Structural Basis for Recruitment of DAPK1 to the KLHL20 E3 Ligase

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自噬与泛素化蛋白降解途径的分子机制及其功能

细胞内所有的蛋白质和大多数的细胞外蛋白都在不断的进行更新,即它们在不断地被降解,并被新合成的蛋白质取代。细胞内蛋白的降解主要通过两个途径,即自噬和泛素蛋白酶体系统。自噬是一种由溶酶体介导的细胞内过多或异常蛋白质的降解机制。在细胞内主要有3种类型的自噬,即分子伴侣介导的自噬、微自噬和巨自噬。泛素蛋白酶体系统是由泛素介导的一种高度复杂的蛋白降解机制,它参与降解细胞内许多蛋白质并且这个过程具有高度特异性。细胞内蛋白质的降解参与调节许多细胞过程,包括细胞周期、DNA修复、细胞生长和分化、细胞质量的控制、病原生物的感染反应和细胞凋亡等。许多严重的人类疾病被认为是由于蛋白质降解系统的紊乱而引起的。文章综述了自噬和泛素化途径及其分子机制,以及蛋白质降解系统紊乱的病理学意义。     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin‐specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation‐induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.     

The COP9 signalosome coerces autophagy and the ubiquitin-proteasome system to police the heart

We demonstrated for the first time that the COP9 signalosome (COPS) controls the degradation of a surrogate and a bona fide misfolded protein in the cytosol of cardiomyocytes likely via supporting ubiquitination by CUL/cullin-RING ligases, and that Cops8 hypomorphism exacerbates cardiac proteinopathy in mice, in which autophagic impairment appears to be in play. It will be extremely imprtant to investigate cardiac ablation of another Cops gene to decipher whether COPS8 deficiency phenotypes are attributable to the COPS or unique to COPS8.     

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase

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Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis

Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.     

High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy

Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine—a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

Phosphoproteomic identification of ULK substrates reveals VPS15‐dependent ULK/VPS34 interplay in the regulation of autophagy

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK‐dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK‐dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation‐independent accumulation of ULK substrates and kinase activity‐regulated recruitment of autophagy proteins to ubiquitin‐positive structures.     

BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells

Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.     

Carboxyl terminus of Hsp70‐interacting protein (CHIP) is required to modulate cardiac hypertrophy and attenuate autophagy during exercise

The carboxyl terminus of Hsp70‐interacting protein (CHIP) is a ubiquitin ligase/cochaperone critical for the maintenance of cardiac function. Mice lacking CHIP (CHIP?/?) suffer decreased survival, enhanced myocardial injury and increased arrhythmias compared with wild‐type controls following challenge with cardiac ischaemia reperfusion injury. Recent evidence implicates a role for CHIP in chaperone‐assisted selective autophagy, a process that is associated with exercise‐induced cardioprotection. To determine whether CHIP is involved in cardiac autophagy, we challenged CHIP?/? mice with voluntary exercise. CHIP?/? mice respond to exercise with an enhanced autophagic response that is associated with an exaggerated cardiac hypertrophy phenotype. No impairment of function was identified in the CHIP?/? mice by serial echocardiography over the 5 weeks of running, indicating that the cardiac hypertrophy was physiologic not pathologic in nature. It was further determined that CHIP plays a role in inhibiting Akt signalling and autophagy determined by autophagic flux in cardiomyocytes and in the intact heart. Taken together, cardiac CHIP appears to play a role in regulating autophagy during the development of cardiac hypertrophy, possibly by its role in supporting Akt signalling, induced by voluntary running in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy

The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.     

Auto-ubiquitination of NEDD4-1 Recruits USP13 to Facilitate Autophagy through Deubiquitinating VPS34

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Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

A new method to measure autophagy flux in the nervous system

A current need in the neuroscience field is a simple method to monitor autophagic activity in vivo in neurons. Until very recently, most reports have been based on correlative and static determinations of the expression levels of autophagy markers in the brain, generating conflicting interpretations. Autophagy is a fundamental process mediating the degradation of diverse cellular components, including organelles and protein aggregates at basal levels, whereas alterations in the process (i.e., autophagy impairment) operate as a pathological mechanism driving neurodegeneration in most prevalent diseases. We have recently described a new simple method to deliver and express an autophagy flux reporter through the peripheral and central nervous system of mice by the intracerebroventricular delivery of adeno-associated viruses (AAV) into newborn mice. We obtained a wide expression of a monomeric tandem mCherry-GFP-LC3 construct in neurons through the nervous system and demonstrated efficient and accurate measurements of LC3 flux after pharmacological stimulation of the pathway or in disease settings of axonal damage. Here we discuss the possible applications of this new method to assess autophagy activity in neurons in vivo.     

A new method to measure autophagy flux in the nervous system

A current need in the neuroscience field is a simple method to monitor autophagic activity in vivo in neurons. Until very recently, most reports have been based on correlative and static determinations of the expression levels of autophagy markers in the brain, generating conflicting interpretations. Autophagy is a fundamental process mediating the degradation of diverse cellular components, including organelles and protein aggregates at basal levels, whereas alterations in the process (i.e., autophagy impairment) operate as a pathological mechanism driving neurodegeneration in most prevalent diseases. We have recently described a new simple method to deliver and express an autophagy flux reporter through the peripheral and central nervous system of mice by the intracerebroventricular delivery of adeno-associated viruses (AAV) into newborn mice. We obtained a wide expression of a monomeric tandem mCherry-GFP-LC3 construct in neurons through the nervous system and demonstrated efficient and accurate measurements of LC3 flux after pharmacological stimulation of the pathway or in disease settings of axonal damage. Here we discuss the possible applications of this new method to assess autophagy activity in neurons in vivo.     

Measurement of autophagy flux in the nervous system in vivo

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.