Autophagosomes For Degradation Research Articles

S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy

p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to theproduction of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Disruption of axonal transport in neurodegeneration.

Neurons are markedly compartmentalized, which makes them reliant on axonal transport to maintain their health. Axonal transport is important for anterograde delivery of newly synthesized macromolecules and organelles from the cell body to the synapse and for the retrograde delivery of signaling endosomes and autophagosomes for degradation. Dysregulation of axonal transport occurs early in neurodegenerative diseases and plays a key role in axonal degeneration. Here, we provide an overview of mechanisms for regulation of axonal transport; discuss how these mechanisms are disrupted in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, hereditary spastic paraplegia, amyotrophic lateral sclerosis, and Charcot-Marie-Tooth disease; and discuss therapeutic approaches targeting axonal transport.

Autophagy is Disrupted in the Livers of Obese Mice Exposed to Asparaginase

Amino acid insufficiency activates general control nonderepressible 2 (GCN2), resulting in preferential synthesis of Activating Transcription Factor 4 (ATF4) to promote cellular protection and recovery. Asparaginase is an enzyme which breaks down asparagine and glutamine and is used to treat acute lymphoblastic leukemia in children and adults. Patients who are overweight or obese carry greater risk of developing hepatotoxicity during treatment and we reported that diet‐induced obesity predisposes mice to liver steatosis and dysfunction during asparaginase exposure. To understand the mechanism behind these risks, we subjected liver‐specific ATF4 knock‐out mice (ls‐Atf4KO) and their wild type controls, to a high‐fat diet (60% fat by kcal) or low fat chow (~18% fat by kcal) for 12 weeks before exposing them to 8 daily intraperitoneal injections of asparaginase (3 international units per gram body weight) or saline vehicle. Untargeted metabolomics were conducted on the livers of mice (n=6 per group; 3 male and 3 female). Asparaginase exposure decreased liver asparagine independent of diet or genotype. The liver metabolomes in lean mice were largely unaffected by asparaginase or loss of ATF4. In contrast, the liver metabolomes of obese mice exposed to asparaginase showed striking changes in sphingolipids that correlated with elevations in endoplasmic reticulum (ER) stress as indicated by the phosphorylation of protein kinase R‐like ER‐resident kinase (PERK). Increased synthesis of sphingolipids occurs during the induction of autophagy. Consistent with a sphingolipid signature of increased autophagosome formation, microtubule‐associated protein 1 light chain 3 beta‐II (LC3‐II) was elevated in obese, ls‐Atf4KO mice exposed to asparaginase. Additionally, ubiquitin‐binding protein 62 (p62), a cargo protein which binds to toxic waste and is then incorporated into the autophagosome for degradation, was elevated in obese mice exposed to asparaginase. This increase in p62 levels indicates a block in fusion of the autophagosome with the lysosome. Levels of p62 were highest in obese ls‐Atf4KO mice administered asparaginase. Overall, these data suggest that asparaginase minimally affects the liver metabolome in lean, wild‐type mice. In contrast, asparaginase causes marked shifts in the liver metabolome of obese mice which coincides with ER stress and autophagy dysfunction. Loss of ATF4 exacerbates autophagy disruption in obese mice exposed to asparaginase, highlighting its role in remediating ER stress.

Autophagy Induced by Palmitic Acid Regulates Neutrophil Adhesion Through the Granule-Dependent Degradation of αMβ2 Integrin in Dairy Cows With Fatty Liver.

β2 integrins are critical for neutrophil firm adhesion, trans-endothelial migration, and the recruitment to the inflamed tissue. Autophagy is implicated in cell migration and tumor metastasis through facilitating the turnover of β1 integrins; however, whether autophagy is able to control neutrophil migration by promoting the degradation of β2 integrins is unexplored. Here, we show that high blood levels of palmitic acid (PA) strongly triggered neutrophil autophagy activation, leading to adhesion deficiency in dairy cows with fatty liver. The three neutrophil granule subtypes, namely, azurophil granules (AGs), specific granules (SGs), and gelatinase granules (GGs), were engulfed by the autophagosomes for degradation, resulting in an increased vacuolation in fatty liver dairy cow neutrophils. Importantly, the adhesion-associated molecules CD11b and CD18 distributed on AGs, SGs, and GGs were degraded with the three granule subtypes by autophagy. Moreover, FGA, Hsc70, and TRIM21 mediated the degradation of cytosolic oxidized–ubiquitinated CD11b and CD18. Collectively, our results demonstrate that high blood PA triggers neutrophil autophagy-dependent vacuolation and granule-dependent adhesion deficiency, decreasing neutrophil mobility, and impairing the innate immune system of dairy cow with fatty liver. This theory extends the category of autophagy in maintaining granule homeostasis and provides a novel strategy to improve the immune of dairy cows with metabolic disease.

Discovery of Novel Regulators of Autophagy in Animals

The clearance of organelles by autophagy is important for cell health, and defects in this process have been associated with age-associated degenerative disorders. Ubiquitination of proteins enables their recognition by cargo receptors that facilitate the delivery of both protein aggregates and organelles to forming autophagosomes for degradation. We have investigated developmentally programmed autophagy to identify novel regulators of organelle clearance. We identified an Atg7-independent autophagy program that is required for cell size reduction and clearance of mitochondria. We have used this system to screen for new factors regulate ubiquitin-dependent autophagy and clearance of organelles. We screened a collection of putative ubiquitin binding domain encoding genes, and identified the novel gene Vps13D. Vps13D is an essential gene that is necessary for autophagy, mitochondrial size, mitochondrial clearance, and is associated with human movement disorders. We have used genetics and biochemistry to identify factors that link Vps13D, mitochondrial biology and autophagy.

AC6 regulates the microtubule-depolymerizing kinesin KIF19A to control ciliary length in mammals

Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders.

Abstract 276: Mir-139 regulates autophagy in prostate cancer cells through Beclin-1 and mTOR signaling proteins

Abstract Background: Prostate cancer represents the second leading cause of cancer death in men and develops as a result of the accumulation of genetic and epigenetic alterations. We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using NGS-based whole miRNome sequencing and qPCR-based validation analysis. We have shown that expression of miR-139, one of the downregulated miRNAs from this panel, was associated with improved prognosis in patients with localized prostate cancer. Furthermore, miR-139 overexpression inhibited prostate cancer cell growth through signaling pathways mediated in part through downregulation of AXL and IGF1R. Here, we further investigated the molecular mechanisms by which miR-139 could inhibit prostate cancer cell growth. Methods: We examined miR-139 transfected PC3, DU145 and LNCaP cells by morphology as well as by cell-based assays, confocal microscopy and immunoblotting. Results and Discussion: We found that treatment of prostate cancer cells with miR-139 resulted in phenotypic changes characteristic of autophagic cells. Conversion of the microtubule-associated protein light chain 3 (LC3-I to LC3-II) was used to monitor autophagy. We observed that the treatment of PC3 and LNCaP cells with miR-139 increased the conversion of LC3-I to LC3-II protein that was specifically inhibited by addition of miR-139 antagomir. The upregulation of LC3 II was further confirmed by confocal microscopy of PC3 cells treated with miR-139. Mammalian target of rapamycin (mTOR) and Beclin1 are two important autophagy-related molecules playing significant roles in different stages. mTOR negatively regulates autophagy machinery. Treatment of cells with miR-139 inhibited both activation and expression of mTOR. We also observed increased levels of phospho-Beclin 1 in PC3 cells treated with mir-139. Beclin 1 is a scaffold protein that assembles components for promoting or inhibiting autophagy and its phosphorylation controls autophagy. The cargo adaptor protein, p62/SQSTM1, interacts with autophagic substrates, delivering them to the autophagosome for degradation, and is degraded during autophagy. We found that during early treatment of cells with miR-139, p62 expression is inhibited, while at later stages, p62 expression accumulates, suggesting that autophagic flux may be blocked. These results suggest that miR-139 is regulating autophagy in prostate cancer cells at least in part through the mTOR and Beclin-1 proteins. Citation Format: Tania C. Benatar, Robert Nam, Christopher Sherman, Yutaka Amemiya, Arun Seth. Mir-139 regulates autophagy in prostate cancer cells through Beclin-1 and mTOR signaling proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 276.

Microglia clear neuron-released \u03b1-synuclein via selective autophagy and prevent neurodegeneration

Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. Herein, we report a neuroprotective role for microglia in the clearance of neuron-released α-synuclein. Neuronal α-synuclein activates microglia, which in turn engulf α-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Synucleinphagy requires the presence of microglial Toll-like receptor 4 (TLR4), which induces transcriptional upregulation of p62/SQSTM1 through the NF-κB signaling pathway. Induction of p62, an autophagy receptor, is necessary for the formation of α-synuclein/ubiquitin-positive puncta that are degraded by autophagy. Finally, disruption of microglial autophagy in mice expressing human α-synuclein promotes the accumulation of misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration. Our study thus identifies a neuroprotective function of microglia in the clearance of α-synuclein via TLR4-NF-κB-p62 mediated synucleinphagy.

Celastrol Inhibits Dopaminergic Neuronal Death of Parkinson's Disease through Activating Mitophagy.

Parkinson’s disease (PD) is a neurodegenerative disease, which is associated with mitochondrial dysfunction and abnormal protein accumulation. No treatment can stop or slow PD. Autophagy inhibits neuronal death by removing damaged mitochondria and abnormal protein aggregations. Celastrol is a triterpene with antioxidant and anti-inflammatory effects. Up until now, no reports have shown that celastrol improves PD motor symptoms. In this study, we used PD cell and mouse models to evaluate the therapeutic efficacy and mechanism of celastrol. In the substantia nigra, we found lower levels of autophagic activity in patients with sporadic PD as compared to healthy controls. In neurons, celastrol enhances autophagy, autophagosome biogenesis (Beclin 1↑, Ambra1↑, Vps34↑, Atg7↑, Atg12↑, and LC3-II↑), and mitophagy (PINK1↑, DJ-1↑, and LRRK2↓), and these might be associated with MPAK signaling pathways. In the PD cell model, celastrol reduces MPP+-induced dopaminergic neuronal death, mitochondrial membrane depolarization, and ATP reduction. In the PD mouse model, celastrol suppresses motor symptoms and neurodegeneration in the substantia nigra and striatum and enhances mitophagy (PINK1↑ and DJ-1↑) in the striatum. Using MPP+ to induce mitochondrial damage in neurons, we found celastrol controls mitochondrial quality by sequestering impaired mitochondria into autophagosomes for degradation. This is the first report to show that celastrol exerts neuroprotection in PD by activating mitophagy to degrade impaired mitochondria and further inhibit dopaminergic neuronal apoptosis. Celastrol may help to prevent and treat PD.

Emerging Mechanisms of Cellular Iron Transport and Trafficking

Iron is an essential nutrient required by every cell in the human body, yet it can also be a potent cellular toxin. Iron is essential because enzymes that require iron co-factors (namely, heme, iron-sulfur clusters, mono- and dinuclear iron centers) are involved in virtually every major metabolic process in the cell. Iron deficiency continues to be the most common nutritional deficiency in the world, while iron overload is a feature of an increasing number of human diseases, including genetic disorders such as hereditary hemochromatosis, thalassemias, and Friedreich ataxia, as well as chronic inflammatory diseases of the liver, such as hepatitis C and steatohepatitis. The biochemical mechanisms by which iron causes toxicity are not completely known. Previously, we identified Poly rC-binding protein 1 (Pcbp1) as a protein that directly binds and delivers iron to ferritin, the major iron storage protein in mammalian cells. Pcbp1 and its paralog Pcbp2 are multifunctional adaptors that bind cellular RNA, DNA, proteins, and iron, altering the fate of their binding partners. Ferritin is a large polypeptide, containing 24 subunits of H- and L-chains assembled into a hollow sphere. Reduced (ferrous) iron enters the sphere through pores formed between the subunits and is oxidized on the interior surface to form nanocrystals of ferric oxyhydroxides. Pcbp1 is the first example of an iron chaperone- a protein that specifically binds iron ions and delivers them to target proteins, such as ferritin, through direct protein-protein interactions. In cultured cells, Pcbp1 is also required for efficient iron delivery to non-heme enzymes in addition to ferritin. These enzymes include the mononuclear iron-containing prolyl hydroxylases that regulate HIF and the dinuclear iron-containing deoxyhypusine hydroxylase, which is required for the modification of lysine to hypusine. In the adult human, 70% of total body iron is present in circulating erythrocytes, which are produced by the bone marrow at a rate of 2 million reticulocytes per second. We examined the roles of Pcbp1 and Ncoa4 in this extraordinary flux of iron through the erythron. Ncoa4 is the cargo receptor that recruits ferritin into the autophagosome for degradation in the lysosome. Using an in vitro model of erythroid differentiation, we showed that depletion of Pcbp1 or Ncoa4 impeded trafficking of iron through ferritin, which impaired the synthesis of heme and hemoglobin. Mice with tamoxifen-induced Pcbp1 deficiency exhibited a microcytic anemia typical of iron deficiency, with activation of compensatory erythropoiesis. The role of ferritin in erythropoiesis has been controversial, but our studies indicate that iron flux through ferritin is an obligatory process in the early stages of erythroid terminal differentiation. Our in vitro studies of erythrocytes revealed that the interactions of Pcbp1 and Ncoa4 with ferritin changed during differentiation, with maximal binding of Pcbp1 at early stages and Ncoa4 at late stages. These binding activities are regulated by cellular iron in mechanistically distinct ways. Ongoing studies of Pcbp1 deficiency in other murine tissues suggest the importance of iron chaperones in maintaining the bioavailable pool of iron in mammalian cells. DisclosuresNo relevant conflicts of interest to declare.

Structural insights into pro-aggregation effects of C. elegans CRAM-1 and its human ortholog SERF2

Toxic protein aggregates are key features of progressive neurodegenerative diseases. In addition to “seed” proteins diagnostic for each neuropathy (e.g., Aβ1–42 and tau in Alzheimer’s disease), aggregates contain numerous other proteins, many of which are common to aggregates from diverse diseases. We reported that CRAM-1, discovered in insoluble aggregates of C. elegans expressing Q40::YFP, blocks proteasomal degradation of ubiquitinated proteins and thus promotes aggregation. We now show that CRAM-1 contains three α-helical segments forming a UBA-like domain, structurally similar to those of mammalian adaptor proteins (e.g. RAD23, SQSTM1/p62) that shuttle ubiquitinated cargos to proteasomes or autophagosomes for degradation. Molecular modeling indicates that CRAM-1, through this UBA-like domain, can form tight complexes with mono- and di-ubiquitin and may thus prevent tagged proteins from interacting with adaptor/shuttle proteins required for degradation. A human ortholog of CRAM-1, SERF2 (also largely disordered), promotes aggregation in SH-SY5Y-APPSw human neuroblastoma cells, since SERF2 knockdown protects these cells from amyloid formation. Atomistic molecular-dynamic simulations predict spontaneous unfolding of SERF2, and computational large-scale protein-protein interactions predict its stable binding to ubiquitins. SERF2 is also predicted to bind to most proteins screened at random, although with lower average stability than to ubiquitins, suggesting roles in aggregation initiation and/or progression.

Approaches for Studying Autophagy in Caenorhabditis elegans.

Macroautophagy (hereafter referred to as autophagy) is an intracellular degradative process, well conserved among eukaryotes. By engulfing cytoplasmic constituents into the autophagosome for degradation, this process is involved in the maintenance of cellular homeostasis. Autophagy induction triggers the formation of a cup-shaped double membrane structure, the phagophore, which progressively elongates and encloses materials to be removed. This double membrane vesicle, which is called an autophagosome, fuses with lysosome and forms the autolysosome. The inner membrane of the autophagosome, along with engulfed compounds, are degraded by lysosomal enzymes, which enables the recycling of carbohydrates, amino acids, nucleotides, and lipids. In response to various factors, autophagy can be induced for non-selective degradation of bulk cytoplasm. Autophagy is also able to selectively target cargoes and organelles such as mitochondria or peroxisome, functioning as a quality control system. The modification of autophagy flux is involved in developmental processes such as resistance to stress conditions, aging, cell death, and multiple pathologies. So, the use of animal models is essential for understanding these processes in the context of different cell types throughout the entire lifespan. For almost 15 years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. This review presents a rapid overview of physiological processes involving autophagy in Caenorhabditis elegans, the different assays used to monitor autophagy, their drawbacks, and specific tools for the analyses of selective autophagy.

Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins

The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.

Abstract B15: Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells.

Abstract Purpose: The purpose of this study was to determine whether transcriptional repression of the adaptor protein SQSTM1/p62, which plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation, could potentiate multiple myeloma (MM) cell death in response to agents (e.g., BH3-mimetics) that elicit a cytoprotective autophagic response. Methods: To this end, various multiple myeloma cell lines (e.g., U266, RPMI8226, and bortezomib-resistant PS-R U266 cells) were exposed to the pan BH3-mimetic obatoclax (Teva) in the presence or absence of pan-CDK inhibitors e.g., flavopiridol (Telero), dinaciclib (Merck), after which the induction of autophagy (LC3-II expression, GFP-LC3 puncta by confocal microscopy) and apoptosis (Annexin V staining by flow cytometry) were monitored. Autophagic flux was determined using cells transfected with a pBABE mCherry-EGFP-LC3B plasmid, followed by observation of autophagosomes and autolysosomes. Accumulation of ubiquitinated protein aggregates was assessed by filter trap (retardation) assays and immunofluorescent staining using an anti-ubiquitin antibody. The functional significance of perturbations in protein expression (e.g., p62, CDK9, cyclin T, Bik) was determined using cells in which target gene expression was down-regulated by shRNA. Alterations in the appearance of autophagic vacuoles (AV) were observed using electron microscopy. Lysosome biogenesis was analyzed by LysoTracker Probe. Gene expression was determined by quantitative real-time PCR (qPCR), and protein-protein interactions characterized by co-immunoprecipitation. The in vivo effects of combined treatment were assessed in mouse MM flank xenograft models. Results: Here we report that whereas p62 levels fluctuated in a time-dependent manner during autophagy induced by obatoclax, inhibition (e.g., by CDK inhibitors administered at sub-micromolar concentrations) or knockdown of CDK9 or cyclin T1 transcriptionally suppressed p62 expression, but did not affect autophagic flux or modify lysosome biogenesis. These interventions, or shRNA directly targeting SQSTM1/p62, resulted in cargo-loading failure and inefficient autophagy, phenomena recently described in Huntington's disease neurons. Inefficient autophagy was characterized by the formation of “empty” AVs accompanied by the accumulation of ubiquitinated proteins, presumably due to failure of loading into autophagosomes for degradation. These events led to accumulation of the BH3-only protein NBK/Bik on ER, most likely by blocking loading and autophagic degradation of Bik. Whereas Bik up-regulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., by shRNA targeting Ulk1, Beclin-1, or Atg5, or pharmacologically by 3-MA or spautin-1), arguing that Bik accumulation primarily stems from inefficient autophagy. Moreover, NBK/Bik knock-down markedly attenuated apoptosis in vitro and in in vivo. Finally, similar interactions between a CDK inhibitor and obatoclax were observed in vivo in MM mouse models. Together, these findings identify a novel cross-link between autophagy and apoptosis, wherein targeting p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation which triggers apoptosis. Conclusions: Collectively, these findings highlight a novel approach to target the cargo-loading process during cytoprotective autophagy by the transcriptional down-regulation of the adaptor protein p62, rather than direct inhibition of autophagy at either early or late stages (e.g., by chloroquine). Together, these findings provide new mechanistic insights into and a theoretical foundation for developing a autophagy-targeting therapeutic strategy based on induction of inefficient autophagy in MM. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Richard Jones, Orlowski Robert, Steven Grant. Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B15.

WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism

Nucleation-promoting factors (NPFs) control the spatio-temporal activity of Arp2/3 complex in cells]. Thus, WASP and the WAVE complex direct the formation of branched actin networks at the leading edge during cell motility and endo/exocytosis, whereas the WASH complex is involved in endosomal transport. Less understood are WHAMM and JMY, two NPFs with similar domain architecture. JMY is found in the nucleus and the cytosol and is involved in transcriptional regulation, cell motility, and trans-Golgi transport. WHAMM was reported to bind microtubules and to be involved in ER to cis-Golgi transport. Here, we show that WHAMM directs the activity of Arp2/3 complex for autophagosome biogenesis through an actin-comet tail motility mechanism. Macroautophagy--the process by which cytosolic material is engulfed into autophagosomes for degradation and/or recycling--was recently shown to involve actin, but the mechanism is unknown. We found that WHAMM forms puncta that colocalize and comigrate with the autophagy markers LC3, DFCP1, and p62 through a WHAMM-dependent actin-comet tail mechanism. Under starvation, WHAMM and actin are observed at the interface between neighboring autophagosomes, whose number and size increase with WHAMM expression. Interfering with actin polymerization, inhibiting Arp2/3 complex, knocking down WHAMM, or blocking its interaction with Arp2/3 complex through mutagenesis all inhibit comet tail formation and reduce the size and number of autophagosomes. Finally, JMY shows similar localization to WHAMM and could be involved in similar processes. These results reveal a link between Arp2/3-complex-dependent actin assembly and autophagy.

The role of ATG5-dependent autophagy during Rickettsia australis infection. (INC4P.339)

Abstract Rickettsiae are obligate intracellular bacteria that replicate in the cytoplasm of eukaryotic cells. We found that degradative autophagy is induced upon the infection of murine bone marrow macrophages (BMMs) with R. australis and the bacteria are specifically targeted by autophagosomes for degradation. Inhibition of autophagy using 3-methylamine (3-MA) significantly increased bacterial burden in BMMs. Consistently, rickettsial loads in livers of infected Atg5flox/flox Lyz-Cre mice were significantly greater than those of Atg5flox/flox littermates. As a consequence, host survival was significantly reduced in autophagy-deficient Atg5flox/flox Lyz-Cre animals, suggesting that macrophage autophagy mediates host cellular immunity against rickettsiae in vivo. In contrast, limited level of autophagy was induced in human endothelial cells, which are the major targets of rickettsiae. Taken together, our results suggest that R. australis induces autophagy in murine macrophages and plays a role in bacterial clearance both in vitro and in vivo. However, levels of autophagy in human endothelial cells early during infections appear to be lower compared to those in murine macrophages.

Abstract 4: Macrophage p62/SQSTM1 Ameliorates Atherosclerosis by Sequestering Inclusion Bodies and Mediating Mitophagy

Protein and organelle turnover is critical for cellular homeostasis and is prominently mediated by autophagy. Disruptions in autophagy lead to accumulation of protein aggregates and dysfunctional organelles such as mitochondria. Recent evidence suggests that the chaperone protein p62 is a critical link for targeting polyubiquitinated protein aggregates/damaged mitochondria to autophagosomes for degradation. Herein we describe a p62-centric mechanism of handling protein aggregates and dysfunctional mitochondria in atherosclerosis. Macrophages deficient in autophagy (ATG5-/-) or rendered deficient by incubation with atherogenic lipids have significantly increased levels of p62. This coincides with 1) the accumulation of polyubiquitinated proteins co-localizing with p62 and present as cytoplasmic inclusion bodies, and 2) p62 co-localization with mitochondrial markers. Aortas from atherosclerotic (ApoE-/-) mice also have progressive and marked elevations in p62, polyubiquitinated proteins, and mitochondrial reactive oxygen species that predominantly co-localize with plaque macrophages, a process further exacerbated in the autophagy-deficient setting. The formation of cytoplasmic inclusions and maintenance of adequate mitochondrial function appears to be dependent on p62. Lipid-loaded p62-null macrophages show polyubiquitinated protein accumulation present in a diffuse/disrupted cytoplasmic pattern. These macrophages also develop larger dysmorphic mitochondria with increased polarization and decreased oxidative phosphorylation capacity. As a result, p62-null macrophages display apoptotic susceptibility to atherogenic lipids and increased IL-1β secretion likely through mitochondrial-dependent inflammasome activation. Consistent with our in vitro observations, mice with either whole-body p62-deficiency or transplanted with p62-deficient bone marrow show significantly increased atherosclerotic plaque burden and lesion complexity with increased apoptosis and necrotic cores. Taken together, these data demonstrate a previously unrecognized atheroprotective role for macrophage p62 by facilitating the formation of inclusion bodies and maintaining healthy mitochondria.

Structural basis of Atg conjugation systems essential for autophagy

Autophagy is an evolutionarily-conserved, intracellular degradation system for which two ubiquitin-like modifiers, Atg8 and Atg12, play essential roles. After processed by Atg4, the exposed C-terminal glycine of Atg8 is activated by Atg7 (E1) and is then transferred to Atg3 (E2), and is finally conjugated with a phospholipid, phosphatidylethanolamine (PE) through an amide bond. Whereas, Atg12 is activated by the same E1, Atg7, without processing, and is then transferred to Atg10 (E2), and is finally conjugated with Atg5 through an isopeptide bond. Atg12-Atg5 conjugates, together with Atg16, function as an E3-like enzyme to facilitate the conjugation reaction between Atg8 and PE. During autophagy, Atg8-PE conjugates play a critical role in selective cargo recognition in addition to autophagosome formation. We determined the structures of all these Atg proteins and their complexes mainly by X-ray crystallography, and performed structure-based biochemical analyses on them [1,2]. These studies established the molecular mechanisms of Atg8 and Atg12 modification reactions that have many unique features compared with canonical ubiquitin-like systems. Furthermore, we found a conserved motif named the Atg8-family interacting motif (AIM), through which Atg8 recognizes specific cargoes and selectively incorporates them into autophagosomes for degradation [3].

Paternal Mitochondrial Destruction after Fertilization Is Mediated by a Common Endocytic and Autophagic Pathway in Drosophila

Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. We investigated the fate of Drosophila paternal mitochondria after fertilization. We demonstrate that the sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. We further provide evidence for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, we show that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria.

Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

The mitochondrion-associated protein LRPPRC (leucine-rich pentatricopeptide repeat-containing) interacts with one of the microtubule-associated protein family members MAP1S (microtubule-associated protein 1 small form), originally named C19ORF5 (chromosome 19 open reading frame 5), to form a complex. MAP1S interacts with LC3 (light chain 3), the mammalian homologue of yeast autophagy marker ATG8 and one of the most important autophagy markers in mammalian cells, and helps the attachment of autophagosomes with microtubules for trafficking and recruitment of substrate mitochondria into autophagosomes for degradation. MAP1S activates autophagosomal biogenesis and degradation to remove misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress oxidative stress-induced genomic instability and tumorigenesis. Previously, various studies have attributed LRPPRC nucleic acid-associated functions. Instead, in the present study, we show that LRPPRC associates with mitochondria, interacts with Beclin 1 and Bcl-2 and forms a ternary complex to maintain the stability of Bcl-2. Suppression of LRPPRC leads to reduction in mitochondrial potential and reduction in Bcl-2. Lower levels of Bcl-2 lead to release of more Beclin 1 to form the Beclin 1–PI3KCIII (class III phosphoinositide 3-kinase) complex to activate autophagy and accelerate the turnover of dysfunctional mitochondria through the PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) pathway. The activation of autophagy induced by LRPPRC suppression occurs upstream of the ATG5–ATG12 conjugate-mediated conversion of LC3-I into LC3-II and has been confirmed in multiple mammalian cell lines with multiple autophagy markers including the size of GFP–LC3 punctate foci, the intensity of LC3-II and p62 protein and the size of the vacuolar structure. The activated autophagy enhances the removal of mitochondria through lysosomes. LRPPRC therefore acts to suppress the initiation of basal levels of autophagy to clean up dysfunctional mitochondria and other cellular debris during the normal cell cycle.