异体自噬的操控:病原微生物对宿主翻译后修饰的调节作用

自噬是一种广泛存在于真核细胞中的溶酶体依赖性分解代谢途径,涉及细胞分化、饥饿耐受和免疫防御等生物学功能.其中,异体自噬被定义为真核细胞特异性识别并清除胞内病原微生物的过程,是免疫细胞行使宿主防御的重要方式.然而,许多病原微生物已经"开发"了特殊的毒力因子(包括效应蛋白质和表面蛋白质等),衍生出多种逃避或劫持自噬作用的策...     

The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.     

Differential regulation of autophagy during metabolic stress in astrocytes and neurons

ABSTRACT

Macroautophagy/autophagy is a key homeostatic process that targets cytoplasmic components to the lysosome for breakdown and recycling. Autophagy plays critical roles in glia and neurons that affect development, functionality, and viability of the nervous system. The mechanisms that regulate autophagy in glia and neurons, however, are poorly understood. Here, we define the molecular underpinnings of autophagy in primary cortical astrocytes in response to metabolic stress, and perform a comparative study in primary hippocampal neurons. We find that inducing metabolic stress by nutrient deprivation or pharmacological inhibition of MTOR (mechanistic target of rapamycin kinase) robustly activates autophagy in astrocytes. While both paradigms of metabolic stress dampen MTOR signaling, they affect the autophagy pathway differently. Further, we find that starvation-induced autophagic flux is dependent on the buffering system of the starvation solution. Lastly, starvation conditions that strongly activate autophagy in astrocytes have less pronounced effects on autophagy in neurons. Combined, our study reveals the complexity of regulating autophagy in different paradigms of metabolic stress, as well as in different cell types of the brain. Our findings raise important implications for how neurons and glia may collaborate to maintain homeostasis in the brain.     

自体吞噬———Ⅱ型程序性死亡

自噬 (autophagy) 是广泛存在于真核细胞内的一种溶酶体依赖性的降解途径,在饥饿的条件下,它可以调节细胞内长寿命蛋白和细胞器的降解,降解产物再被细胞重新利用 . 因此自噬在细胞发育、细胞免疫、组织重塑及对环境适应等方面有着十分重要的作用 . 近来发现,自噬还参与降解病原微生物、抵御感染的过程,称之为异噬 . 对自噬的分子机制和调节以及其在生理病理过程中的作用进行相应讨论 .     

Galectins control MTOR and AMPK in response to lysosomal damage to induce autophagy

The Ser/Thr protein kinase MTOR (mechanistic target of rapamycin kinase) regulates cellular metabolism and controls macroautophagy/autophagy. Autophagy has both metabolic and quality control functions, including recycling nutrients at times of starvation and removing dysfunctional intracellular organelles. Lysosomal damage is one of the strongest inducers of autophagy, and yet mechanisms of its activation in response to lysosomal membrane damage are not fully understood. Our recent study has uncovered a new signal transduction system based on cytosolic galectins that elicits autophagy by controlling master regulators of metabolism and autophagy, MTOR and AMPK, in response to lysosomal damage. Thus, intracellular galectins are not, as previously thought, passive tags recognizing damage to guide selective autophagy receptors, but control the activation state of AMPK and MTOR in response to endomembrane damage.

Abbreviations: MTOR: mechanistic target of rapamycin kinase; AMPK: AMP-activated protein kinase / Protein Kinase AMP-Activated; SLC38A9: Solute Carrier Family 38 Member 9; APEX2: engineered ascorbate peroxidase 2; RRAGA/B: Ras Related GTP Binding A or B; LAMTOR1: Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 1; LGALS8: Lectin, Galactoside-Binding, Soluble, 8 / Galectin 8; LGALS9: Lectin, Galactoside-Binding, Soluble, 9 / Galectin 9; TAK1: TGF-Beta Activated Kinase 1 / Mitogen-Activated Protein Kinase Kinase Kinase 7 (MAP3K7); STK11/LKB1: Serine/Threonine Kinase 11 / Liver Kinase B1; ULK1: Unc-51 Like Autophagy Activating Kinase 1.     

Autophagy in CD4+ T-cell immunity and tolerance

Autophagy is a homeostatic process that enables eukaryotic cells to deliver cytoplasmic constituents for lysosomal degradation, to recycle nutrients and to survive during starvation. In addition to these primordial functions, autophagy has emerged as a key mechanism in orchestrating innate and adaptive immune responses to intracellular pathogens. Autophagy restricts viral infections as well as replication of intracellular bacteria and parasites and delivers pathogenic determinants for TLR stimulation and for MHC class II presentation to the adaptive immune system. Apart from its role in defense against pathogens, autophagy-mediated presentation of self-antigens in the steady state could have a crucial role in the induction and maintenance of CD4(+) T-cell tolerance. This review describes the mechanisms by which the immune system utilizes autophagic degradation of cytoplasmic material to regulate adaptive immune responses.     

SAR405, a PIK3C3/Vps34 inhibitor that prevents autophagy and synergizes with MTOR inhibition in tumor cells

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a prosurvival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents. We recently described the discovery of inhibitors of PIK3C3/Vps34 (phosphatidylinositol 3-kinase, catalytic subunit type 3), the lipid kinase component of the class III phosphatidylinositol 3-kinase (PtdIns3K). This PtdIns3K isoform has attracted significant attention in recent years because of its role in autophagy. Following chemical optimization we identified SAR405, a low molecular mass kinase inhibitor of PIK3C3, highly potent and selective with regard to other lipid and protein kinases. We demonstrated that inhibiting the catalytic activity of PIK3C3 disrupts vesicle trafficking from late endosomes to lysosomes. SAR405 treatment also inhibits autophagy induced either by starvation or by MTOR (mechanistic target of rapamycin) inhibition. Finally our results show that combining SAR405 with everolimus, the FDA-approved MTOR inhibitor, results in a significant synergy on the reduction of cell proliferation using renal tumor cells. This result indicates a potential therapeutic application for PIK3C3 inhibitors in cancer.     

Bacterial xenophagy and its possible role in cancer: A potential antimicrobial strategy for cancer prevention and treatment

Macroautophagy/autophagy is a conserved catabolic process through which cellular excessive or dysfunctional proteins and organelles are transported to the lysosome for terminal degradation and recycling. Over the past few years increasing evidence has suggested that autophagy is not only a simple metabolite recycling mechanism, but also plays a critical role in the removal of intracellular pathogens such as bacteria and viruses. When autophagy engulfs intracellular pathogens, the pathway is called ‘xenophagy’ because it leads to the elimination of foreign microbes. Recent studies support the idea that xenophagy can be modulated by bacterial infection. Meanwhile, convincing evidence indicates that xenophagy may be involved in malignant transformation and cancer therapy. Xenophagy can suppress tumorigenesis, particularly during the early stages of tumor initiation. However, in established tumors, xenophagy may also function as a prosurvival pathway in response to microenvironment stresses including bacterial infection. Therefore, bacterial infection-related xenophagy may have an effect on tumor initiation and cancer treatment. However, the role and machinery of bacterial infection-related xenophagy in cancer remain elusive. Here we will discuss recent developments in our understanding of xenophagic mechanisms targeting bacteria, and how they contribute to tumor initiation and anticancer therapy. A better understanding of the role of xenophagy in bacterial infection and cancer will hopefully provide insight into the design of novel and effective therapies for cancer prevention and treatment.     

Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2

Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that occurs constitutively in cells, but can also be induced by stressors such as nutrient starvation or protein aggregation. Autophagy has been implicated in multiple disease mechanisms including neurodegeneration and cancer, with both tumor suppressive and oncogenic roles. Uncoordinated 51-like kinase 1 (ULK1) is a critical autophagy protein near the apex of the hierarchal regulatory pathway that receives signals from the master nutrient sensors MTOR and AMP-activated protein kinase (AMPK). In mammals, ULK1 has a close homolog, ULK2, although their functional distinctions have been unclear. Here, we show that ULK1 and ULK2 both function to support autophagy activation following nutrient starvation. Increased autophagy following amino acid or glucose starvation was disrupted only upon combined loss of ULK1 and ULK2 in mouse embryonic fibroblasts. Generation of PtdIns3P and recruitment of WIPI2 or ZFYVE1/DFCP1 to the phagophore following amino acid starvation was blocked by combined Ulk1/2 double knockout. Autophagy activation following glucose starvation did not involve recruitment of either WIPI1 or WIPI2 to forming autophagosomes. Consistent with a PtdIns3P-independent mechanism, glucose-dependent autophagy was resistant to wortmannin. Our findings support functional redundancy between ULK1 and ULK2 for nutrient-dependent activation of autophagy and furthermore highlight the differential pathways that respond to amino acid and glucose deprivation.     

Xenophagy in herpes simplex virus replication and pathogenesis

Autophagy functions in part as an important host defense mechanism to engulf and degrade intracellular pathogens, a process that has been termed xenophagy. Xenophagy is detrimental to the invading microbe in terms of replication and pathogenesis and many pathogens either dampen the autophagic response, or utilize the pathway to enhance their life cycle. Herpes simplex virus type 1 (HSV-1) counteracts the induction of xenophagy through its neurovirulence protein, ICP34.5. ICP34.5 binds protein phosphatase 1alpha to counter PKR-mediated phosphorylation of eIF2alpha, and also binds the autophagy-promoting protein Beclin 1. Through these interactions, ICP34.5 prevents translational arrest and down-regulates the formation of autophagosomes. Whereas autophagy antagonism promotes neurovirulence, it has no impact on the replication of HSV-1 in permissive cultured cells. As discussed in this article, this work raises a number of questions as to the mechanism of ICP34.5-mediated inhibition of autophagy, as well as to the role of autophagy antagonism in the lifecycle of HSV-1.     

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.     

Subversion of Autophagy in Adherent Invasive Escherichia coli-Infected Neutrophils Induces Inflammation and Cell Death

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.     

Mediatory molecules that fuse autophagosomes and lysosomes

Autophagy functions to degrade intracellular foreign microbial invaders by a process that is termed xenophagy (antimicrobial autophagy). Xenophagosomes undergo a stepwise maturation process culminating in a fusion event with lysosomes, after which the cargos are degraded. Recent investigations by our laboratory demonstrate that endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are involved in the fusion between xenophagosomes and lysosomes. Knockdown of the combinational SNARE proteins Vti1b and VAMP8 with siRNAs disturbs the colocalization of LC3 with LAMP-1. We also find that the invasive efficiency of group A Streptococcus into cells is not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency is significantly diminished, indicating that xenophagy is functionally impaired. In addition, knockdown of these SNAREs inhibits the fusion of canonical autophagosomes with lysosomes. Together, these findings indicate that VAMP8 and Vti1b mediate fusion with lysosomes in both antimicrobial and canonical autophagy.     

Coronavirus NSP6 restricts autophagosome expansion

Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.     

Coronavirus NSP6 restricts autophagosome expansion

Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.     

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ^TM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.     

Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.     

Role of autophagy in breast cancer

Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1(+/-) mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.