WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

Autocrine stimulation by insulin-like growth factor I is involved in the growth, tumorigenicity and chemoresistance of human esophageal carcinoma cells

Insulin-like growth factor I (IGF-I) receptor (IGF-IR)-mediated signals are known to be involved in cell growth and transformation and prevention of apoptosis. In this study, we demonstrated the coexpression of IGF-I and IGF-IR in human esophageal carcinoma tissues. We also demonstrated the IGF-I autocrine system in esophageal carcinoma cell lines. Both the CE48T/VGH and CE81T/VGH cell lines showed proliferative responses to IGF-I stimulation. Autokinase activity of IGF-IR in these cells can be triggered by the exogenous addition of IGF-I. In addition, an IGF-I peptide antagonist, JB1, specifically inhibited ligand-induced receptor autophosphorylation in a dose-dependent manner. Under serum-free conditions, JB1 also reduced the degree of IGF-IR phosphorylation and cell numbers. Furthermore, the addition of JB1 decreased the number of CE81T/VGH colonies formed in methyl cellulose agar and the size and the incidence of tumors which grew in mice with severe combined immunodeficiency. These results imply that an IGF-I autocrine system in human esophageal carcinoma cells could stimulate tumor growth. Finally, we found that IGF-I prevented the apoptosis of CE81T/VGH cells induced by chemotherapeutic drugs, such as cisplatin, 5-fluorouracil and camptothecin. Thus, interruption of IGF-IR function may provide a way to retard tumor growth and increase the sensitivity of esophageal carcinoma to chemotherapy.     

Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.     

Platelet-Derived Growth Factor Is an Autocrine Stimulator for the Growth and Survival of Human Esophageal Carcinoma Cell Lines

Platelet-derived growth factors (PDGF) are important mitogens for mesenchyme-derived cells. Neither PDGF nor PDGF receptors (PDGFR) are expressed in epithelial cells under normal physiological conditions. However, we have found that PDGF-BB induces c-junexpression and promotes the growth of the human esophageal carcinoma cell line CE48T/VGH. Scatchard analysis revealed the presence of 6 × 10⁵binding sites for PDGF-BB per cell, with a Kd of 9.7 nM. Furthermore, our data indicate that CE48T/VGH expresses β type PDGFR (PDGFRβ) within vitroauto-kinase activity. We have also found that CE48T/VGH expresses the mRNA of the PDGF-A and PDGF-B chains and secretes PDGF molecules. Addition of anti-PDGF neutralizing antibody significantly decreased cell numbers of CE48T/VGH under serum-free conditions. The detached cells underwent apoptosis characterized by micronucleation. These results suggest that expression of the PDGF autocrine system may not only provide the growth advantage but also prevent the apoptosis for CE48T/VGH.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

Arecoline and the 30–100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified ≥10 kDa fraction (ANE ≥ 10 K) and subsequently to the 30–100 kDa fraction (ANE 30–100 K). ANE and ANE ≥10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE ≥ 10 K, and ANE 30–100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30–100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser²⁴⁴⁸. In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser²⁴⁴⁸ phosphorylation are capable of triggering apoptosis and autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Shyun-Yeu Liu, Mei-Huei Lin, Yu-Rung Hsu and Ya-Yun Shih contributed equally to this work.     

Atg14L and Rubicon: Yin and yang of Beclin 1-mediated autophagy control

Emerging evidence suggests that Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, functions to coordinate two important cellular pathways: autophagy and apoptosis. Beclin 1 is a component of the Vps34/class III phosphatidylinositol 3-kinase (PtdIns3K) protein complex. However, the Beclin 1-Vps34/PtdIns3K protein complex formation and its function in autophagy regulation remain to be elucidated. Through an integrated approach that combines mouse genetics and biochemistry, we identified two novel Beclin 1 interacting proteins, Atg14L and Rubicon. We found that Atg14L and Rubicon play opposing roles in autophagy regulation by forming distinct complexes with Beclin 1, modulating the Vps34/PtdIns3K activity and targeting distinct steps of the autophagic process.     

Autophagy induction by a natural ingredient of areca nut

We recently identified an autophagy-inducing areca nut ingredient (AIAI) in the partially purified 30-100 kDa fraction of areca nut extract (ANE), designated as ANE 30-100K. Before disintegration, most ANE 30-100K-treated cells exhibit rounding morphology, cytoplasmic clearance, and nuclear shrinkage, distinct from arecoline- and cisplatin-induced cellular apoptosis. This unique death pattern is verified to be autophagy by LC3-I cleavage, acidic vesicles, and autophagic vacuoles. As analyzed by Molish's Test, Selinowaff's Test, and thin-layer chromatography, most of the ANE 30-100K constituents are carbohydrates, whereas the protein content of this fraction is less than 1% as assessed by protein assay reagent. The cytotoxicity of ANE 30-100K is further shown to be sensitive to cellulase and proteinase K digestion suggesting AIAI in ANE 30-100K to be a proteoglycan (or glycoprotein). Thus, although ANE contains apoptosis-inducing ingredients such as arecoline, it predominantly triggers autophagic cell death by this natural AIAI.     

A Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1

Autophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy.Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1), antiaging (2–4), development (1, 5), protection of the genome (6), and regulation of cell size (7). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8–10), cardiomyopathy (11), and neurodegenerative disorders (12).Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg)² are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13, 14).In mammalian systems, autophagosomes fuse with lysosomes to generate autophagolysosomes, which undergo a maturation process by fusing with endocytic compartments and lysosomes (15). Because it is not known how the Atg1 homolog acts in mammals, a different mechanism may be involved in regulating autophagy. Beclin 1/Atg6, microtubule-associated protein 1 light chain 3 (LC3)/Atg8, Atg5, Atg12, and Atg13 are essential for autophagosome formation in mammalian species (5, 16–20). Atg7 and Atg3 are required in the conjugation reaction between Atg12 and Atg5 and in the lipidation of LC3. During the formation of autophagosomes in mammalian cells, LC3 is lipidated via a ubiquitylation-like system (17, 21), generating a soluble form, LC3-I. LC3-I is further modified to a membrane-bound form, LC3-II, which is subsequently localized to autophagosomes and autolysosomes until being degraded by the lysosome.Beclin 1 was initially isolated as a B-cell lymphoma-2 (Bcl2)-interacting tumor suppressor in mammalian cells (22). Overexpression of Bcl2 attenuates the formation of the kinase complex Beclin 1-class III phosphatidylinositol 3-kinase (PI3KC3) essential for the formation of autophagosomes (23). The UV radiation resistance-associated gene tumor suppressor and the activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra 1) were identified as new Beclin 1-binding partners that also regulate autophagy by regulating the Beclin 1-PI3KC3 kinase complex. Association of Beclin 1 with PI3KC3 is negatively regulated by Bcl2 (22) and positively regulated by UV radiation resistance-associated gene tumor suppressor and Ambra 1 (24, 25). Beclin 1 is homoallelically deleted in many human tumors. A decreased Beclin 1 level causes defective autophagy and breast cancer, but restoration of Beclin 1 induces autophagy and inhibits tumorigenicity of human breast cancer cells (18). These reports evidence the dependence on Beclin 1 for a functional autophagy mechanism.Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells (26, 27). In contrast to yeast, mammalian cells regulate autophagy via both class I and class III PI3K. Class I PI3K plays an inhibitory role, whereas class III PI3K kinase complex, which includes Beclin 1, plays a stimulatory role in autophagy by promoting the nucleation of autophagic vesicles (28, 29). A recent study also indicates that hVps15 is required in regulation of class III PI3K in mammalian cells (30). However, the signaling mechanisms controlling autophagy-essential proteins, in particular Beclin 1, and the opposing consequences of autophagy remain to be resolved.Our present studies identified and positioned a non-canonical MEK/ERK pathway downstream of AMPK and upstream of TSC and mTOR. This MEK/ERK module regulated autophagy via regulating the Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR pathway. Moderately enhanced Beclin 1 by transient or moderate activation of MEK/ERK and subsequent inhibition on mTORC1 and mTORC2 individually caused protective autophagy. Strongly pronounced Beclin 1 by sustained or strong activation of MEK/ERK followed by dual inhibition on mTORC1 and mTORC2 caused destructive autophagy. Our results thus reveal interesting Beclin 1 thresholds in regulating autophagy.     

Binding Rubicon to cross the Rubicon

Beclin 1 is an antitumor protein, required for mammalian autophagy, but its precise molecular function is poorly understood. Mass spectrometry analysis reveals that two novel proteins, Atg14L and Rubicon, associate with Beclin 1, together with a known Beclin 1-binding protein, UVRAG. The interactions of Atg14L and UVRAG with the Beclin 1-Vps34 (class III PI3-kinase)-Vps15 core complex are mutually exclusive; Rubicon associates with a subpopulation of UVRAG-containing complexes. The Atg14L complex, which positively regulates autophagy at an early step, localizes to the phagophore/isolation membrane, autophagosome and endoplasmic reticulum. In contrast, the Rubicon-UVRAG complex localizes to the late endosome/lysosome and negatively regulates both autophagy at a later step and the endocytic pathway. Thus, the Beclin 1-Vps34-Vps15 complex functions in autophagy and the endocytic pathway, but its function in a given context depends on the identity of its interacting subunits.     

Autophagy induction by a natural ingredient of areca nut

We recently identified an autophagy-inducing areca nut ingredient (AIAI) in the partially purified 30-100 kDa fraction of areca nut extract (ANE), designated as ANE 30-100K. Before disintegration, most ANE 30-100K-treated cells exhibit rounding morphology, cytoplasmic clearance, and nuclear shrinkage, distinct from arecoline- and cisplatin-induced cellular apoptosis. This unique death pattern is verified to be autophagy by LC3-I cleavage, acidic vesicles and autophagic vacuoles. As analyzed by Molish’s Test, Selinowaff’s Test, and thin-layer chromatography, most of the ANE 30-100K constituents are carbohydrates, whereas the protein content of this fraction is less than 1% as assessed by Protein Assay reagent. The cytotoxicity of ANE 30-100K is further shown to be sensitive to cellulase and proteinase K digestion suggesting AIAI in ANE 30-100K to be a proteoglycan (or glycoprotein). Thus, although ANE contains apoptosis-inducing ingredients such as arecoline, it predominantly triggers autophagic cell death by this natural AIAI.

Addendum to: Liu SY, Lin MH, Hsu YR, Shih YY, Chiang WF, Lee CH, Chou TH, Liu YC. Arecoline and the 30-100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: A pilot study. J Biomed Sci 2008; doi:10.1007/s11373-008-9273-8.     

The CD40-autophagy pathway is needed for host protection despite IFN-Γ-dependent immunity and CD40 induces autophagy via control of P21 levels

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40(-/-) mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1(+/-) mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40(-/-) mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ.     

电针对快速衰老小鼠肝脏自噬及脂质代谢的影响

目的: 研究电针对快衰老小鼠(SAMP8)肝脏中自噬相关因子LC3-Ⅱ、Beclin1、Atg7、P62表达的影响,探讨电针改善衰老小鼠肝脏脂质代谢的机制。方法: 30周龄雄性SAMP8小鼠随机分为模型组、药物组、电针组,每组7只,以同周龄抗快速老化SAMR1小鼠7只作为对照组。对照组和模型组动物常规饲养4周,不进行任何干预;药物组采用雷帕霉素按(10 mg·kg^-1·d^-1)腹腔注射干预(1次/日,连续每周6 d);电针组予双侧“肾俞”“太冲”穴电针治疗(15 min/次,1次/日,连续每周6 d)。检测小鼠的血清脂质代谢情况、肝脏脂质沉积情况、肝脏自噬体的分布、肝脏LC3-Ⅱ、Beclin1、Atg7、P62蛋白表达与mRNA表达的变化。结果: 与对照组比较,模型组小鼠血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)显著升高(P＜0.01);模型组肝脏脂滴沉积明显,自噬体减少,自噬相关因子LC3-Ⅱ、Beclin1、Atg7蛋白及mRNA表达水平明显下降(P＜0.01),P62蛋白及mRNA表达水平明显增高(P＜0.01)。与模型组相比,电针组与药物组小鼠血清TG、TC、LDL含量明显降低(P＜0.01);肝脏脂滴沉积减轻,自噬体增多,LC3-Ⅱ、Beclin1、Atg7的蛋白及mRNA表达水平均显著升高(P＜0.01),P62蛋白及mRNA表达水平显著下降(P＜0.01)。电针组肝脏Beclin1、Atg7蛋白及mRNA表达水平均与药物组无明显差异(P＞0.05)。结论: 电针能够减轻肝脏脂质代谢紊乱,可能与调节肝脏自噬相关因子LC3-Ⅱ、Beclin1、Atg7、P62的表达变化,从而促进SAMP8小鼠肝脏自噬有关。     

Pivotal role of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in apoptosis and autophagy

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C(2)-ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21(+/+) mouse embryonic fibroblasts (MEFs). On the other hand, C(2)-ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21(-/-) MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21(+/+) MEFs treated with C(2)-ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C(2)-ceramide-treated p21(-/-) MEFs. gamma-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.     

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.     

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.