Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

Expression profile and molecular function of beclin-1 in Epinephelus akaara in response to immune stimuli and oxidative stress.

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H2O2-mediated apoptosis, as evidenced by the MTT assay, analysis of mRNA expression levels of apoptotic genes, and AO-EtBr staining. Overall, our study demonstrated the protective role of EaBECN-1 against viral pathogenesis and oxidative stress through autophagy, increasing our understanding of the role of beclin-1 in fish.

Genetic Analyses of the Arabidopsis ATG1 Kinase Complex Reveal Both Kinase-Dependent and Independent Autophagic Routes during Fixed-Carbon Starvation.

Under nutrient and energy-limiting conditions, plants up-regulate sophisticated catabolic pathways such as autophagy to remobilize nutrients and restore energy homeostasis. Autophagic flux is tightly regulated under these circumstances through the AuTophaGy-related1 (ATG1) kinase complex, which relays upstream nutrient and energy signals to the downstream components that drive autophagy. Here, we investigated the role(s) of the Arabidopsis (Arabidopsis thaliana) ATG1 kinase during autophagy through an analysis of a quadruple mutant deficient in all four ATG1 isoforms. These isoforms appear to act redundantly, including the plant-specific, truncated ATG1t variant, and like other well-characterized atg mutants, homozygous atg1abct quadruple mutants display early leaf senescence and hypersensitivity to nitrogen and fixed-carbon starvations. Although ATG1 kinase is essential for up-regulating autophagy under nitrogen deprivation and short-term carbon starvation, it did not stimulate autophagy under prolonged carbon starvation. Instead, an ATG1-independent response arose requiring phosphatidylinositol-3-phosphate kinase (PI3K) and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), possibly through phosphorylation of the ATG6 subunit within the PI3K complex by the catalytic KIN10 subunit of SnRK1. Together, our data connect ATG1 kinase to autophagy and reveal that plants engage multiple pathways to activate autophagy during nutrient stress, which include the ATG1 route as well as an alternative route requiring SnRK1 and ATG6 signaling.plantcell;31/12/2973/FX1F1fx1.

14-3-3ζ binds to and stabilizes phospho-beclin 1S295 and induces autophagy in hepatocellular carcinoma cells.

Data from The Cancer Genome Atlas (TCGA) indicate that the expression levels of 14-3-3ζ and beclin 1 (a key molecule involved in cellular autophagy) are up-regulated and positively correlated with each other (R = .5, P < .05) in HCC tissues. Chemoresistance developed in hepatoma cancer cells is associated with autophagy initiation. This study aimed to explore 14-3-3ζ's role in regulating autophagy in HCC cells, with a focus on beclin 1. The co-localization of 14-3-3ζ and beclin 1 was detectable in primary HCC tissues. To simulate in vivo tumour microenvironment (hypoxia), CSQT-2 and HCC-LM3 cells were exposed to 2% oxygen for 24 hours. The protein levels of 14-3-3ζ and phospho-beclin 1S295 peaked at 12 hours following hypoxia. Meanwhile, the strongest autophagy flux occurred: LC3II was increased, and p62 was decreased significantly. By sequencing the coding area of BECN 1 gene of CSQT-2 and HCC-LM3 cells, we found that the predicted translational products of BECN 1 gene contained RLPS295 VP (R, arginine; L, leucine; P, proline; S, serine; V, valine), a classic 14-3-3ζ binding motif. CO-IP results confirmed that 14-3-3ζ bound to beclin 1, and this connection was markedly weakened when S295 was mutated into A295 (alanine). Further, 14-3-3ζ overexpression prevented phospho-beclin 1S295 from degradation and enhanced its binding to VPS34, whilst its knockdown accelerated the degradation. Additionally, 14-3-3ζ enhanced the chemoresistance of HCC cells to cis-diammined dichloridoplatium by activating autophagy. Our work reveals that 14-3-3ζ binds to and stabilizes phospho-beclin 1S295 and induces autophagy in HCC cells to resist chemotherapy.

Peptide-Based Autophagic Gene and Cisplatin Co-delivery Systems Enable Improved Chemotherapy Resistance.

Cisplatin-based chemotherapy is a widely used first-line strategy for numerous cancers. However, drug resistances are often inevitable accompanied by the long-term use of cisplatin in vivo, significantly hampering its therapeutic efficacy and clinical outcomes. Among others, autophagy induction is one of the most common causes of tumor resistance to cisplatin. Herein, a self-assembled nanoprodrug platform was developed with the synergistic effect of cisplatin and RNAi to fight against cisplatin-resistant lung cancer. The nanoprodrug platform consists of three molecular modules, including prodrug complex of Pt(IV)-peptide-bis(pyrene), DSPE-PEG, and cRGD-modified DSPE-PEG. The Pt(IV) is immobilized with peptide via amide bonds, allowing the Pt(IV) to be loaded with a loading efficiency of >95% and rapid-release active platinum ions (Pt(II)) in the presence of glutathione (GSH). Meanwhile, the peptide of the prodrug complex could efficiently deliver Beclin1 siRNA ( Beclin1 is an autophagy initiation factor) to the cytoplasm, thereby leading to autophagy inhibition. In addition, incorporation of DSPE-PEG and cRGD-modified DSPE-PEG molecules improves the biocompatibility and cellular uptake of the nanoprodrug platform. In vivo results also indicate that the nanoprodrug platform significantly inhibits the growth of a cisplatin-resistant tumor on xenograft mice models with a remarkable inhibition rate, up to 84% after intravenous injection.

Regulation of Beclin 1-Mediated Autophagy by Oncogenic Tyrosine Kinases.

Beclin 1 is a major regulator of autophagy, and it is a core component of the class III PI3K complexes. Beclin 1 is a highly conserved protein and its function is regulated in a number of ways, including post-translational modifications. Several studies indicate that receptor and non-receptor tyrosine kinases regulate autophagy activity in cancer, and some suggest the importance of Beclin 1 tyrosine phosphorylation in this process. Here we summarize the current knowledge of the mechanism whereby some oncogenic tyrosine kinases regulate autophagy through Beclin 1.

Identification of Beclin-1 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%-82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.

Beclin-1 is involved in the regulation of antimicrobial peptides expression in Chinese mitten crab Eriocheir sinensis.

Beclin-1, the mammalian ortholog of yeast Atg6, plays essential roles in the regulation of various processes, including autophagy, apoptosis, embryonic development and immune responses in vertebrates. However, the information about Beclin-1 in invertebrates especially in crustaceans is still very limited. In the present study, a novel Beclin-1 (designated as EsBeclin-1) was identified from Chinese mitten crab Eriocheir sinensis. The open reading frame of EsBeclin-1 cDNA was of 1,275 bp, encoding a typical APG6 domain. The deduced amino acid sequence of EsBeclin-1 shared high similarity ranging from 42.9% to 63.6% with the previously identified Beclins. In the phylogenetic tree, EsBeclin-1 was firstly clustered with Drosophila melanogaster Atg6 and then assigned into the branch of invertebrate Beclin-1. The mRNA transcripts of EsBeclin-1 were highly expressed in hepatopancreas, hemocytes and gill. After lipopolysaccharide (LPS) and Aeromonas hydrophila stimulations, the relative mRNA expression of EsBeclin-1 in hemocytes was significantly increased from 3 to 24 h with the peak level of 4.70-fold (p < 0.01) and 2.91-fold (p < 0.01) at 6 h, respectively. EsBeclin-1 protein was diffusely distributed in the cytoplasm of crab hemocytes under normal conditions, whereas it displayed predominantly punctuate distribution after LPS stimulation. After EsBeclin-1 was interfered with specific EsBeclin-1-dsRNA, the mRNA transcripts of some antimicrobial peptides, including EsALF2, EsLYZ, EsCrus and EsCrus2 in crab hemocytes were significantly decreased at 6 h post LPS stimulation. These results implicated that EsBeclin-1 played a role in regulating the antimicrobial peptides expressions in the immune responses of E. sinensis.

Hydroxamic Acid Derivatives of ß-Carboline/Hydroxycinnamic Acid Hybrids Inducing Apoptosis and Autophagy through the PI3K/Akt/mTOR Pathways.

Naturally occurring ß-carbolines are known to have antitumor activities but with limited effectiveness. In order to improve their efficacy, a series of new hydroxamic-acid-containing ß-carbolines connected via a hydroxycinnamic acid moitey (12a-f) were developed to incorporate histone deacetylase (HDAC) inhibition for possible synergistic effects. When evaluated in in vitro assays, most of the analogues showed significant antitumor activities against four human cancer cells. In particular, 12b showed the highest cytotoxic potency of the series, including drug-resistant Bel7402 cells, but had minimal effect on normal hepatic LO2 cells. These compounds also showed excellent inhibitory effects against HDAC1/6, which appear to contribute greatly to their antiproliferative properties. Compound 12b enhanced the acetylation levels of histone H3 and α-tubulin and induced greater cancer cell apoptosis than the FDA-approved HDAC inhibitor SAHA by regulating expression of apoptotic proteins Bax, Bcl-2, and caspase 3. Importantly, 12b also induced a significant amount of autophagic flux activity in Bel7402 cells by increasing the expression of Beclin-1 and LC3-II proteins and decreasing that of LC3-I and p62. Finally, 12b significantly inhibited PI3K/Akt/mTOR signaling, an important cell-growth-promoting pathway aberrantly activated in many cancers. Together, the results suggest that these hydroxamic-acid-containing ß-carboline derivatives may be new leads for the discovery of agents for the treatment of human carcinoma cancers.

Structural transitions in conserved, ordered Beclin 1 domains essential to regulating autophagy.

Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositol-3-kinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiled-coil domain (CCD). However, the last 16 CCD residues, composing an "overlap helix" (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the C-terminal ß-α repeated, autophagy-specific domain (BARAD). Here, using CD spectroscopy, isothermal titration calorimetry, and small-angle X-ray scattering, we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the two-domain CCD-BARAD. Last, we used cellular assays to demonstrate that mutation of these OH-interface residues abrogates starvation-induced up-regulation of autophagy but does not affect basal autophagy. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains (i.e. the CCD or the BARAD). Our findings have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes.

Peptides derived from evolutionarily conserved domains in Beclin-1 and Beclin-2 enhance the entry of lentiviral vectors into human cells.

Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolutionarily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34+ hematopoietic stem/progenitor cells while maintaining their capacity to engraft in vivo into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.

Beclin-1 is involved in tongue sole Cynoglossus semilaevis immune defense against bacterial infection.

In mammals, beclin-1 is a key player that regulates autophagic activity. In fish, the immune function of beclin-1 is essentially unknown. In this study, we analyzed the involvement of tongue sole (Cynoglossus semilaevis) beclin-1 (named CsBECN1) in antibacterial immunity. CsBECN1 is composed of 451 amino acid residues and shares 84.5-95.1% overall sequence identities with other teleost beclin-1. CsBECN1 possesses a typical Bcl-2 homology domain 3 and an Atg6 domain. Expression of CsBECN1 occurred in multiple tissues and was upregulated during bacterial infection. Knockdown of CsBECN1 significantly enhanced bacterial dissemination in the tissues of tongue sole, whereas overexpression of CsBECN1 significantly reduced bacterial dissemination. Taken together, these results indicate that CsBECN1 is required for the antibacterial immunity of tongue sole.

The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity.

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 µM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 µM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.

Characterisation of the conformational preference and dynamics of the intrinsically disordered N-terminal region of Beclin 1 by NMR spectroscopy.

Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.

Prediction of post translation modifications at the contact site between Anaplasma phagocytophilum and human host during autophagosome induction using a bioinformatic approach.

Autophagy is crucial for maintaining physiological homeostasis, but its role in infectious diseases is not yet adequately understood. The binding of Anaplasma translocated substrate-1 (ATS1) to the human Beclin1 (BECN1) protein is responsible for the modulation of autophagy pathway. ATS1-BECN1 is a novel type of interaction that facilitates Anaplasma phagocytophilum proliferation, leading to intracellular infection via autophagosome induction and segregation from the lysosome. Currently, there is no report of post translational modifications (PTMs) of BECN1 or cross-talk required for ATS-BECN1 complex formation. Prediction/modeling of the cross-talk between phosphorylation and other PTMs (O-ß-glycosylation, sumoylation, methylation and palmitoylation) has been attempted in this study, which might be responsible for regulating function after the interaction of ATS1 with BECN1. PTMs were predicted computationally and mapped onto the interface of the docked ATS1-BECN1 complex. Results show that BECN1 phosphorylation at five residues (Thr91, Ser93, Ser96, Thr141 and Ser234), the interplay with O-ß-glycosylation at three sites (Thr91, Ser93 and Ser96) with ATS1 may be crucial for attachment and, hence, infection. No other PTM site at the BECN1 interface was predicted to associate with ATS1. These findings may have significant clinical implications for understanding the etiology of Anaplasma infection and for therapeutic studies.

Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.

Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy.

Inhibition of autophagy enhances heat-induced apoptosis in human non-small cell lung cancer cells through ER stress pathways.

The occurrence and mechanisms of autophagy induced by heat stress are not well known in lung cancer cells. Here, we have demonstrated that heat stress induces autophagy in A549 and NCI-H460 cells through morphological and biochemical analyses. The inhibition of autophagy by chloroquine, 3-methyladenine and Beclin 1 siRNA enhanced heat-induced apoptosis. Moreover, the combination of chloroquine and heat stress inhibited tumor growth and enhanced apoptosis in vivo experiments. In addition, heat-induced autophagy involved the ER stress pathway (PERK- or IRE1-dependent). Further, heat treatment led to the increased phosphorylation of AMPK and the decreased phosphorylation of mTOR in vitro and in vivo. Knockdown of GRP78 inhibited the AMPK-mTOR pathway, and the AMPK inhibitor compound C decreased heat-induced autophagy, suggesting that activation of ER stress was involved in autophagy induction and promotion of the AMPK-mTOR pathway. In conclusion, our data suggested that the heat treatment of lung cancer cells triggered protective autophagy, as mediated by ER stress. Thus, inhibition of autophagy can be a promising strategy to enhance hyperthermia in the treatment of lung cancer patients.

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway.

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex. SUMMARY: In this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.

IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation.

Extracellular cytokines are enriched in the tumor microenvironment and regulate various important properties of cancers, including autophagy. However, the precise molecular mechanisms underlying the link between autophagy and extracellular cytokines remain to be elucidated. In the present study, we demonstrate that IL-6 activates autophagy through the IL-6/JAK2/BECN1 pathway and promotes chemotherapy resistance in colorectal cancer (CRC). Mechanistically, IL-6 triggers the interaction between JAK2 and BECN1, where JAK2 phosphorylates BECN1 at Y333. We demonstrate that BECN1 Y333 phosphorylation is crucial for BECN1 activation and IL-6-induced autophagy by regulating PI3KC3 complex formation. Furthermore, we investigate BECN1 Y333 phosphorylation as a predictive marker for poor CRC prognosis and chemotherapy resistance. Combination treatment with autophagy inhibitors or pharmacological agents targeting the IL-6/JAK2/BECN1 signaling pathway may represent a potential strategy for CRC cancer therapy.