Table of Contents

Volume 6, Issue 7

  October 1, 2010

  Pages 835 - 1005

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EDITOR'S CORNER

Open Access Article

A matter of balance between life and death: Targeting reactive oxygen species (ROS)-induced autophagy for cancer therapy

Spencer B. Gibson
Pages 835 - 837
http://dx.doi.org/10.4161/auto.6.7.13335
Abstract | PDF

Reactive oxygen species (ROS) have been implicated in many biological functions and diseases. Often their role is counterintuitive, where ROS can either promote cell survival or cell death depending on the cellular context. Similarly, autophagy is involved in many biological functions and diseases where it can either promote cell survival or cell death. There is now a growing consensus that ROS controls autophagy in multiple contexts and cell types. Furthermore, alterations in ROS and autophagy regulation contribute to cancer initiation and progression. However, how ROS and autophagy contribute to cancer and how to target either for cancer treatment is controversial. Blocking ROS generation could prevent cancer initiation, whereas blockage of autophagy seems to be required for initiation of cancer. In cancer progression, high levels of ROS correspond with increased metabolism, and under metabolic stress autophagy is required to maintain cellular integrity. In cancer treatment, therapeutic drugs that increase ROS and autophagy have been implicated in their mechanism for cell death, such as 2-methoxyestrodial (2-ME) and arsenic trioxide (As₂O₃), whereas other therapeutic drugs that induce ROS and autophagy seem to have a protective effect. This has led to different approaches to treat cancer patients where autophagy is either activated or inhibited. Both views of ROS and autophagy are valid and reflect the balance within a cell to either survive or die. Understanding this balancing act within a cell is essential to determine whether to block or activate ROS-controlled autophagy for cancer therapy.




REVIEW

Open Access Article

ROS-mediated mechanisms of autophagy stimulation and their relevance in cancer therapy

Michael Dewaele, Hannelore Maes and Patrizia Agostinis
Pages 838 - 854
http://dx.doi.org/10.4161/auto.6.7.12113
Abstract | PDF

Mounting evidence suggests that reactive oxygen species (ROS) are multifaceted signalling molecules implicated in a variety of cellular programs during physiological as well as pathological conditions. Recently, ROS produced endogenously, by deranged metabolism of cancer cells, or exogenously, by ROS-generating drugs, have been shown to promote macroautophagy, a lysosomal pathway of self-degradation with essential prosurvival functions. Several molecular aspects of the modulation of autophagy pathways by ROS have been revealed in the past years and it is now clear that these processes are mutually linked and play a crucial role in cancer progression and in response to cancer therapeutics. In this review we address the molecular mechanisms underlying the activation of autophagy pathways by ROS and focus on the role of autophagy in cancer cells responding to ROS-producing agents, which are utilized as a therapeutic modality to kill cancer cells.




BASIC BRIEF REPORTS

Open Access Article

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Melissa N. Quinsay, Robert L. Thomas, Youngil Lee and Åsa B. Gustafsson
Pages 855 - 862
http://dx.doi.org/10.4161/auto.6.7.13005
Abstract | PDF

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition, and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes, and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation, and mPTP opening.

Open Access Article

UVRAG mutations associated with microsatellite unstable colon cancer do not affect autophagy

Helene Knævelsrud, Terje Ahlquist, Marianne A. Merok, Arild Nesbakken, Harald Stenmark, Ragnhild A. Lothe and Anne Simonsen
Pages 863 - 870
http://dx.doi.org/10.4161/auto.6.7.13033
Abstract | PDF

Reduced levels of autophagy correlate with tumorigenesis, and several inducers of autophagy have been found to be tumor suppressors. One such autophagic inducer is the Beclin 1 binding protein UVRAG, a positive regulator of the class III PI3K/Vps34 complex. UVRAG has been implicated in the formation and maturation of autophagosomes, as well as in endocytic trafficking and suppression of proliferation and in vivo tumorigenicity. In this study we show that approximately one-third of a large series of colon carcinomas with microsatellite instability (MSI ) (n = 102) carry a monoallelic UVRAG mutation, leading to expression of a truncated protein, indicating that this event is involved in tumorigenesis. In order to investigate whether the high incidence of UVRAG mutation in MSI colorectal carcinomas is associated with dysfunctional autophagy we analyzed autophagy levels in several colon cancer cell lines that express wild-type or mutant UVRAG protein. No reduction in autophagy was detected in cell lines expressing mutant UVRAG. Consistent with this, depletion of UVRAG in HE K cells stably expressing GFP-LC3 did not inhibit autophagy, but did decrease epidermal growth factor receptor (EGFR) degradation. Overall our results show that there is no correlation between the presence of the monoallelic UVRAG mutation and inhibition of autophagy. Thus, our data indicate that mechanisms other than autophagy contribute to the tumorigenicity of microsatellite unstable colon carcinomas with monoallelic UVRAG mutation.

Open Access Article

The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations

Sven Geisler, Kira M. Holmström, Angela Treis, Diana Skujat, Stephanie S. Weber, Fabienne C. Fiesel, Philipp J. Kahle and Wolfdieter Springer
Pages 871 - 878
http://dx.doi.org/10.4161/auto.6.7.13286
Abstract | PDF

Mitochondrial dysfunction is an early sign of many neurodegenerative diseases. Very recently, two Parkinson disease (PD) associated genes, PINK1 and Parkin, were shown to mediate the degradation of damaged mitochondria via selective autophagy (mitophagy). PINK1 kinase activity is needed for prompt and efficient Parkin recruitment to impaired mitochondria. PD-associated Parkin mutations interfere with the process of mitophagy at distinct steps. Here we show that whole mitochondria are turned over via macroautophagy. Moreover, disease-associated PINK1 mutations also compromise the selective degradation of depolarized mitochondria. This may be due to the decreased physical binding activity of PD-linked PINK1 mutations to Parkin. Thus, PINK1 mutations abrogate autophagy of impaired mitochondria upstream of Parkin. In addition to compromised PINK1 kinase activity, reduced binding of PINK1 to Parkin leads to failure in Parkin mitochondrial translocation, resulting in the accumulation of damaged mitochondria, which may contribute to disease pathogenesis.




BASIC RESEARCH PAPERS

Open Access Article

Induction of autophagic flux by amino acid deprivation is distinct from nitrogen starvation-induced macroautophagy

Nitai Ecker, Angelica Mor, Dikla Journo and Hagai Abeliovich
Pages 879 - 890
http://dx.doi.org/10.4161/auto.6.7.12753
Abstract | PDF

A number of signaling mechanisms have been implicated in the regulation of autophagic trafficking. Tor kinase activity, cAMP levels, and the GAAC pathway have all been suggested to be involved. Here, we closely analyzed the stimuli that underlie induction of autophagic trafficking in Saccharomyces cerevisiae. We find evidence for the existence of a novel aspect of the autophagic pathway that is regulated by intracellular amino acids, uncoupled from extracellular nutrient levels, and is absolutely dependent on Gcn2 and Gcn4. This requirement for Gcn2 and Gcn4 distinguishes amino-acid starvation induced autophagy from classic macroautophagy: Macroautophagic flux in response to nitrogen starvation is only partly diminished in gcn2Δ and gcn4Δ cells. However this maintenance of autophagic flux in gcn mutants during nitrogen starvation reflects the formation of larger numbers of smaller autophagosomes. We report that gcn2Δ and gcn4Δ cells are defective in the induction of Atg8 and Atg4 upon starvation, and this defect results, during total nitrogen starvation, in the formation of abnormally small autophagosomes, although overall autophagic flux remains close to normal due to a compensatory increase in the overall number of autophagosomes.

Open Access Article

Autophagic degradation of active caspase-8: A crosstalk mechanism between autophagy and apoptosis

Wen Hou, Jie Han, Caisheng Lu, Leslie A. Goldstein and Hannah Rabinowich
Pages 891 - 900
http://dx.doi.org/10.4161/auto.6.7.13038
Abstract | PDF

Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection. The current study has identified such a mechanism that is centered on the regulation of caspase-8 activity. The study took advantage of Bax-/- Hct116 cells that are TRAIL-resistant despite significant DISC processing of caspase-8, and of the availability of a caspase-8-specific antibody that exclusively detects the caspase-8 large subunit or its processed precursor. Utilizing these biological tools, we investigated the expression pattern and subcellular localization of active caspase-8 in TRAIL-mediated autophagy and in the autophagy-to-apoptosis shift upon autophagy inhibition. Our results suggest that the TRAIL-mediated autophagic response counter-balances the TRAIL-mediated apoptotic response by the continuous sequestration of the large caspase-8 subunit in autophagosomes and its subsequent elimination in lysosomes. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.

Open Access Article

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Takahiko Akematsu, Ronald E. Pearlman and Hiroshi Endoh
Pages 901 - 911
http://dx.doi.org/10.4161/auto.6.7.13287
Abstract | PDF

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible "attack me" signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.

Open Access Article

Glucocorticoids downregulate Fyn and inhibit IP₃-mediated calcium signaling to promote autophagy in T lymphocytes

Michael W. Harr, Karen S. McColl, Fei Zhong, Jason K. Molitoris and Clark W. Distelhorst
Pages 912 - 921
http://dx.doi.org/10.4161/auto.6.7.13290
Abstract | PDF

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.

Open Access Article

Autophagy-induced regression of hyaloid vessels in early ocular development

Jeong Hun Kim, Jin Hyoung Kim, Young Suk Yu, Ji Young Mun and Kyu-Won Kim
Pages 922 - 928
http://dx.doi.org/10.4161/auto.6.7.13306
Abstract | PDF

The hyaloid vessel is a transient intraocular circulatory system that undergoes a complete regression as the retina becomes matured with retinal vascularization. If the complete involution of the hyaloid vessels failes, the pathological persistence of these vessels results in persistent hyperplastic primary vitreous (PHPV) associated with severe ocular pathologies. Unfortunately despite its clinical significance, cellular and molecular processes involved in hyaloid regression remain to be elucidated. Herein, we for the first time demonstrated that autophagy could contribute to the regression of hyaloid vessels in early developing retina. In developing retina, hyaloid vessel regression coincided with retinal vascular development; this occurred simultaneous with apoptotic and autophagic processes. Moreover, in vascular endothelial cells under hypoxic conditions, LC3-II conversion was detected along with caspase-3 activation. The autophagy inducer rapamycin induced autophagy-mediated cell death of vascular endothelial cells in a dose-dependent manner. Moreover, rapamycin significantly enhanced the involution of hyaloid vessels in the early developing eye. Therefore, our results suggest that the autophagy pathway would be involved in hyaloid regression that occurs during early ocular development. Furthermore, activation of the autophagy pathway could be considered for a therapeutic approach to PHPV.




TOOLBOX

Open Access Article

Quantitation of "autophagic flux" in mature skeletal muscle

Jeong-Sun Ju, Arun S. Varadhachary, Sara E. Miller and Conrad C. Weihl
Pages 929 - 935
http://dx.doi.org/10.4161/auto.6.7.12785
Abstract | PDF

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure "autophagic flux" in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3II protein may render possible misinterpretations since LC3II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3II, a technique aptly named the "autophagometer". In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a "colchicine block." Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an "in vivo autophagometer" study using colchicine in skeletal muscle.

Open Access Article

Synthetic substrates for measuring activity of autophagy proteases-autophagins (Atg4)

Chih-Wen Shu, Marcin Drag, Miklos Bekes, Dayong Zhai, Guy S. Salvesen and John C. Reed
Pages 936 - 947
http://dx.doi.org/10.4161/auto.6.7.13075
Abstract | PDF

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-L-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A2 (LC3B-PLA2), which upon cleavage releases active PLA2 for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA2 substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases, and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 x 10⁵ M^-1/sec^-1) than Ac-GTFG-AFC peptide (0.92 M^-1/sec^-1), consistent with substrate induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24, and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.




AUTOPHAGIC PUNCTA

Open Access Article

p66^shc's role as an essential mitophaghic molecule in controlling neuronal redox and energetic tone

Amy M. Kleman, Jacquelynn E. Brown, Stephanie L.H. Zeiger, Jane C. Hettinger, Joshua D. Brooks, Benjamin Holt, Jason D. Morrow, Erik S. Musiek, Ginger L. Milne and BethAnn McLaughlin
Pages 948 - 949
http://dx.doi.org/10.4161/auto.6.7.13007
Abstract | PDF

Stroke is the leading cause of adult disability in the U.S. and is now recognized as a global epidemic. There are currently no FDA approved drugs to block the cell death that results from oxygen and glucose deprivation. This void in clinical medicine has sparked an intense interest in understanding endogenous cellular protective pathways that might be exploited for therapeutic development. The work highlighted here describes the critical role between redox tone and energetic stress signaling in mediating mitophagy and determining neuronal cell fate following acute oxygen glucose deprivation.

Open Access Article

The Golgi as a potential membrane source for autophagy

Jiefei Geng and Daniel J. Klionsky
Pages 950 - 951
http://dx.doi.org/10.4161/auto.6.7.13009
Abstract | PDF

In macroautophagy (hereafter autophagy), a morphological hallmark is the formation of double-membrane vesicles called autophagosomes that sequester and deliver cytoplasmic components to the lysosome/vacuole for degradation. This process begins with an initial sequestering compartment, the phagophore, which expands into the mature autophagosome. A tremendous amount of work has been carried out to elucidate the mechanism of how the autophagosome is formed. However, an important missing piece in this puzzle is where the membrane comes from. Independent lines of evidence have shown that pre-existing organelles may continuously supply lipids to support autophagosome formation. In our analysis, we identified several components of the late stage secretory pathway that may redirect Golgi-derived membrane to autophagosome formation in response to starvation conditions.

Open Access Article

Uncovering the roles of PINK1 and Parkin in mitophagy

Noriyuki Matsuda and Keiji Tanaka
Pages 952 - 954
http://dx.doi.org/10.4161/auto.6.7.13039
Abstract | PDF

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, and thus elucidation of the pathogenic mechanism and establishment of a fundamental cure is essential in terms of public welfare. Fortunately, our understanding of the pathogenesis of two types of recessive familial PDs-early-onset familial PD caused by dysfunction of the PTEN induced putative kinase 1 (PINK1) gene and autosomal recessive juvenile Parkinsonism (AR-JP) caused by a mutation in the Parkin gene-has evolved and continues to expand.

Open Access Article

SnoN/SkiL, a TGFβ signaling mediator: A participant in autophagy induced by arsenic trioxide

Fadi Raffoul, Christie Campla and Meera Nanjundan
Pages 955 - 957
http://dx.doi.org/10.4161/auto.6.7.13041
Abstract | PDF

Effective treatment of cancer cells with chemotherapeutic drugs relies on their ability to induce cell death, making the discovery of their mechanisms of action crucial. Arsenic trioxide (As₂O₃), used in the treatment of promyelocytic leukemia (PML), triggers cell death in several solid tumor cell lines including ovarian carcinomas. While As₂O₃ is remarkably cytotoxic in human ovarian cancer cells, its mechanism of action is poorly understood. We recently investigated the effects of As₂O₃ on several transforming growth factor-β (TGFβ) signaling mediators to better understand its cell death mechanism. Indeed, dysregulated (TGFβ) signaling is typical of ovarian cancers. Based on our findings, we propose that As₂O₃ induces a Beclin 1-independent autophagic pathway in ovarian carcinoma cells by modulating SnoN/SkiL expression, implicating SnoN as a novel therapeutic target for ovarian cancers.

Open Access Article

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases

Valeria Crippa, Serena Carra, Paola Rusmini, Daniela Sau, Elena Bolzoni, Caterina Bendotti, Silvia De Biasi and Angelo Poletti
Pages 958 - 960
http://dx.doi.org/10.4161/auto.6.7.13042
Abstract | PDF

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motorneurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signalling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motorneuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.

Open Access Article

Autophagy, proteases and the sense of balance

Sandra Cabrera, Guillermo Mariño, Alvaro F. Fernández and Carlos López-Otín
Pages 961 - 963
http://dx.doi.org/10.4161/auto.6.7.13065
Abstract | PDF

The knowledge of the molecular mechanisms underlying autophagy has considerably improved after the isolation and characterization of autophagy-defective mutants in the yeast Saccharomyces cerevisiae. Two ubiquitin-like conjugation systems are required for yeast autophagy. One of them requires the participation of Atg8 synthesized as a precursor protein, which is cleaved after a Gly residue by a cysteine proteinase called Atg4. The new Gly-terminal residue from Atg8 is activated by Atg7 (an E1-like enzyme) then transferred to Atg3 (an E2-like enzyme) and finally conjugated with membrane-bound phosphatidylethanolamine (PE) through an amide bond. The complex Atg8–PE is also deconjugated by the protease Atg4, facilitating the release of Atg8 from membranes. This modification system, which is essential for the membrane rearrangement dynamics that accompany the initiation and execution of autophagy, is conserved in higher eukaryotes including mammals. We have previously identified and cloned the four human orthologues of the yeast proteinase Atg4, whereas parallel studies have revealed that there are at least six orthologues of yeast Atg8 in mammals (LC3A, LC3B, LC3C, GABARAP, ATG8L/GABARAPL1 and GATE-16/GABARAPL2). Thus, in mammals, the Atg4-Atg8 proteolytic system is composed of four proteinases (autophagins) that may target at least six distinct substrates, contrasting with the simplified yeast system in which one single protease cleaves a sole substrate. Currently, it is unclear why mammals have developed this array of closely related enzymes, as other essential autophagy genes such as Atg3, Atg5 or Atg7 are represented in mammalian cells by a single orthologue. It has been suggested that the multiplication of Atg4 orthologues may reflect a regulatory heterogeneity of functionally redundant proteins or, alternatively, derive from the acquisition of new functions that are not related to autophagy. Our first approach to elucidate this question was based on the generation of autophagin-3/Atg4C-deficient mice, which however presented a minor phenotype. With the generation of autophagin-1/Atg4B-deficient mice, recently reported, we have progressed in our attempt to identify the in vivo physiological and pathological roles of autophagins.

Open Access Article

Autophagic degradation of an oncoprotein

Stig Ove Bøe and Anne Simonsen
Pages 964 - 965
http://dx.doi.org/10.4161/auto.6.7.13066
Abstract | PDF

Acute promyelocytic leukemia (APL) is characterized by a chromosomal t(15;17) translocation that fuses the gene encoding the promyelocytic leukemia protein (PML) to that encoding retinoic acid receptor alpha (RARA). The product of this genetic aberration, the PML/RARA fusion protein, is highly oncogenic and supports malignant transformation and growth of hematopoietic precursor cells at the promyelocytic stage of differentiation. Successful treatment of APL by all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) depends on the ability of these drugs to induce proteolytic degradation of this chimeric protein. In a recently published study we demonstrate that PML/RARA is amenable for degradation by autophagy and that ATRA- and ATO-induced PML/RARA degradation is autophagy dependent. Consequently, autophagic degradation regulates basal turnover as well as therapy-induced elimination of this oncoprotein. In addition, our study reveals an important role of autophagy in promoting granulocytic differentiation of APL cells.

Open Access Article

Induction of autophagy by dual mTORC1-mTORC2 inhibition in BCR-ABL-expressing leukemic cells

Eliza Vakana, Antonella Sassano and Leonidas C. Platanias
Pages 966 - 967
http://dx.doi.org/10.4161/auto.6.7.13067
Abstract | PDF

Over recent years, there have been substantial research advances on the mechanisms by which BCR-ABL transforms hematopoietic cells and promotes leukemic cell growth and survival. Among the diverse signaling cascades activated by BCR-ABL, the mTOR pathway plays a critical role in mRNA translation of genes that promote leukemogenesis and mitogenic responses. We have recently shown that dual targeting of mTORC1 and mTORC2 complexes using a catalytic mTOR inhibitor, OSI-027, results in generation of potent antileukemic effects against BCR-ABL transformed cells. Such effects were also seen in cells expressing the T315I mutation, which is resistant to all currently approved BCR-ABL kinase inhibitors. Our studies also demonstrate that such dual catalytic inhibition of mTORC2 and mTORC1 complexes in BCR-ABL-expressing K562 cells results in induction of autophagy, and that inhibition of the autophagic process using chloroquine promotes apoptosis of these cells. Altogether, our studies suggest that autophagy may be a limiting factor for the induction of apoptosis during dual mTORC2-mTORC1 targeting, in at least some types of BCR-ABL-expressing cells and have raised the potential of combinations of catalytic inhibitors of mTOR with autophagy inhibitors for the treatment of refractory Ph+ leukemias.

Open Access Article

Glutaminolysis yields a metabolic by-product that stimulates autophagy

Christina H. Eng and Robert T. Abraham
Pages 968 - 970
http://dx.doi.org/10.4161/auto.6.7.13082
Abstract | PDF

Autophagy is an intracellular degradative pathway that plays key roles in the homeostatic turnover of long-lived or damaged proteins and organelles, and in the survival of cells during starvation or other stressful conditions. We have uncovered an unexpected link between glutamine (Gln) metabolism and the regulation of autophagy. Our findings indicate that ammonia, generated from Gln deamination in mitochondria, functions as an autocrine- and/or paracrine-acting stimulator of autophagic flux.

Open Access Article

Abnormal mitochondrial autophagy in nephropathic cystinosis

Poonam Sansanwal and Minnie M. Sarwal
Pages 971 - 973
http://dx.doi.org/10.4161/auto.6.7.13099
Abstract | PDF

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with 3 clinical variants of cystinosis: nephropathic, intermediate, and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

Open Access Article

Acinus: A nuclear regulator of autophagy and endocytic trafficking

Helmut Krämer
Pages 974 - 975
http://dx.doi.org/10.4161/auto.6.7.13100
Abstract | PDF

Fusion with lysosomes is the common last step of endocytic trafficking and autophagy. Accordingly, several proteins are required in both pathways for cargoes to reach their destinations. Among these proteins, Drosophila Acinus stands out, as it exerts opposite effects on these two pathways, and thus establishes a new paradigm. Loss of Acinus function destabilizes early endosomes, thereby promoting the delivery of their cargo to lysosomes. By contrast, the maturation of autophagosomes to autolysosomes is inhibited in acn mutant cells. The increase in autophagy upon Acinus overexpression and its location to the nucleus are consistent with Acinus being a novel regulator of autophagy.

Open Access Article

The combination of a histone deacetylase inhibitor with the BH3-mimetic GX15-070 has synergistic antileukemia activity by activating both apoptosis and autophagy

Yue Wei, Tapan Kadia, Weigang Tong, Ming Zhang, Yu Jia , Hui Yang, Yumin Hu, Jean Viallet, Susan O’Brien and Guillermo Garcia-Manero
Pages 976 - 978
http://dx.doi.org/10.4161/auto.6.7.13117
Abstract | PDF

We analyzed the cellular and molecular effects of two different histone deacetylase inhibitors (HDACi), MGCD0103 and vorinostat, in combination with GX15-070, a BH3-mimetic, in acute myeloid leukemia (AML) cell lines and primary AML cells, and demonstrated that the combination has a synergistic antileukemia effect. We observed that in addition to apoptosis, autophagy also accounts for the observed non-apoptotic decrease of cell viability. Mechanistically, we established a role for calpain activity and ER-located caspase signaling in the induction of both autophagy and apoptosis following this combination of drugs. These findings reveal that for this specific combination, autophagy plays a positive role inducing cytotoxicity, and that the involved ER signaling networks, as well as their clinical relevance, should be further studied in both preclinical and clinical trials of leukemia and other malignancies.

Open Access Article

Ubiquilin at a crossroads in protein degradation pathways

Cara Rothenberg and Mervyn J. Monteiro
Pages 979 - 980
http://dx.doi.org/10.4161/auto.6.7.13118
Abstract | PDF

Ubiquilin proteins are conserved across all eukaryotes and function in the regulation of protein degradation. We found that ubiquilin functions to regulate macroautophagy and that the protein is also a substrate of chaperone-mediated autophagy.

Open Access Article

Autophagic receptors Nbr1 and p62 coregulate skeletal remodelling

Sarah L. Waters , Katie Marchbank, Ellen Solomon and Caroline A. Whitehouse
Pages 981 - 983
http://dx.doi.org/10.4161/auto.6.7.13155
Abstract | PDF

Skeletal remodelling is an ongoing process requiring the coordinated action of different cell types to maintain homeostatic control of bone synthesis and degradation. Mutations in p62/SQSTM1 are associated with sporadic and 5q35-linked Paget’s Disease of Bone (PDB), characterized by focal increased bone turnover. These mutations cluster in the ubiquitin associated (UBA) domain and are thought to lead to enhancement of NFκB pathway activation involved in osteoclastogenesis and hyper-responsiveness to receptor activator of nuclear factor-κB ligand (RANKL). The structurally similar selective autophagic receptor, Nbr1, binds to LC3 and p62, and is sequestered into autophagosomes, whereas it accumulates in autophagic-deficient tissues. We have shown that truncation of Nbr1 in a murine model, where it can still interact with p62 but not LC3, leads to increased osteoblast differentiation and activity in vivo. This results in an age-dependent increase in bone mass and bone mineral density. This is a molecular consequence of loss of autophagy receptor function via deletion of its C-terminal UBA domain, and/or modulation of the p38 MAPK cellular signalling pathway.

Open Access Article

Four metazoan autophagy genes regulate cargo recognition, autophagosome formation and autolysosomal degradation

Ye Tian, Haiyan Ren, Yu Zhao, Qun Lu, Xinxin Huang, Peiguo Yang and Hong Zhang
Pages 984 - 985
http://dx.doi.org/10.4161/auto.6.7.13156
Abstract | PDF

The mechanism responsible for induction and maturation of autophagosomes in multicellular organisms is poorly understood. We performed genetic screens in C. elegans and identified three essential autophagy genes, epg-3, -4 and -5, which have highly conserved homologs in mammals, but are absent in yeast. We also identified a nematode-specific gene, epg-2, that is required for degradation of components of the specialized protein aggregates, called PGL granules. epg-2, -3, -4 and -5 define discrete genetic steps of the autophagy pathway. We further demonstrated that mammalian homologs of EPG-3, -4 and -5 are essential for starvation-induced autophagy. Our study establishes C. elegans as a model to identify components of the basal autophagy pathway specific to higher eukaryotes and to further assemble these genes into genetic pathways.

Open Access Article

Traf6 and A20 differentially regulate TLR4-Induced autophagy by affecting the ubiquitination of Beclin 1

Chong-Shan Shi and John H. Kehrl
Pages 986 - 987
http://dx.doi.org/10.4161/auto.6.7.13288
Abstract | PDF

Toll-like receptor 4 (TLR4) signaling triggers autophagy, which has been linked to both adaptive and innate immunity. Engagement of TLR4 recruits to the receptor complex Beclin 1, a key component of a class III phosphatidylinositol 3-kinase complex (PI3KC3) that initiates autophagosome formation. Recently, we found that tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-mediates Lys⁶³ (K63)-linked ubiquitination of Beclin 1 is crucial for TLR4-triggered autophagy in macrophages. We identified two TRAF6-binding motifs in Beclin 1 that facilitate the binding of TRAF6 and the ubiquitination of Beclin 1. A lysine located in the Bcl-2 homology 3 (BH3) domain of Beclin 1 serves as a major site for K63-linked ubiquitination. Opposing TRAF6, the deubiquitinating enzyme A20 reduces the extent of K63-linked ubiquitination of Beclin 1 and limits the induction of autophagy in response to TLR4 signaling. Furthermore, treatment of macrophages with either interferon- or interleukin-1 triggers the K63-linked ubiquitination of Beclin 1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin 1 plays a key role in regulating autophagy during inflammatory responses.

Open Access Article

Anti-neoplastic activity of the cytosolic FoxO1 results from autophagic cell death

Ying Zhao, Lina Wang, Jing Yang, Ping Zhang, Ke Ma, Jingyi Zhou, Wenjuan Liao and Wei-Guo Zhu
Pages 988 - 990
http://dx.doi.org/10.4161/auto.6.7.13289
Abstract | PDF

Although Beclin 1 and mTOR are considered to be the main molecules to modulate the autophagic process, searching for other autophagy-regulating molecules is still an ongoing challenge to scientists. Here we demonstrated that FoxO1, a forkhead O family protein, is a mediator of autophagy. Upon oxidative stress or serum starvation, endogenous FoxO1 was required for autophagy in human cancer cell lines, and this process was independent of FoxO1's transcriptional activity as well as mTOR or Beclin 1. In response to stress, FoxO1 dissociated from an NAD+-dependent histone deacetylase SIRT2, and FoxO1 thus became acetylated, and in turn bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. In particular, cytosolic FoxO1 suppressed tumor xenograft growth in nude mice in an autophagy-dependent manner. Our studies provide evidence that cytosolic FoxO1-induced autophagy is associated with tumor suppression function.

Open Access Article

Impaired autophagy in Lafora disease

Erwin Knecht, Carmen Aguado, Sovan Sarkar, Viktor I. Korolchuk, Olga Criado-García, Santiago Vernia, Patricia Boya, Pascual Sanz, Santiago Rodríguez de Córdoba and David C. Rubinsztein
Pages 991 - 993
http://dx.doi.org/10.4161/auto.6.7.13308
Abstract | PDF

Lafora disease (LD) is a progressive, lethal, autosomal recessive, neurodegenerative disorder that manifests with myoclonus epilepsy. LD is characterized by the presence of intracellular inclusion bodies called Lafora bodies (LB), in brain, spinal cord and other tissues. More than 50% of LD is caused by mutations in EPM2A that encodes laforin. Here we review our recent findings that revealed that laforin regulates autophagy. We consider how autophagy compromise may predispose to LB formation and neurodegeneration in LD, and discuss future investigations suggested by our data.

Open Access Article

Autophagy-independent LC3 function in vesicular traffic

Cornelis A.M. de Haan, Maurizio Molinari and Fulvio Reggiori
Pages 994 - 996
http://dx.doi.org/10.4161/auto.6.7.13309
Abstract | PDF

As protein folding is an imperfect process, the endoplasmic reticulum (ER) contains folding as well as ER-associated degradation (ERAD) machineries. In order to prevent premature interruption of folding, ERAD regulators and effectors such as EDEM1 and OS-9 are selectively cleared from the ER in so-called EDEMosomes to downregulate the degradative activity. The mechanism by which EDEM1 and OS-9 are subjected to rapid turnover, also known as ERAD tuning, shows similarities with, but is clearly distinct from, macroautophagy. Positive strand RNA coronaviruses (CoVs) such as the severe acute respiratory syndrome (SARS)-CoV and mouse hepatitis virus (MHV), induce in infected cells the formation of autophagosome-like, double-membrane vesicles (DMVs) to which their replication and transcription complexes are anchored. While it seems clear that CoVs hijack ER-derived host cell membranes for replication, the mechanism by which these DMVs are assembled has remained completely mysterious.

Open Access Article

Autophagy in pancreatic cancer: An emerging mechanism of cell death

Nameeta Mujumdar and Ashok K. Saluja
Pages 997 - 998
http://dx.doi.org/10.4161/auto.6.7.13334
Abstract | PDF

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1, and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.




VIEWS AND COMMENTARIES

Open Access Article

Under the ROS: Thiol network is the principal suspect for autophagy commitment

Giuseppe Filomeni, Enrico Desideri, Simone Cardaci, Giuseppe Rotilio and Maria Rosa Ciriolo
Pages 999 - 1005
http://dx.doi.org/10.4161/auto.6.7.12754
Abstract | PDF

Low molecular weight and protein sulphydryls undergo reactive oxygen species (ROS)-mediated oxidation. However, differently from the irreversible damages that oxidative conditions yield on biomolecules, the oxidation of reactive cysteines frequently results in reversible modifications, which represent the prototype of the molecular mechanisms underlying redox signaling. Many proteins involved in a wide range of cellular processes have been classified as "redox-sensitive", thereby modulating their function/activity in dependence of the redox state of their critical thiols. Growing pieces of evidence from the last few years are supporting the idea that ROS production also correlates with the occurrence of autophagy. Nonetheless, the cysteine protease Atg4 remains the sole example of a protein whose redox regulation has been completely characterized and demonstrated to be necessary for the progression of autophagy. The principal aim of this commentary is to draw attention on the remarkable number of proteins that can fit the double role of: (i) being involved in autophagy, especially in autophagosome formation; (ii) sensing alterations of the cellular redox state by means of reactive cysteine residues. We will also attempt to provide a hypothetical model to explain the possible functional role of thiols in the occurrence of autophagy and outline a network of redox reactions likely concurring to allow the correct initiation and completion of autophagosomes.