DRAM, a p53-induced modulator of autophagy, is critical for apoptosis

Inactivation of cell death is a major step in tumor development, and p53, a tumor suppressor frequently mutated in cancer, is a critical mediator of cell death. While a role for p53 in apoptosis is well established, direct links to other pathways controlling cell death are unknown. Here we describe DRAM (damage-regulated autophagy modulator), a p53 target gene encoding a lysosomal protein that induces macroautophagy, as an effector of p53-mediated death. We show that p53 induces autophagy in a DRAM-dependent manner and, while overexpression of DRAM alone causes minimal cell death, DRAM is essential for p53-mediated apoptosis. Moreover, analysis of DRAM in primary tumors revealed frequent decreased expression often accompanied by retention of wild-type p53. Collectively therefore, these studies not only report a stress-induced regulator of autophagy but also highlight the relationship of DRAM and autophagy to p53 function and damage-induced programmed cell death.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

DAMP-mediated autophagy contributes to drug resistance

Damage-associated molecular pattern molecules (DAMPs) are cellularly derived molecules that can initiate and perpetuate immune responses following trauma, ischemia and other types of tissue damage in the absence of pathogenic infection. High mobility group box 1 (HMGB1) is a prototypical DAMP and is associated with the hallmarks of cancer. Recently we found that HMGB1 release after chemotherapy treatment is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia. Overexpression of HMGB1 by gene transfection rendered leukemia cells resistant to cell death; whereas depletion or inhibition of HMGB1 and autophagy by RNA interference or pharmacological inhibitors increased the sensitivity of leukemia cells to chemotherapeutic drugs. HMGB1 release sustains autophagy as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, degradation of p62 and accumulation of autophagosomes and autolysosomes. Moreover, these data suggest a role for HMGB1 in the regulation of autophagy through the PI3KC3-MEKERK: pathway, supporting the notion that HMGB1-induced autophagy promotes tumor resistance to chemotherapy.     

The early secretory pathway contributes to autophagy in yeast

Autophagy is a starvation response in eukaryotes by which the cell delivers cytoplasmic components to the vacuole for degradation, and is mediated by a double membrane structure called the autophagosome. We have previously proposed that the specific combination of COPII like components, including Sec24p, is required for autophagy (Ishihara, N. et al. (2001) Mol. Biol. Cell, 12: 3690-3702). The autophagic defect in sec24 deleted mutant cells was, however, suppressed upon the recovery of its secretory flow by the overexpression of its homologue, Sfb2p. We have also reported that the autophagic defect is not observed in sec13 and sec31 mutants, a phenomenon that can be explained by the fact that starvation stress suppresses the secretory defect of these mutants. These observations indicate that the active flow in the early secretory pathway plays an important role in autophagy; that is, autophagy proceeds in the presence, but not in the absence of the early secretory flow. Both autophagy and its closely related cytoplasm to vacuole-targeting (Cvt) pathway occur through a pre-autophagosomal structure (PAS), and since the PAS and the functional Cvt pathway exist in all sec mutants, the early secretory pathway must be involved specifically in autophagy, subsequent to PAS formation.     

The intertransmembrane region of Kaposi's sarcoma-associated herpesvirus modulator of immune recognition 2 contributes to B7-2 downregulation

Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor virus, encodes two homologous membrane-associated E3 ubiquitin ligases, modulator of immune recognition 1 (MIR1) and MIR2, to evade host immunity. Both MIR1 and MIR2 downregulate the surface expression of major histocompatibility complex class I (MHC I) molecules through ubiquitin-mediated endocytosis followed by lysosomal degradation. Since MIR2 additionally downregulates a costimulatory molecule (B7-2) and an integrin ligand (intercellular adhesion molecule 1 [ICAM-1]), MIR2 is thought to be a more important molecule for immune evasion than MIR1; however, the molecular basis of the MIR2 substrate specificity remains unclear. To address this issue, we determined which regions of B7-2 and MIR2 are required for MIR2-mediated B7-2 downregulation. Experiments with chimeras made by swapping domains between human B7-2 and CD8α, a non-MIR2 substrate, and between MIR1 and MIR2 demonstrated a significant contribution of the juxtamembrane (JM) region of B7-2 and the intertransmembrane (ITM) region of MIR2 to MIR2-mediated downregulation. Structure prediction and mutagenesis analyses indicate that Phe119 and Ser120 in the MIR2 ITM region and Asp244 in the B7-2 JM region contribute to the recognition of B7-2 by MIR2. This finding provides new insight into the molecular basis of substrate recognition by MIR family members.     

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.     

Host cell autophagy contributes to Plasmodium liver development

Autophagy plays an important role in the defence against intracellular pathogens. However, some microorganisms can manipulate this host cell pathway to their advantage. In this study, we addressed the role of host cell autophagy during Plasmodium berghei liver infection. We show that vesicles containing the autophagic marker LC3 surround parasites from early time‐points after invasion and throughout infection and colocalize with the parasitophorous vacuole membrane. Moreover, we show that the LC3‐positive vesicles that surround Plasmodium parasites are amphisomes that converge from the endocytic and autophagic pathways, because they contain markers of both pathways. When the host autophagic pathway was inhibited by silencing several of its key regulators such as LC3, Beclin1, Vps34 or Atg5, we observed a reduction in parasite size. We also found that LC3 surrounds parasites in vivo and that parasite load is diminished in a mouse model deficient for autophagy. Together, these results show the importance of the host autophagic pathway for parasite development during the liver stage of Plasmodium infection.     

Alcohol-induced autophagy contributes to loss in skeletal muscle mass

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.     

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca²⁺ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca²⁺ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca²⁺ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca²⁺-store content and enhanced agonist-induced Ca²⁺ release. The mechanism involved the upregulation of intralumenal ER Ca²⁺-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca²⁺-buffering capacity and reduced the ER Ca²⁺ leak. Second, starvation led to Ins(1,4,5)P₃R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P₃Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P₃-binding domain of the Ins(1,4,5)P₃R. The starvation-induced Ins(1,4,5)P₃R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P₃Rs in ⁴⁵Ca²⁺-flux assays, indicating a direct regulation of Ins(1,4,5)P₃R activity by Beclin 1. Finally, we found that Ins(1,4,5)P₃R-mediated Ca²⁺ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P₃R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca²⁺ signaling and autophagy stimulation as a proximal event in response to starvation.     

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.     

The induction of autophagy by mechanical stress

The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.     

The induction of autophagy by mechanical stress

The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.