Atg8 Family Research Articles

No ATG8s, no problem? How LC3/GABARAP proteins contribute to autophagy.

The ATG8 family LC3/GABARAP proteins are attached to the membrane of nascent autophagosomes, but their functions during autophagy are unclear. In this issue, Nguyen et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201607039) show that LC3/GABARAP proteins are not essential for autophagosome formation but are critical for autophagosome-lysosome fusion.

Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.

Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members divided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome-lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome-lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.

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.

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors.

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Changes in 30K protein synthesis during delayed degeneration of the silk gland by a caspase-dependent pathway in a Bombyx (silkworm) mutant.

The silk gland in silkworm (Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval-pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval-pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals.

Molecular mechanisms of autophagy in plants: Role of ATG8 proteins in formation and functioning of autophagosomes

Autophagy is an efficient way of degradation and removal of unwanted or damaged intracellular components in plant cells. It plays an important role in recycling of intracellular structures (during starvation, removal of cell components formed during plant development or damaged by various stress factors) and in programmed cell death. Morphologically, autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which are essential for the isolation and degradation of cytoplasmic components. Among autophagic (ATG) proteins, ATG8 from the ubiquitin-like protein family plays a key role in autophagosome formation. ATG8 is also involved in selective autophagy, fusion of autophagosome with the vacuole, and some other intracellular processes not associated with autophagy. In contrast to yeasts that carry a single ATG8 gene, plants have multigene ATG8 families. The reason for such great ATG8 diversity in plants remains unclear. It is also unknown whether all members of the ATG8 family are involved in the formation and functioning of autophagosomes. To answer these questions, the identification of the structure and the possible functions of plant proteins from ATG8 family is required. In this review, we analyze the structures of ATG8 proteins from plants and their homologs from yeast and animal cells, interactions of ATG8 proteins with functional ligands, and involvement of ATG8 proteins in different metabolic processes in eukaryotes.

SLC27A4 regulate ATG4B activity and control reactions to chemotherapeutics-induced autophagy in human lung cancer cells.

Autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses in eukaryotic cells. Dysregulation of this system is linked with numerous human diseases, including cancers. ATG4B, a cysteine protease required for autophagy, cleaves the C-terminal amino acid of ATG8 family proteins to reveal a C-terminal glycine which is necessary for ATG8 proteins conjugation to phosphatidylethanolamine (PE) and insertion to autophagosome precursor membranes. However, the mechanism governing the protein stability of ATG4B in human cancer cells is not fully understood. In this study, tandem affinity purification/mass spectrometry (TAP/MS) were applied to the investigation of the interaction between ATG4B and potential candidate proteins. Then, co-immunoprecipitation (Co-IP) and GST-pull down assays indicated that the candidate protein-SLC27A4 directly interacts with ATG4B in lung cancer cell lines. Intriguingly, we also found that ATG4B protein expression was increased in parallel with SLC27A4 in lung cancer cell lines as well as lung tumor tissues. However, relevant functional research of SLC27A4 in autophagy or oncotherapy has not been investigated before. In this study, we hypothesized that SLC27A4 might act as a mediator of ATG4B, in some respects, through the protein binding directly. Further, we found that the high expression level of SLC7A4 increased the ATG4B stability and was conducive to rapid reaction to everolimus (RAD001)-induced autophagy in human lung cancer cells. As expected, the results showed that SLC27A4 could help to maintain the protein stability and intracellular concentration of ATG4B, thereby triggering rapid autophagy through releasing ATG4B to cytoplasm under conditions of reduced nutrient availability or during stress of chemotherapy in lung cancer cells. Reduced SLC27A4 by si-RNA also showed the enhanced therapeutic efficiency of everolimus, doxorubicin, and cisplatin in human lung cancer cell lines. Collectively, this study may help researchers better understand the mechanism of autophagy vitality in human cancers and SLC27A4/ATG4B complex might act as a new potential therapeutic target of lung tumor chemotherapy.

Reticulophagy and nucleophagy: New findings and unsolved issues

Autophagy targets various intracellular components ranging from proteins and nucleic acids to organelles for their degradation in lysosomes or vacuoles. In selective types of autophagy, receptor proteins play central roles in target selection. These proteins bind or localize to specific targets, and also interact with Atg8 family proteins on forming autophagosomal membranes, leading to the efficient sequestration of the targets by the membranes. Our recent study revealed that yeast cells actively degrade the endoplasmic reticulum (ER) and even part of the nucleus via selective autophagy under nitrogen-deprived conditions. We identified novel receptors, Atg39 and Atg40, specific to these pathways. Here, we summarize our findings on ‘reticulophagy’ (or ‘ER-phagy’) and ‘nucleophagy’, and discuss key issues that remain to be solved in future studies.

Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy.

Autophagy is a major pathway for the clearance of harmful material from the cytoplasm. During autophagy, cytoplasmic material is delivered into the lysosomal system by organelles called autophagosomes. Autophagosomes form in a de novo manner and, in the course of their formation, isolate cargo material from the rest of the cytoplasm. Cargo specificity is conferred by autophagic cargo receptors that selectively link the cargo to the autophagosomal membrane decorated with ATG8 family proteins such as LC3B. Here we show that the human cargo receptor p62/SQSTM-1 employs oligomerization to stabilize its interaction with LC3B and linear ubiquitin when they are clustered on surfaces. Thus, oligomerization enables p62 to simultaneously select for the isolation membrane and the ubiquitinated cargo. We further show in a fully reconstituted system that the interaction of p62 with ubiquitin and LC3B is sufficient to bend the membrane around the cargo.

Conformational Polymorphism in Autophagy-Related Protein GATE-16

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.

Abstract 996: Retinoic acids activate autophagy in breast cancer cells through the FOXO3A-RARA pathway and blocking autophagy enhances retinoic acid-mediated cell death

Abstract A series of pre-clinical studies showed promising anti-cancer effects of retinoic acids (RA) in breast cancer models. However, clinical studies were mostly disappointing possibly also due to the trial design and emerging resistances. One way to confer resistance to a given treatment is activation of protective autophagy. Autophagy is a cellular recycling mechanism that is characterized by the formation of double-membraned autophagosomes. Autophagosomes engulf bulk cytoplasm or selected contents thereof and transfer them to lysosomes for degradation. Activation of autophagy upon anti-cancer treatment may allow prolonged survival of the tumor cells. We therefore asked if autophagy is activated by all-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, in mammary tumor cells and if autophagy might protect the cells from the pro-apoptotic effects of ATRA. We found that ATRA induces autophagic flux in ATRA-sensitive SKBR3 but not in -resistant MD-MBA- 453 human breast cancer cells as seen by increased LC3B-II protein abundance and turnover of long-lived proteins. Moreover, using RA receptor alpha (RARA), RARB and RARG agonists as well as RARA knockdown breast cancer cells we demonstrated that activation of autophagy by RA depends on RARA expression. RA-treatment also caused induction of the transcription factor FOXO3A, a known transcriptional target of RARA. Since the expression of several autophagy genes namely the ATG8 family members, Beclin-1 and PI3KC3 are controlled by FOXO3A, we tested if FOXO3A might link ATRA-mediated signaling to autophagy activation. Indeed, depleting FOXO3A in SKBR3 cells resulted in significantly decreased autophagic activity and autophagy gene expression upon RA treatment. Importantly, blocking autophagy by knocking down the key autophagy genes ATG5 or ATG7 in breast cancer cells resulted in significantly increased apoptosis under ATRA treatment. Of note, inhibition of autophagy also attenuated cyto-differentiation similarly to the phenotype seen in RARA knockdown breast cancer cells. In summary, our data demonstrate for the first time that retinoic acids induce autophagy in breast cancer cells via the FOXO3A-RARA axis. Furthermore, inhibition of autophagy results in enhanced ATRA-mediated apoptosis representing a potential new treatment strategy for a selected group of breast cancer patients. Citation Format: Anna M. Schlaefli, Enrico Garattini, Mario P. Tschan. Retinoic acids activate autophagy in breast cancer cells through the FOXO3A-RARA pathway and blocking autophagy enhances retinoic acid-mediated cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2015-996

Abstract 2910: The dual role of autophagy in cadmium induced prostate carcinogenesis

Abstract Cadmium chloride (Cd) and cadmium products cause both acute and chronic cytotoxicity and Cd exposure correlates with the risk of cancer, especially prostate cancer in humans. On the cellular and molecular level, Cd compounds may promote transformation of healthy cells to a malignant state by inducing expression of pro-survival signaling molecules. However, the precise molecular mechanism by which Cd affects cellular transformation in different tissue types, especially in prostate, is yet to be delineated. Autophagy is an intracellular lysosomal degradation process by which cells remove damaged organelles and maintain the integrity of intracellular components. In cancer cells, autophagy promotes cancer cell survival as well as cell death. It is unclear of how, and under what circumstances, these dual and opposite roles of autophagy affect both anti-oncogenic and oncogenic functions. It is possible that uncharacterized molecular switch that influences the anti-oncogenic function of autophagy in healthy cells to become oncogenic. In our studies, we found that exposure of normal prostate epithelial cells (RWPE-2) in culture to Cd (10μM) results in growth inhibition. By contrast, the growth of Cd-transformed prostate epithelial (CTPE) cells was not inhibited by the same Cd treatment. While analyzing the molecular link between Cd induced toxicity and generation of reactive oxygen species (ROS) generation in RWPE-2 and CTPE cells; we found there was no significant change in the ROS induction in both cell types. In addition, no changes in the expression of autophagy targeted genes (Atg family proteins) were seen in Cd treated in both non-transformed and transformed cells. However, microtubule-associated protein 1A/1B-light chain 3B (LC-3B), which is directly involved in autophagy initiation was upregulated in Cd-treated non-transformed cells, but not in Cd-treated transformed cells. LC-3B activation also correlated with growth inhibition in non-transformed cells, while impairing LC-3B activation in RWPE-2 cells causes resistance to CD induced growth inhibition. Our ongoing study will determine whether induction of autophagy via LC-3B is a key event that facilitates Cd-mediated conversion of non-transformed prostate epithelial cells to a malignant state. We strongly believe our studies will define a new concept of LC-3B mediated autophagy in Cd-induced prostate carcinogenesis. Citation Format: Trinath P. Das, Arokya MS John, Suman Suman, Murali Ankem, Chendil Damodaran. The dual role of autophagy in cadmium induced prostate carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2015-2910

Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.

Macroautophagy is a highly regulated intracellular degradation process which has been extensively studied over the last decade. This pathway has been initially described as a non selective process inducing the degradation of parts of the cytoplasm as well as organelles at random. Nevertheless, over the last few years, new research highlighted the existence of a more selective autophagy pathway specifically recruiting some organelles or aggregates to the autophagosomes in order to induce their degradation. These selective autophagy pathways such as aggrephagy, mitophagy, pexophagy or xenophagy, involve the intervention of a cargo, the material to be degraded, cargo adapters, the molecules allowing the recruitment of the cargo to the autophagosome, and the proteins of the ATG8 family which link the cargo adapters to the autophagosome. One of the main questions which now remain is to develop new techniques and protocols able to discriminate between these different types of induced autophagy. In our work, we studied the possibility to use the P-LISA technique, which has been recently developed to study endogenous in vivo protein interactions, as a new technique to characterize the ATG proteins specifically involved in bulk or selective autophagy. In this manuscript, we indeed demonstrate that this technique allows the study of endogenous ATG protein interactions in cells following autophagy induction, but more interestingly that this technique might be used to characterize the ATG proteins involved in selective autophagy.

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs' previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through "PI4P shuttling." Importantly, PI4KIIα's role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP2) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome's fusion machinery.

Polyamines modulate the roscovitine-induced celldeath switch decision autophagy vs. apoptosis in MCF-7 and MDA-MB-231 breast cancer cells.

Current clinical strategies against breast cancer mainly involve the use of anti‑hormonal agents to decrease estrogen production; however, development of resistance is a major problem. The resistance phenotype depends on the modulation of cell‑cycle regulatory proteins, cyclins and cyclin‑dependent kinases. Roscovitine, a selective inhibitor of cyclin‑dependent kinases, shows high therapeutic potential by causing cell‑cycle arrest in various cancer types. Autophagy is a type of cell death characterized by the enzymatic degradation of macromolecules and organelles in double‑ or multi‑membrane autophagic vesicles. This process has important physiological functions, including the degradation of misfolded proteins and organelle turnover. Recently, the switch between autophagy and apoptosis has been proposed to constitute an important regulator of cell death in response to chemotherapeutic drugs. The process is regulated by several proteins, such as the proteins of the Atg family, essential for the initial formation of the autophagosome, and PI3K, important at the early stages of autophagic vesicle formation. Polyamines (PAs) are small aliphatic amines that play major roles in a number of eukaryotic processes, including cell proliferation. The PA levels are regulated by ornithine decarboxylase (ODC), the rate‑limiting enzyme in PA biosynthesis. In this study, we aimed to investigate the role of PAs in roscovitine‑induced autophagic/apoptotic cell death in estrogen receptor‑positive MCF‑7 and estrogen receptor‑negative MDA‑MB‑231 breast cancer cells. We show that MDA‑MB‑231 cells are more resistant to roscovitine than MCF‑7 cells. This difference was related to the regulation of autophagic key molecules in MDA‑MB‑231 cells. In addition, we found that exogenous PAs have a role in the cell death decision between roscovitine‑induced apoptosis or autophagy in MCF‑7 and MDA‑MB‑231 breast cancer cells.

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.

Mulan E3 ubiquitin ligase interacts with multiple E2 conjugating enzymes and participates in mitophagy by recruiting GABARAP

Mulan is an E3 ubiquitin ligase embedded in the outer mitochondrial membrane (OMM) with its RING finger facing the cytoplasm and a large domain located in the intermembrane space (IMS). Mulan is known to have an important role in cell growth, cell death, and more recently in mitophagy. The mechanism of its function is poorly understood; but as an E3 ligase it is expected to interact with specific E2 ubiquitin conjugating enzymes and these complexes will bind and ubiquitinate specific substrates. The unique topology of Mulan can provide a direct link of communicating mitochondrial signals to the cytoplasm. Our studies identified four different E2 conjugating enzymes (Ube2E2, Ube2E3, Ube2G2 and Ube2L3) as specific interactors of Mulan. Each of these E2 conjugating enzymes was fused to the RING finger domain of Mulan and used in a modified yeast two-hybrid screen. Several unique interactors for each Mulan–E2 complex were isolated. One such specific interactor of Mulan–Ube2E3 was the GABARAP (GABAA receptor-associated protein). GABARAP is a member of the Atg8 family of proteins that plays a major role in autophagy/mitophagy. The interaction of GABARAP with Mulan–Ube2E3 required an LC3-interacting region (LIR) located in the RING finger domain of Mulan as well as the presence of Ube2E3. The isolation of four different E2 conjugating enzymes, as specific partners of Mulan E3 ligase, suggests that Mulan is involved in multiple biological pathways. In addition, the interaction of GABARAP with Mulan–Ube2E3 supports the role of Mulan as an important regulator of mitophagy and provides a plausible mechanism for its function in this process.

Molecular cloning and characterization of two novel autophagy-related genes belonging to the ATG8 family from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Rhipicephalus (Boophilus) microplus is an obligate haematophagous arthropod and the major problem for cattle industry due to economic losses it causes. The parasite shows a remarkable adaptability to changing environmental conditions as well as an exceptional ability to survive long-term starvation. This ability has been related to a process of intracellular protein degradation called autophagy. This process in ticks is still poorly understood and only few autophagy-related (ATG) genes have been characterized. The aim of the present study was to examine the ESTs database, BmiGI, of R. microplus searching for ATG homologues. We predicted five putative ATG genes, ATG3, ATG4, ATG6 and two ATG8s. Further characterization led to the identification of RmATG8a and RmATG8b, homologues of GABARAP and MAP1LC3, respectively, and both of them belonging to the ATG8 family. PCR analyses showed that the expression level of RmATG8a and RmATG8b was higher in egg and larval stages when compared to ovary and midgut from adult ticks. This up-regulation coincides with the period in which ticks are in a starvation state, suggesting that autophagy is active in R. microplus.

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19.

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.

The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells

AbstractPrevious studies demonstrated that imatinib mesylate (IM) induces autophagy in chronic myeloid leukemia (CML) and that this process is critical to cell survival upon therapy. However, it is not known if the autophagic process differs at basal levels between CML patients and healthy individuals and if pretreatment CML cells harbor unique autophagy characteristics that could predict patients’ clinical outcomes. We now demonstrate that several key autophagy genes are differentially expressed in CD34+ hematopoietic stem/progenitor cells, with the highest transcript levels detected for ATG4B, and that the transcript and protein expression levels of ATG4 family members, ATG5 and BECLIN-1 are significantly increased in CD34+ cells from chronic-phase CML patients (P < .05). Importantly, ATG4B is differentially expressed in pretreatment CML stem/progenitor cells from subsequent IM responders vs IM nonresponders (P < .05). Knockdown of ATG4B suppresses autophagy, impairs the survival of CML stem/progenitor cells and sensitizes them to IM treatment. Moreover, deregulated expression of ATG4B in CD34+ CML cells inversely correlates with transcript levels of miR-34a, and ATG4B is shown to be a direct target of miR-34a. This study identifies ATG4B as a potential biomarker for predicting therapeutic response in treatment-naïve CML stem/progenitor cells and uncovers ATG4B as a possible drug target in these cells.