that starvation or mTOR inhibition promotes VPS34-dependent ribophagic flux, which unlike yeast, is largely ATG8 conjugation independent and occurs concomitantly with other cytosolic protein autophagic flux reporters8,9. Ribophagic flux was not induced upon inhibition of translational elongation or nascent chain uncoupling, but was induced in a comparatively selective manner upon proteotoxic stress via arsenite10 or chromosome mis-segregation11 dependent upon VPS34 and ATG8 conjugation. Unexpectedly, agents typically used to induce selective autophagy also promoted increased ribosome and cytosolic protein reporter flux, suggesting significant bulk or “by-stander” autophagy during what is often considered selective autophagy12,13. These results emphasize the importance of monitoring non-specific cargo flux when assessing selective autophagy pathways.

Downregulation of SIRT1 signaling underlies hepatic autophagy impairment in glycogen storage disease type Ia.

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.

A comparative in-silico analysis of autophagy proteins in ciliates.

Autophagy serves as a turnover mechanism for the recycling of redundant and/or damaged macromolecules present in eukaryotic cells to re-use them under starvation conditions via a double-membrane structure known as autophagosome. A set of eukaryotic genes called autophagy-related genes (ATGs) orchestrate this highly elaborative process. The existence of these genes and the role they play in different eukaryotes are well-characterized. However, little is known of their role in some eukaryotes such as ciliates. Here, we report the computational analyses of ATG genes in five ciliate genomes to understand their diversity. Our results show that Oxytricha trifallax is the sole ciliate which has a conserved Atg12 conjugation system (Atg5-Atg12-Atg16). Interestingly, Oxytricha Atg16 protein includes WD repeats in addition to its N-terminal Atg16 domain as is the case in multicellular organisms. Additionally, phylogenetic analyses revealed that E2-like conjugating protein Atg10 is only present in Tetrahymena thermophila. We fail to find critical autophagy components Atg5, Atg7 and Atg8 in the parasitic ciliate Ichthyophthirius multifiliis. Contrary to previous reports, we also find that ciliate genomes do not encode typical Atg1 since all the candidate sequences lack an Atg1-specific C-terminal domain which is essential for Atg1 complex formation. Consistent with the absence of Atg1, ciliates also lack other members of the Atg1 complex. However, the presence of Atg6 in all ciliates examined here may rise the possibility that autophagosome formation could be operated through Atg6 in ciliates, since Atg6 has been shown as an alternative autophagy inducer. In conclusion, our results highlight that Atg proteins are partially conserved in ciliates. This may provide a better understanding for the autophagic destruction of the parental macronucleus, a developmental process also known as programmed nuclear death in ciliates.

Apple autophagy-related protein MdATG3s afford tolerance to multiple abiotic stresses

The efficient degradation system of autophagy in plant cells has important roles in removing and recycling intracellular components during normal development or under environmental stresses. Formation of autophagosomes requires the conjugation of ubiquitin-like protein ATG8 to phosphatidylethanolamine (PE). We isolated two ubiquitin-conjugating enzyme E2-like ATG3 homologues from Malus domestica − MdATG3a and MdATG3b − that are crucial for ATG8-PE conjugation. Both share a conserved N-terminal, as well as the catalytic and C-terminal domains of ATG3 with HPC and FLKF motifs. Each promoter was isolated from genomic DNA and contained several cis-acting elements that are involved in responses to environmental stresses or hormones. In addition to having the same cellular localization in the nucleus and cytoplasm, MdATG3a and MdATG3b showed similar expression patterns toward leaf senescence, nitrogen starvation, drought, salinity, and oxidative stress at the transcriptional level. Ectopic expression of either in Arabidopsis conferred tolerance to osmotic or salinity stress and also improved growth performance under nitrogen- or carbon-starvation. Callus lines of ‘Orin’ apple that over-expressed MdATG3b also displayed better growth performance when nutrient supplies were limited. These overall results demonstrate that, as important autophagy genes, overexpression of MdATG3s can afford tolerance to multiple abiotic stresses at the cellular and whole-plant level.

Dynamics of Atg5-Atg12-Atg16L1 Aggregation and Deaggregation.

Macroautophagy is a physiological process that is implicated in various pathological conditions, including neurodegenerative diseases and cancer. The execution of canonical autophagy is regulated by a core signaling cascade and it involves two well-characterized, ubiquitin-like conjugation systems-the Atg5/Atg12/Atg16L1 and the Atg8-phosphatidyl ethanolamine (PE), which are both catalyzed by Atg7. The conjugation of Atg5-Atg12 and the subsequent interaction with the positive regulator Atg16L1 are essential for the conjugation of Atg8 to PE and the subsequent formation of autophagosomes. The interaction between Atg5-Atg12 complex and Atg16L1 is highly dynamic, induced upon activation of the autophagic process, and required for the recruitment of the At5-Atg12 complex to sites of autophagosome formation. Monitoring the Atg5-Atg12-Atg16L1 aggregation and deaggregation may be used not only as means to study the dynamics of autophagy, but in another important point, it may provide important insights on the basic molecular mechanisms of autophagy in physiological and pathological settings. In this chapter, we describe methods of monitoring the Atg5-Atg12-Atg16L1 aggregation and deaggregation, with emphasis on prostate cancer.

Closing the loop.

The ATG conjugation system is not essential for autophagosome formation in mammals

Mechanism of cargo-directed Atg8 conjugation during selective autophagy.

Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.

In Vitro Reconstitution of Atg8 Conjugation and Deconjugation.

Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation. Among the factors for autophagosome formation are the autophagy-related (Atg) proteins belonging to the Atg8 conjugation system. In this system, the ubiquitin-like Atg8 protein is conjugated to the membrane lipid phosphatidylethanolamine present in autophagosomal membranes. Atg8 can also be removed from membranes by Atg4-mediated deconjugation. Here, we describe in vitro systems that recapitulate the enzymatic reactions occurring in vivo by presenting expression and purification strategies for all the components of the Saccharomyces cerevisiae Atg8 conjugation system. We also present protocols for in vitro Atg8 conjugation and deconjugation reactions employing small and giant unilamellar vesicles.

Ectopic expression of an autophagy-associated MdATG7b gene from apple alters growth and tolerance to nutrient stress in Arabidopsis thaliana

Plants are often subjected to unfavorable growing conditions, such as a limited supply of nutrients. Macroautophagy (autophagy) is an intracellular recycling process that targets unwanted proteins or damaged organelles, thus providing the nitrogen and carbon needed for survival and new plant growth during different phases of development and nutrient deprivation. During the formation of autophagosomes, AuTophaGy-related protein 7 (ATG7) is involved in both the ATG8 and ATG12 conjugation pathways. We isolated two ATG7 orthologs—MdATG7a and MdATG7b—from ‘Golden Delicious’ apple (Malus domestica Borkh.). Both were differentially responsive to leaf senescence, nitrogen depletion, and oxidative stresses at the transcriptional level. Heterologous expression of MdATG7b in Arabidopsis accelerated vegetative growth and facilitated the transition into reproductive phase, while also increasing the number of lateral branches, alleviating the negative effects of salt stress on seed germination and conferring tolerance to nitrogen- and carbon starvation. These results suggest that, as one of the important autophagy-related genes, MdATG7b enhances plant tolerance to nitrogen and carbon limitations, and it might be used as an important candidate for breeding crops with improved tolerance to nutrient-starvation stress.

Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites.

ABSTRACTPlasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity.

Dietary compound gossypetin inhibits bone resorption through down-regulating lysosomal cathepsin K activity and autophagy-related protein induction in actin ring-bearing osteoclasts

Gossypetin, usually isolated from the flowers and the calyx of Hibiscus sabdariffa, possesses anti-microbial and anti-atherosclerotic effects. However, anti-osteoporotic effects of gossypetin have not been elucidated. Gossypetin attenuated RANKL-induced multinucleated osteoclast formation with enhanced TRAP activity and blunted bone resorption active in osteoclasts. Gossypetin inhibited the actin ring formation and αvβ3 integrin induction for sealing zones. This compound suppressed the induction of CAII, V-ATPase, ClC-7 and Ae2, all required for secretion of proton and chloride ions into resorption lacunae. Furthermore, gossypetin reduced lysosomal cathepsin K transcription and MMP-9 activity, blunting accumulation of lysosomes in osteoclasts displaying an actin ring. The presence of gossypetin deterred the induction of Rab7, and Atg12–Atg5 conjugate and Atg7 involved in LC3 lipidation, all prerequisites to osteoclast ruffled border formation. These observations demonstrate for the first time that gossypetin was effective in retarding ruffled border formation and acidification in a sealed microenvironment of osteoclast resorption lacunae.

Induction of Autophagy interferes the tachyzoite to bradyzoite transformation of Toxoplasma gondii.

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.

Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay.

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

FGFR3/fibroblast growth factor receptor 3 inhibits autophagy through decreasing the ATG12–ATG5 conjugate, leading to the delay of cartilage development in achondroplasia

FGFR3 (fibroblast growth factor receptor 3) is a negative regulator of endochondral ossification. Gain-of-function mutations in FGFR3 are responsible for achondroplasia, the most common genetic form of dwarfism in humans. Autophagy, an evolutionarily conserved catabolic process, maintains chondrocyte viability in the growth plate under stress conditions, such as hypoxia and nutritional deficiencies. However, the role of autophagy and its underlying molecular mechanisms in achondroplasia remain elusive. In this study, we found activated FGFR3 signaling inhibited autophagic activity in chondrocytes, both in vivo and in vitro. By employing an embryonic bone culture system, we demonstrated that treatment with autophagy inhibitor 3-MA or chloroquine led to cartilage growth retardation, which mimics the effect of activated-FGFR3 signaling on chondrogenesis. Furthermore, we found that FGFR3 interacted with ATG12–ATG5 conjugate by binding to ATG5. More intriguingly, FGFR3 signaling was found to decrease the protein level of ATG12–ATG5 conjugate. Consistently, using in vitro chondrogenic differentiation assay system, we showed that the ATG12–ATG5 conjugate was essential for the viability and differentiation of chondrocytes. Transient transfection of ATG5 partially rescued FGFR3-mediated inhibition on chondrocyte viability and differentiation. Our findings reveal that FGFR3 inhibits the autophagic activity by decreasing the ATG12–ATG5 conjugate level, which may play an essential role in the pathogenesis of achondroplasia.