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Core autophagy genes and human diseases
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ScienceDirect Core autophagy genes and human diseases Yan G Zhao1 and Hong Zhang2,3 Autophagy involves the formation of double-membrane autophagosomes and their delivery to lysosomes for degradation. In response to various endogenous and exogenous stimuli, autophagy recycles cellular constituents and removes cytotoxic threats such as protein aggregates and damaged organelles to maintain cellular homeostasis. Dysfunctional autophagy has been linked with multiple human diseases, including neurodegenerative diseases, tumorigenesis, diabetes, and immune diseases. Here we focus on human genetic disorders caused by hypomorphic or regulatory mutations in early acting autophagy genes or by mutations in genes acting at autophagosome maturation. Protein aggregates assembled via liquid–liquid phase separation (LLPS) exhibit distinct biophysical properties that are modulated by disease-related mutations. Abnormal phase transition of protein aggregates affects their removal and is associated with the pathogenesis of various neurodegenerative diseases.
fuse with vesicles originating from the endolysosomal compartment to form amphisomes and eventually degradative autolysosomes (Figure 1). Degraded molecules are then transported to the cytosol for reuse [1–4]. Under starvation and other stress conditions, autophagy functions as a cell-survival mechanism. Autophagy also selectively removes misfolded and toxic proteins and/or damaged organelles to maintain cellular homeostasis [4–7]. Perturbations of autophagy are linked with multiple pathological changes in humans, including neurodegenerative diseases, cancer, inflammation, and metabolic and developmental disorders [4–7]. We mainly focus on recent reports of genetic associations of core autophagy genes with human diseases (Table 1). We also discuss how phase separation and transition modulate autophagic degradation of protein aggregates and how these processes are involved in neurodegenerative diseases.
Autophagic machinery Addresses 1 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA 2 National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China 3 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, PR China Corresponding authors: Zhao, Yan G ([email protected]), Zhang, Hong ([email protected])
Current Opinion in Cell Biology 2019, 61:117–125 This review comes from a themed issue on Differentiation and disease Edited by Sara Wickstrom and Yingzi Yang
https://doi.org/10.1016/j.ceb.2019.08.003 0955-0674/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Autophagy is an evolutionarily conserved lysosomemediated catabolic process. The hallmark of autophagy is the formation of double-membrane autophagosomes that engulf a portion of cytosol and deliver it to lysosomes for degradation [1–4]. Autophagosome formation starts with de novo initiation and nucleation of isolation membranes (IMs), which expand and close. Nascent autophagosomes www.sciencedirect.com
A group of Atg (autophagy-related) genes identified from yeast genetic screens laid the groundwork for our understanding of the molecular mechanism of autophagosome formation [1–5]. Most of these ATG proteins are conserved in mammals. Upon autophagy induction, the Atg1 complex, containing Atg1/ULK1(mammalian counterpart), Atg17/FIP200 and Atg13, translocates to the autophagosome formation sites on the ER. The class III phosphatidylinositol 3-kinase VPS34 complex, consisting of VPS34, Atg6/Beclin 1, VPS15, and ATG14, is further recruited to generate phosphatidylinositol 3-phosphate (PI(3)P). The Atg1 and Vps34 complexes are required for initiation and nucleation of IMs. ATG9-positive vesicles are proposed to serve as one of the membrane sources for IM initiation and elongation. ATG9 trafficking is regulated by the PI(3)P effector ATG18 and its binding partner ATG2. Expansion of IMs requires two ubiquitin-like conjugation systems. The E1-like enzyme Atg7 and E2-like enzyme Atg10 catalyze the conjugation of the ubiquitin-like protein Atg12 with Atg5, which further interacts with Atg16L1. Atg8 is conjugated to phosphatidylethanolamine (PE) by Atg7 and the E2-like enzyme Atg3 [1–5]. Mammalian autophagy is more complex and contains steps that are not present in yeast. Autophagosomes form at the single peri-vacuolar site in yeast, while in mammals, multiple autophagosomes are generated simultaneously throughout the cytoplasm and closely contact the ER during IM initiation and elongation. Yeast autophagosomes directly fuse with the vacuole, while nascent mammalian autophagosomes detach from the ER and mature by fusion with endolysosomal vesicles before forming degradative autolysosomes [2,8,9]. Genetic Current Opinion in Cell Biology 2019, 61:117–125
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Figure 1
ER ATG2
ULK1 complex
Isolation membrane
Ω
WDR45
WDR45B
ATG14 Beclin 1
WDR45B
VPS34
WIPI2
EPG5
ATG12 ATG16L
ATG5 Lysosome Autophagosome
ATG8s pro-ATG8s PI(3)P
ATG4s
Rab7
Autolysosome
Current Opinion in Cell Biology
The role of core autophagy genes in the autophagy pathway. Upon autophagy induction, the most upstream complex – the ULK1 complex – is translocated to the autophagosome formation sites on the ER for the nucleation of isolation membranes (IMs). The VPS34/Beclin 1/ATG14 PI(3) kinase complex is then recruited, and generates PI(3)P de novo at the ER subdomains, called omegasomes, as well as on the IMs. The PI(3)P effector WIPI2 interacts with the ATG5/12/16L1 complex for ATG8sPE conjugation and IM elongation. ATG4s are cysteine proteinases that cleave the C-terminal of pro-ATG8s into mature ATG8s, and also cleave the ATG8-PE conjugates for recycling of ATG8s from closed autophagosomes, amphisomes and autolysosomes. WDR45 forms a complex with ATG2, and is also involved in autophagosome biogenesis. The detailed molecular role of the WDR45–ATG2 complex in autophagosome formation remains unknown. After closure, autophagosomes fuse with endocytic vesicles and lysosomes to form degradative autolysosomes. Fusion of autophagosomes/amphisomes with late endosomes/lysosomes requires the concerted actions of SNAREs, Rabs and tether proteins, such as EPG5. ER, endoplasmic reticulum; V, omegasome.
screens in Caenorhabditis elegans have identified a set of metazoan-specific autophagy genes, known as EPG (ectopic P granule) genes, that act at these unique autophagy steps [10]. The ER-localized transmembrane protein EPG3 modulates the ER-IM contact [11], while EPG5 confers the specificity of fusion of autophagosomes/amphisomes with late endosomes/lysosomes [12] (Figure 1). Human genetic studies reveal that mutations in ATG and EPG genes are linked with predisposition to various diseases.
WIPIs and neurological diseases Yeast Atg18 has four mammalian homologs, WIPI1-4, which are further classified into WIPI1/2 (WD-repeat protein interacting with phosphoinositides 1/2) and WDR45B/45 (also known as WIPI3/4) subgroups [13]. The single C. elegans homologs of each group are ATG-18 and EPG-6, respectively, and they Current Opinion in Cell Biology 2019, 61:117–125
function at different steps of autophagy [13]. WIPI2 directly interacts with ATG16 and defines the action site for the ATG16–ATG12–ATG5 complex that functions as an E3 for LC3 lipidation [14]. C. elegans EPG-6 but not ATG-18 binds to ATG-2 [13]. Similarly, WDR45 shows stronger interaction with ATG2A/B than WIPI1 and WIPI2 [15]. The role of WDR45 and WDR45B in autophagy has yet to be determined. Cryo-electron microscopy analysis reveals that WDR45 is a spherical protein attached to one end of the bar-like ATG2A/B to form a club-shaped structure, resembling the seahorse shape of HOPS complex [15,16]. The WDR45–ATG2 complex tethers a PI(3) P-containing vesicle with another PI(3)P-free vesicle and mediates direct lipid transfer [17,18,19]. This complex may function to bridge IMs/autophagosomes with the ER or other vesicles, thus providing phospholipids for IM expansion. www.sciencedirect.com
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Table 1 Core autophagy genes and related-human diseases Autophagy gene WDR45
WDR45B
WIPI2
EPG5
ATG16L1
ATG5
ATG4A/B/C/D
BECN1
Function
Related human diseases
Disease-related mouse models
Interacts with ATG2 and may function in isolation membrane expansion [13,15,16,17,18,19]. It is also involved in sensing upstream signaling and regulating the autophagosome size [91]. Interacts with FIP200. It may regulate autophagosome size, and mTOR activity via interaction with TSC on lysosomes [91].
BPAN [20 ,21 ,22 ], Rett syndrome [25,26], West syndrome [27], Xlinked ID [28,29]
Nestin-Wdr45 knockout mice exhibit axon swelling, and learning and memory defects [30].
ID [33–35]
Conventional Wdr45b knockout mice show motor deficiency and cognitive impairment. Pathologically, the mutant mice exhibit cerebellar atrophy and axon swelling [36].
Directly binds to PI(3)P and recruits the ATG5-12-16L1 complex for LC3 lipidation at autophagosome formation sites [14]. Acts as a Rab7 effector to tether autophagosomes/amphisomes with late endosomes/lysosomes for fusion [12].
CP [37], global developmental disorder [38]
Vici syndrome [39]
EPG5 knockout mice display selective motor neuron loss, recapitulating ALS phenotypes [40]. The knockout mice also show retinal atrophy, callosal agenesis and muscle degeneration [42,43]. Atg16L1T300A knock-in mice show morphological changes in Paneth and goblet cells, impaired xenophagy and increased cytokine production [50,51].
Forms an E3-like complex with ATG12–ATG5 that promotes LC3 lipidation for isolation membrane expansion [1–5]. It also has autophagy-independent functions such as secretion [51]. Conjugates with ATG12, then further forms a complex with ATG16L, which functions as an E3 for LC3 lipidation [1–5]. It is also involved in LC3-associated phagocytosis [62].
CD [45]
Cleaves pro-ATG8s to expose the glycine residue for conjugation and also cleaves ATG8s from PE conjugates on autophagosomal structures [2,65]. Component of the PI(3) kinase complex for de novo PI(3)P generation [1–5]. Critical for both autophagic and endocytic pathways [1–5,72].
Kashin–Beck Disease (ATG4C) [66], CD (ATG4A/B/D) [65,67,68]
SLE [56–60], ataxia and developmental delay [63], CP [64]
Breast and ovarian cancer [6]
Knockout of Atg5 in thymic epithelial cells leads to defects in negative thymic selection and autoimmunity [61]. Macrophage-specific Atg5 knockout affects LAP and degradation of dying cells, increases cytokine production, and causes an SLE-like phenotype [62]. Atg4b knockout mice shows abnormal Paneth cells and are more sensitive to experimental colitis [65].
Heterozygous deletion of Becn1 increases the frequency of spontaneous tumorigenesis [70].
Abbreviations: BPAN, b-propeller protein-associated neurodegeneration; ID, intellectual disability; CD, Crohn’s disease; COPD, chronic obstructive pulmonary disease; SLE, systemic lupus erythematosus; CP, cerebral palsy; TSC, Tuberous sclerosis 1; LAP, LC3-associated phagocytosis.
De novo mutations in the X-chromosome gene WDR45 have been linked with one subtype of NBIA (neurodegeneration with brain iron accumulation), namely protein-associated neurodegeneration b-propeller (BPAN), also known as SENDA (static encephalopathy of childhood with neurodegeneration in adulthood) [20,21,22]. NBIA is a heterogenous single-gene disorder involving iron deposits in various brain regions such as globus pallidus and substantia nigra [23]. Whether iron accumulation is a primary cause or consequence in NBIA remains controversial [24]. BPAN features static www.sciencedirect.com
encephalopathy in infancy or early childhood with sudden-onset parkinsonism, dystonia and cognitive decline in adolescence or early adulthood. BPAN patients show reduced autophagic activity [20,21,22]. Furthermore, mutations in WDR45 are also associated with a broader disease spectrum, including MECP2 mutation-negative Rett syndrome, West syndrome and X-linked intellectual disability, which share some clinical similarities [25–29]. The phenotypic diversity could be due to the time when somatic WDR45 mutations occur and/or other genetic and environmental modifiers. Current Opinion in Cell Biology 2019, 61:117–125
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Mice with central nervous system-specific knockout of Wdr45 exhibit extensive swollen axons and impaired learning and memory, mimicking key features of BPAN [30]. The knockout mice, like those with knockouts of genes involved in other types of NBIA such as Pank2 or Pla2g6, show no signs of iron deposition in the brain [30–32].
degeneration and pathological muscle changes [42,43]. Vici syndrome patients suffer from recurrent infections such as pulmonary infections. However, Epg5 KO mice show elevated basal lung inflammation [44]. The deficits in Epg5 knockout mice appear to be less severe than Vici syndrome patients, which could be due to differential environmental stress and genetic differences [40].
WDR45B mutations cause intellectual disability (ID), which is a common neurodevelopmental disorder [33–35]. Wdr45b deficiency in mice leads to motor defects and cognitive malfunction, similar to ID patients [36]. Wdr45b mutant mice show age-related cerebellar atrophy and Purkinje cell loss, with axon swelling [36]. Accumulation of p62 and ubiquitin-positive aggregates, indicative of defective autophagy, is observed in multiple cerebral and cerebellar regions of Wdr45 and Wdr45b single knockout mice, but the defect is much weaker compared to mice deficient in the core autophagy genes Atg5 and Atg7 [4,30,36]. This is probably due to genetic redundancy, as Wdr45b/45 double knockout mice die one day after birth. They show dramatic autophagy defects in the brain, but not in other tissues, which suggests that Wdr45b/45 function tissue-specifically [36].
ATG16L1 and Crohn’s disease
The WIPI2 missense mutation Y246C is potentially linked with cerebral palsy (CP), a developmental disorder characterized by impaired coordination, poor muscle tone and movement problems [37]. The WIPI2 missense mutation V249M is associated with global developmental disorder and it affects autophagy potentially by weakening the PI(3)P-binding activity of WIPI2 and/or the interaction of WIPI2 with ATG16L1 [38].
EPG5 and Vici syndrome Vici syndrome is a rare recessively inherited multi-system disorder with severe early onset neurodevelopmental and neurodegenerative deficits, whose key features include agenesis of the corpus callosum, developmental delay, microcephaly, cataracts, cardiomyopathy, and combined immunological abnormalities [39]. It is causatively linked with EPG5 mutations [39]. EPG5 functions as a tether protein by interacting with LC3 on autophagosomes/ amphisomes and Rab7 on late endosomes/lysosomes. EPG5 promotes assembly of the autophagosomal fusion machinery, the STX17/SNAP29/VAMP8 SNARE complex [17]. EPG5 depletion causes accumulation of nondegradative autolysosomes in Vici syndrome patients and model organisms [17,40,41]. Depletion of EPG5 impairs endocytic trafficking and recycling, which may also contribute to disease pathogenesis [40]. Systematic Epg5 knockout mice show selective motor neuron loss in the 5th layer of the cerebral cortex and spinal cord and develop late-onset hind-limb paralysis [40]. Epg5 KO mice display some features of Vici syndrome, including callosal agenesis, photoreceptor Current Opinion in Cell Biology 2019, 61:117–125
Crohn’s disease (CD) is a major type of inflammatory bowel disease (IBD), characterized by recurrent inflammation of the gastrointestinal tract. Genome-wide association studies (GWASs) have identified multiple SNPs in ATG16L1 which confer increased susceptibility to CD [45]. The most common variant rs2241880 causes a threonine-to-alanine mutation (T300A) in ATG16L1 [45]. Within ATG16L1, the T300A mutation site is localized at or close to the C-terminal WD-repeat domain (WDD), which is absent in yeast [46]. The WDD is dispensable for bulk autophagy, but provides a platform for interaction with other proteins for selective autophagy processes, such as xenophagy (a type of selective autophagy for removing invading pathogens), which is impaired by the T300A mutation [47,48]. The T300A mutation causes CASP3/caspase 3-dependent cleavage of ATG16L1, which may lead to separation of the ATG5-12-interacting structure and the WDD [46,49]. T300A affects other ATG16L1-mediated functions, including secretion of antimicrobial proteins from Paneth cells and inflammasome-mediated cytokine production [50,51,52]. ATG16L1 is also involved in repair of plasma membranes following disruption by bacterial pore-forming toxins [53]. Interestingly, T300A is beneficial in other contexts, such as smoking-related COPD (chronic obstructive pulmonary disease) and colorectal cancer [54,55].
ATG5 and systemic lupus erythematosus (SLE) SLE is a common heterogeneous autoimmune disease, characterized by abnormal immune response and production of autoantibodies against double-stranded DNA. GWAS revealed that SNPs in ATG5 and ATG7 are risk factors for SLE [56–59]. Mutations in several trans-acting regulatory loci of ATG5 that elevate the expression level of ATG5 have also been associated with lupus nephritis, an inflammatory kidney disease caused by SLE [60]. ATG5 is required for maintaining normal innate immune function, including cytokine production, xenophagy and antigen processing, and is also indispensable for T and B cell development and activation [7,61]. ATG5 regulates LC3-associated phagocytosis (LAP), a subtype of phagocytosis requiring the LC3 conjugation system. Without LAP, mice develop SLE-like phenotypes and defects in dead cell clearance [62]. ATG5 mutations are also found in neurodegenerative disorders. A homozygous E122D mutation, which attenuates www.sciencedirect.com
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ATG12–ATG5 conjugation and compromises autophagic activity, was identified in patients with ataxia and developmental delay [63]. A variant of ATG5 with lower plasma protein levels is associated with CP [64].
removal of protein aggregates by the autophagic machinery has been extensively studied; however, the properties of protein aggregates that affect their removal remain largely unknown.
The cysteine proteinase Atg4 cleaves Atg8s to expose the C-terminal glycine for conjugation, and also cleaves Atg8PE from closed autophagic structures [2]. Mammals have four Atg4 orthologs, ATG4A/B/C/D [65]. GWASs identified that ATG4C SNPs are associated with Kashin–Beck Disease (KBD), a chronic degenerative disorder of the bones and joints, and ATG4A/B/D SNPs are associated with susceptibility to CD [65–68]. Atg4b knockout mice also show Paneth cell abnormalities and increased sensitivity to experimental colitis [65].
In response to various stresses, stress granules (SGs) are assembled via LLPS to sequester non-polysomal mRNA, translational machinery subunits and signaling molecules for stress adaptation. Autophagy mediates the clearance of SGs to maintain proteostasis [79,80–85]. Accumulation of SGs has been associated with ALS and other neurodegenerative diseases. Consistent with this, autophagy adaptor proteins (p62 and OPTN) and autophagy regulatory protein (C9ORF72, VCP, UBQLN2, CHMP2A, and TBK1) are mutated in ALS and other neurodegenerative diseases [86]. EPG5 knockout mice, which exhibit key features of ALS, also accumulate cytoplasmic TDP-43 aggregates in neurons [40].
Beclin 1 and cancer Autophagy can suppress or promote tumorigenesis, depending on context. Autophagy inhibits tumor initiation, possibly through its role in the antioxidative response. During tumor progression, autophagy enables tumor cell survival through starvation and metabolic stresses. Monoallelic deletion of Beclin 1 (BECN1) is observed in various human carcinomas [6]. Recent studies showed that the apparent tumor-promoting effect of BECN1 deletion in breast and ovarian cancers may be attributed to co-deletion of the nearby tumor suppressor gene BRCA1 [69]. Reduced Beclin 1 levels are also detected in tumor tissues with poor prognosis [6]. In mice, absence of Becn1 is embryonic lethal, while monoallelic deletion of Becn1 causes spontaneous hepatocellular carcinomas [70]. Liver-specific Atg5 and Atg7 knockout mice develop benign hepatoma [71]. The autophagyindependent roles of Beclin 1, including apoptosis, phagocytosis and endocytic trafficking, may contribute to the malignancy. For example, Beclin 1 regulates epidermal growth factor receptor (EGFR) degradation by affecting maturation of early endosomes [72]. The breast cancer tumor protein HER2 suppresses autophagy by interacting with and inhibiting Beclin 1. Induction of autophagy or disruption of HER2-Beclin1 binding reduces HER2-positive tumor growth [73].
Stress granules, autophagy, and neurodegenerative diseases Mutations in RNA-binding proteins such as TDP-43, hnRNPAs, MATR3, and TIA1 have been linked with the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS) and Parkinson’s disease. The disease mutationcontaining proteins are prone to aggregate and accumulate as cytosolic or nuclear inclusions in neurons [74,75]. Protein aggregates can be assembled via liquid–liquid phase separation (LLPS), a process that concentrates proteins into liquid droplets with confined boundaries in a hydrated intracellular environment [76–78]. Liquid protein droplets can transition into gel-like and solid-state aggregates. The www.sciencedirect.com
Mutations in ALS disease proteins such as SOD1, FUS, and hnRNPAs result in abnormal liquid-to-solid phase transition of SGs [82,85,87]. Degradation of the C. elegans P granule components PGL-1 and PGL-3 provides insights into how the biophysical properties of protein aggregates modulate their autophagic degradation. Oocyte-derived PGL-1 and PGL-3 proteins that segregate into somatic cells during early asymmetric cell divisions are removed by autophagy [88], a process requiring the concerted actions of the receptor protein SEPA-1, the scaffold protein EPG-2, and EPG-11-mediated posttranslational arginine methylation [88,89]. SEPA-1 facilitates LLPS of PGL-1/3, ensuring that low levels of PGL1/-3 partition into granules for autophagy [90]. EPG-2 controls the PGL-1/3 droplet size and also makes the droplets transition into gel-like structures, which may serve as a more stable PAS-like structure for autophagosome formation around the granule [90]. Arginine methylation of PGL-1/-3 by EPG-11 inhibits LLPS and also their autophagic degradation [90]. Under heat stress conditions, mTORC1-mediated phosphorylation of PGL-1/3 is increased and LLPS of PGL-1/3 is enhanced. The level of EPG-2 is insufficient to make PGL granules amenable to degradation, and therefore PGL accumulation occurs to confer heat stress adaptation [90]. This provides a framework to understand how the size and biophysical properties of protein aggregates are specified to accommodate autophagosome size and autophagic flux.
Conclusion Numerous disease-linked SNPs and mutations in core autophagy genes involved in autophagosome formation and maturation, and also in genes encoding selective autophagy adaptor, are causatively linked to the pathogenesis of multiple human diseases [4–7]. As terminally differentiated cells, neurons appear to be the most vulnerable type of cell to autophagy dysfunction, and this also explains why neurological disorders are the most Current Opinion in Cell Biology 2019, 61:117–125
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common diseases caused by mutations in autophagy genes [5]. Given the critical role of autophagy in xenophagy and other immunity-related processes, autophagy genes are also frequently linked with inflammatory diseases [7]. The distinct phenotypes caused by different autophagy gene mutations may be a result of their role at distinct steps in the autophagy pathway, and/or the tissuespecific requirement or the autophagy-independent function of specific genes. Genes involved in later autophagosome–lysosome fusion steps, such as CHAMP2B and VCP, which encode endosomal trafficking factors, and EPG5, cause accumulation of nondegradative autophagic structures and selective degeneration of neurons [40]. Blockages at later steps of autophagy and/or piling up of nonfunctional autolysosomes are more toxic to certain types of neuron, resulting in selective neuronal damage. Since mice deficient in key autophagy genes die perinatally [4], it is unlikely that human patients carry complete loss-of-function mutations. The disease-linked mutations are either hypomorphic [37,38,45,56–60,63,64], or the function of the gene may be partially compensated by its homologs, such as WDR45/45B [36]. The neuralspecific defect in Wdr45/45b double knockout mice suggests that the autophagy machinery in different tissues may have unique factors or regulators [36]. From a therapeutic perspective, exploring the molecular mechanism of autophagy gene function in the autophagy pathway will be beneficial for the treatment of autophagy gene-related diseases. For example, induction of autophagosome formation by mTOR inhibitors or AMPK agonists may aggravate the conditions in patients with defects in later autophagic steps. The toxicity of aberrant protein aggregates and damaged organelles in the cytosol or in autophagosomal vesicle-enclosed compartments may be different and require distinct or even opposite treatments. Elucidating the effect of abnormal phase separation and transition on autophagic degradation of protein aggregates will also be of great importance to understand the pathogenesis of various diseases and to explore new therapies.
Conflict of interest statement Nothing declared.
Acknowledgements We are grateful to Dr. Isabel Hanson for editing work. Work in the authors’ lab is supported by the National Natural Science Foundation of China (NSFC) (grant number 31630048, 31421002, 31561143001 to H.Z. and 31671430 to Y. G.Z.), Beijing Municipal Science and Technology Committee (Z181100001318003), the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (grant number XDB19000000), the Key Research Program of Frontier Sciences, CAS (grant number QYZDY-SSW-SMC006) and the Orphan Disease Center’s Million Dollar Bike Ride pilot grant programs (MDBR-18-104-BPAN and MDBR-19-101-BPAN). Current Opinion in Cell Biology 2019, 61:117–125
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47. Travassos LH, Carneiro LA, Ramjeet M, Hussey S, Kim YG, Magalhaes JG, Yuan L, Soares F, Chea E, Le BL et al.: Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Nat Immunol 2010, 11:55-62. 48. Boada-Romero E, Serramito-Go´mez I, Sacrista´n MP, Boone DL, Xavier RJ, Pimentel-Muin˜os FX: The T300A Crohn’s disease risk polymorphism impairs function of the WD40 domain of ATG16L1. Nat Commun 2016, 7:11821. 49. Murthy A, Li Y, Peng I, Reichelt M, Katakam AK, Noubade R, Roose-Girma M, DeVoss J, Diehl L, Graham RR et al.: A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3. Nature 2014, 506:456-462. Multiple studies identified that the missense variant T300A in ATG16L1 confers predisposition to Crohn’s disease, but it was unclear how this mutation affects the function of ATG16L1. This work demonstrates that the T300A mutation increases the sensitivity of ATG16L1 to caspase 3mediated cleavage, causing decreased ATG16L1 levels and autophagy function. 50. Lassen KG, Kuballa P, Conway KL, Patel KK, Becker CE, Peloquin JM, Villablanca EJ, Norman JM, Liu TC, Heath RJ et al.: Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc Natl Acad Sci U S A 2014, 111:7741-7746. 51. Bel S, Pendse M, Wang Y, Li Y, Ruhn KA, Hassell B, Leal T, Winter SE, Xavier RJ, Hooper LV: Paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine. Science 2017, 357:1047-1052. Paneth cells secrete antimicrobial proteins, including lysozyme, to limit intestinal bacteria. This work demonstrates that lysozyme is secreted by secretory autophagy, a noncanonical secretory pathway. The T300A mutation in ATG16L1 blocks the secretory autophagy pathway and contributes to the pathogenesis of Crohn’s disease. 52. Samie M, Lim J, Verschueren E, Baughman JM, Peng I, Wong A, Kwon Y, Senbabaoglu Y, Hackney JA, Keir M et al.: Selective autophagy of the adaptor TRIF regulates innate inflammatory signaling. Nat Immunol 2018, 19:246-254. 53. Tan JMJ, Mellouk N, Osborne SE, Ammendolia DA, Dyer DN, Li R, Brunen D, van Rijn JM, Huang J, Czuczman MA et al.: An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread. Nat Microbiol 2018, 3:1472-1485. 54. Chen CZ, Ou CY, Wang RH, Lee CH, Lin CC, Chang HY, Hsiue TR: Association of Egr-1 and autophagy-related gene polymorphism in men with chronic obstructive pulmonary disease. J Formos Med Assoc 2015, 114:750-755. 55. Grimm WA, Messer JS, Murphy SF, Nero T, Lodolce JP, Weber CR, Logsdon MF, Bartulis S, Sylvester BE, Springer A et al.: The Thr300Ala variant in ATG16L1 is associated with improved survival in human colorectal cancer and enhanced production of type I interferon. Gut 2016, 65:456-464. 56. Gateva V, Sandling JK, Hom G, Taylor KE, Chung SA, Sun X, Ortmann W, Kosoy R, Ferreira RC, Nordmark G et al.: A largescale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus. Nat Genet 2009, 41:1228-1233. 57. Zhou XJ, Lu XL, Lv JC, Yang HZ, Qin LX, Zhao MH, Su Y, Li ZG, Zhang H: Genetic association of PRDM1-ATG5 intergenic region and autophagy with systemic lupus erythematosus in a Chinese population. Ann Rheum Dis 2011, 70:1330-1337. 58. Dang J, Li J, Xin Q, Shan S, Bian X, Yuan Q, Liu N, Ma X, Li Y, Liu Q: Genegene interaction of ATG5, ATG7, BLK and BANK1 in systemic lupus erythematosus. Int J Rheum Dis 2016, 19:1284-1293. 59. Ciccacci C, Perricone C, Alessandri C, Latini A, Politi C, Delunardo F, Pierdominici M, Conti F, Novelli G, Ortona E et al.: Evaluation of ATG5 polymorphisms in Italian patients with systemic lupus erythematosus: contribution to disease susceptibility and clinical phenotypes. Lupus 2018, 27:1464-1469. 60. Zhang YM, Cheng FJ, Zhou XJ, Qi YY, Hou P, Zhao MH, Zhang H: Detecting genetic associations between ATG5 and lupus nephritis by trans-eQTL. J Immunol Res 2015, 2015:1-7. Current Opinion in Cell Biology 2019, 61:117–125
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Current Opinion in Cell Biology 2019, 61:117–125
ScienceDirect Core autophagy genes and human diseases Yan G Zhao1 and Hong Zhang2,3 Autophagy involves the formation of double-membrane autophagosomes and their delivery to lysosomes for degradation. In response to various endogenous and exogenous stimuli, autophagy recycles cellular constituents and removes cytotoxic threats such as protein aggregates and damaged organelles to maintain cellular homeostasis. Dysfunctional autophagy has been linked with multiple human diseases, including neurodegenerative diseases, tumorigenesis, diabetes, and immune diseases. Here we focus on human genetic disorders caused by hypomorphic or regulatory mutations in early acting autophagy genes or by mutations in genes acting at autophagosome maturation. Protein aggregates assembled via liquid–liquid phase separation (LLPS) exhibit distinct biophysical properties that are modulated by disease-related mutations. Abnormal phase transition of protein aggregates affects their removal and is associated with the pathogenesis of various neurodegenerative diseases.
fuse with vesicles originating from the endolysosomal compartment to form amphisomes and eventually degradative autolysosomes (Figure 1). Degraded molecules are then transported to the cytosol for reuse [1–4]. Under starvation and other stress conditions, autophagy functions as a cell-survival mechanism. Autophagy also selectively removes misfolded and toxic proteins and/or damaged organelles to maintain cellular homeostasis [4–7]. Perturbations of autophagy are linked with multiple pathological changes in humans, including neurodegenerative diseases, cancer, inflammation, and metabolic and developmental disorders [4–7]. We mainly focus on recent reports of genetic associations of core autophagy genes with human diseases (Table 1). We also discuss how phase separation and transition modulate autophagic degradation of protein aggregates and how these processes are involved in neurodegenerative diseases.
Autophagic machinery Addresses 1 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA 2 National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China 3 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, PR China Corresponding authors: Zhao, Yan G ([email protected]), Zhang, Hong ([email protected])
Current Opinion in Cell Biology 2019, 61:117–125 This review comes from a themed issue on Differentiation and disease Edited by Sara Wickstrom and Yingzi Yang
https://doi.org/10.1016/j.ceb.2019.08.003 0955-0674/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Autophagy is an evolutionarily conserved lysosomemediated catabolic process. The hallmark of autophagy is the formation of double-membrane autophagosomes that engulf a portion of cytosol and deliver it to lysosomes for degradation [1–4]. Autophagosome formation starts with de novo initiation and nucleation of isolation membranes (IMs), which expand and close. Nascent autophagosomes www.sciencedirect.com
A group of Atg (autophagy-related) genes identified from yeast genetic screens laid the groundwork for our understanding of the molecular mechanism of autophagosome formation [1–5]. Most of these ATG proteins are conserved in mammals. Upon autophagy induction, the Atg1 complex, containing Atg1/ULK1(mammalian counterpart), Atg17/FIP200 and Atg13, translocates to the autophagosome formation sites on the ER. The class III phosphatidylinositol 3-kinase VPS34 complex, consisting of VPS34, Atg6/Beclin 1, VPS15, and ATG14, is further recruited to generate phosphatidylinositol 3-phosphate (PI(3)P). The Atg1 and Vps34 complexes are required for initiation and nucleation of IMs. ATG9-positive vesicles are proposed to serve as one of the membrane sources for IM initiation and elongation. ATG9 trafficking is regulated by the PI(3)P effector ATG18 and its binding partner ATG2. Expansion of IMs requires two ubiquitin-like conjugation systems. The E1-like enzyme Atg7 and E2-like enzyme Atg10 catalyze the conjugation of the ubiquitin-like protein Atg12 with Atg5, which further interacts with Atg16L1. Atg8 is conjugated to phosphatidylethanolamine (PE) by Atg7 and the E2-like enzyme Atg3 [1–5]. Mammalian autophagy is more complex and contains steps that are not present in yeast. Autophagosomes form at the single peri-vacuolar site in yeast, while in mammals, multiple autophagosomes are generated simultaneously throughout the cytoplasm and closely contact the ER during IM initiation and elongation. Yeast autophagosomes directly fuse with the vacuole, while nascent mammalian autophagosomes detach from the ER and mature by fusion with endolysosomal vesicles before forming degradative autolysosomes [2,8,9]. Genetic Current Opinion in Cell Biology 2019, 61:117–125
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Figure 1
ER ATG2
ULK1 complex
Isolation membrane
Ω
WDR45
WDR45B
ATG14 Beclin 1
WDR45B
VPS34
WIPI2
EPG5
ATG12 ATG16L
ATG5 Lysosome Autophagosome
ATG8s pro-ATG8s PI(3)P
ATG4s
Rab7
Autolysosome
Current Opinion in Cell Biology
The role of core autophagy genes in the autophagy pathway. Upon autophagy induction, the most upstream complex – the ULK1 complex – is translocated to the autophagosome formation sites on the ER for the nucleation of isolation membranes (IMs). The VPS34/Beclin 1/ATG14 PI(3) kinase complex is then recruited, and generates PI(3)P de novo at the ER subdomains, called omegasomes, as well as on the IMs. The PI(3)P effector WIPI2 interacts with the ATG5/12/16L1 complex for ATG8sPE conjugation and IM elongation. ATG4s are cysteine proteinases that cleave the C-terminal of pro-ATG8s into mature ATG8s, and also cleave the ATG8-PE conjugates for recycling of ATG8s from closed autophagosomes, amphisomes and autolysosomes. WDR45 forms a complex with ATG2, and is also involved in autophagosome biogenesis. The detailed molecular role of the WDR45–ATG2 complex in autophagosome formation remains unknown. After closure, autophagosomes fuse with endocytic vesicles and lysosomes to form degradative autolysosomes. Fusion of autophagosomes/amphisomes with late endosomes/lysosomes requires the concerted actions of SNAREs, Rabs and tether proteins, such as EPG5. ER, endoplasmic reticulum; V, omegasome.
screens in Caenorhabditis elegans have identified a set of metazoan-specific autophagy genes, known as EPG (ectopic P granule) genes, that act at these unique autophagy steps [10]. The ER-localized transmembrane protein EPG3 modulates the ER-IM contact [11], while EPG5 confers the specificity of fusion of autophagosomes/amphisomes with late endosomes/lysosomes [12] (Figure 1). Human genetic studies reveal that mutations in ATG and EPG genes are linked with predisposition to various diseases.
WIPIs and neurological diseases Yeast Atg18 has four mammalian homologs, WIPI1-4, which are further classified into WIPI1/2 (WD-repeat protein interacting with phosphoinositides 1/2) and WDR45B/45 (also known as WIPI3/4) subgroups [13]. The single C. elegans homologs of each group are ATG-18 and EPG-6, respectively, and they Current Opinion in Cell Biology 2019, 61:117–125
function at different steps of autophagy [13]. WIPI2 directly interacts with ATG16 and defines the action site for the ATG16–ATG12–ATG5 complex that functions as an E3 for LC3 lipidation [14]. C. elegans EPG-6 but not ATG-18 binds to ATG-2 [13]. Similarly, WDR45 shows stronger interaction with ATG2A/B than WIPI1 and WIPI2 [15]. The role of WDR45 and WDR45B in autophagy has yet to be determined. Cryo-electron microscopy analysis reveals that WDR45 is a spherical protein attached to one end of the bar-like ATG2A/B to form a club-shaped structure, resembling the seahorse shape of HOPS complex [15,16]. The WDR45–ATG2 complex tethers a PI(3) P-containing vesicle with another PI(3)P-free vesicle and mediates direct lipid transfer [17,18,19]. This complex may function to bridge IMs/autophagosomes with the ER or other vesicles, thus providing phospholipids for IM expansion. www.sciencedirect.com
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Table 1 Core autophagy genes and related-human diseases Autophagy gene WDR45
WDR45B
WIPI2
EPG5
ATG16L1
ATG5
ATG4A/B/C/D
BECN1
Function
Related human diseases
Disease-related mouse models
Interacts with ATG2 and may function in isolation membrane expansion [13,15,16,17,18,19]. It is also involved in sensing upstream signaling and regulating the autophagosome size [91]. Interacts with FIP200. It may regulate autophagosome size, and mTOR activity via interaction with TSC on lysosomes [91].
BPAN [20 ,21 ,22 ], Rett syndrome [25,26], West syndrome [27], Xlinked ID [28,29]
Nestin-Wdr45 knockout mice exhibit axon swelling, and learning and memory defects [30].
ID [33–35]
Conventional Wdr45b knockout mice show motor deficiency and cognitive impairment. Pathologically, the mutant mice exhibit cerebellar atrophy and axon swelling [36].
Directly binds to PI(3)P and recruits the ATG5-12-16L1 complex for LC3 lipidation at autophagosome formation sites [14]. Acts as a Rab7 effector to tether autophagosomes/amphisomes with late endosomes/lysosomes for fusion [12].
CP [37], global developmental disorder [38]
Vici syndrome [39]
EPG5 knockout mice display selective motor neuron loss, recapitulating ALS phenotypes [40]. The knockout mice also show retinal atrophy, callosal agenesis and muscle degeneration [42,43]. Atg16L1T300A knock-in mice show morphological changes in Paneth and goblet cells, impaired xenophagy and increased cytokine production [50,51].
Forms an E3-like complex with ATG12–ATG5 that promotes LC3 lipidation for isolation membrane expansion [1–5]. It also has autophagy-independent functions such as secretion [51]. Conjugates with ATG12, then further forms a complex with ATG16L, which functions as an E3 for LC3 lipidation [1–5]. It is also involved in LC3-associated phagocytosis [62].
CD [45]
Cleaves pro-ATG8s to expose the glycine residue for conjugation and also cleaves ATG8s from PE conjugates on autophagosomal structures [2,65]. Component of the PI(3) kinase complex for de novo PI(3)P generation [1–5]. Critical for both autophagic and endocytic pathways [1–5,72].
Kashin–Beck Disease (ATG4C) [66], CD (ATG4A/B/D) [65,67,68]
SLE [56–60], ataxia and developmental delay [63], CP [64]
Breast and ovarian cancer [6]
Knockout of Atg5 in thymic epithelial cells leads to defects in negative thymic selection and autoimmunity [61]. Macrophage-specific Atg5 knockout affects LAP and degradation of dying cells, increases cytokine production, and causes an SLE-like phenotype [62]. Atg4b knockout mice shows abnormal Paneth cells and are more sensitive to experimental colitis [65].
Heterozygous deletion of Becn1 increases the frequency of spontaneous tumorigenesis [70].
Abbreviations: BPAN, b-propeller protein-associated neurodegeneration; ID, intellectual disability; CD, Crohn’s disease; COPD, chronic obstructive pulmonary disease; SLE, systemic lupus erythematosus; CP, cerebral palsy; TSC, Tuberous sclerosis 1; LAP, LC3-associated phagocytosis.
De novo mutations in the X-chromosome gene WDR45 have been linked with one subtype of NBIA (neurodegeneration with brain iron accumulation), namely protein-associated neurodegeneration b-propeller (BPAN), also known as SENDA (static encephalopathy of childhood with neurodegeneration in adulthood) [20,21,22]. NBIA is a heterogenous single-gene disorder involving iron deposits in various brain regions such as globus pallidus and substantia nigra [23]. Whether iron accumulation is a primary cause or consequence in NBIA remains controversial [24]. BPAN features static www.sciencedirect.com
encephalopathy in infancy or early childhood with sudden-onset parkinsonism, dystonia and cognitive decline in adolescence or early adulthood. BPAN patients show reduced autophagic activity [20,21,22]. Furthermore, mutations in WDR45 are also associated with a broader disease spectrum, including MECP2 mutation-negative Rett syndrome, West syndrome and X-linked intellectual disability, which share some clinical similarities [25–29]. The phenotypic diversity could be due to the time when somatic WDR45 mutations occur and/or other genetic and environmental modifiers. Current Opinion in Cell Biology 2019, 61:117–125
120 Differentiation and disease
Mice with central nervous system-specific knockout of Wdr45 exhibit extensive swollen axons and impaired learning and memory, mimicking key features of BPAN [30]. The knockout mice, like those with knockouts of genes involved in other types of NBIA such as Pank2 or Pla2g6, show no signs of iron deposition in the brain [30–32].
degeneration and pathological muscle changes [42,43]. Vici syndrome patients suffer from recurrent infections such as pulmonary infections. However, Epg5 KO mice show elevated basal lung inflammation [44]. The deficits in Epg5 knockout mice appear to be less severe than Vici syndrome patients, which could be due to differential environmental stress and genetic differences [40].
WDR45B mutations cause intellectual disability (ID), which is a common neurodevelopmental disorder [33–35]. Wdr45b deficiency in mice leads to motor defects and cognitive malfunction, similar to ID patients [36]. Wdr45b mutant mice show age-related cerebellar atrophy and Purkinje cell loss, with axon swelling [36]. Accumulation of p62 and ubiquitin-positive aggregates, indicative of defective autophagy, is observed in multiple cerebral and cerebellar regions of Wdr45 and Wdr45b single knockout mice, but the defect is much weaker compared to mice deficient in the core autophagy genes Atg5 and Atg7 [4,30,36]. This is probably due to genetic redundancy, as Wdr45b/45 double knockout mice die one day after birth. They show dramatic autophagy defects in the brain, but not in other tissues, which suggests that Wdr45b/45 function tissue-specifically [36].
ATG16L1 and Crohn’s disease
The WIPI2 missense mutation Y246C is potentially linked with cerebral palsy (CP), a developmental disorder characterized by impaired coordination, poor muscle tone and movement problems [37]. The WIPI2 missense mutation V249M is associated with global developmental disorder and it affects autophagy potentially by weakening the PI(3)P-binding activity of WIPI2 and/or the interaction of WIPI2 with ATG16L1 [38].
EPG5 and Vici syndrome Vici syndrome is a rare recessively inherited multi-system disorder with severe early onset neurodevelopmental and neurodegenerative deficits, whose key features include agenesis of the corpus callosum, developmental delay, microcephaly, cataracts, cardiomyopathy, and combined immunological abnormalities [39]. It is causatively linked with EPG5 mutations [39]. EPG5 functions as a tether protein by interacting with LC3 on autophagosomes/ amphisomes and Rab7 on late endosomes/lysosomes. EPG5 promotes assembly of the autophagosomal fusion machinery, the STX17/SNAP29/VAMP8 SNARE complex [17]. EPG5 depletion causes accumulation of nondegradative autolysosomes in Vici syndrome patients and model organisms [17,40,41]. Depletion of EPG5 impairs endocytic trafficking and recycling, which may also contribute to disease pathogenesis [40]. Systematic Epg5 knockout mice show selective motor neuron loss in the 5th layer of the cerebral cortex and spinal cord and develop late-onset hind-limb paralysis [40]. Epg5 KO mice display some features of Vici syndrome, including callosal agenesis, photoreceptor Current Opinion in Cell Biology 2019, 61:117–125
Crohn’s disease (CD) is a major type of inflammatory bowel disease (IBD), characterized by recurrent inflammation of the gastrointestinal tract. Genome-wide association studies (GWASs) have identified multiple SNPs in ATG16L1 which confer increased susceptibility to CD [45]. The most common variant rs2241880 causes a threonine-to-alanine mutation (T300A) in ATG16L1 [45]. Within ATG16L1, the T300A mutation site is localized at or close to the C-terminal WD-repeat domain (WDD), which is absent in yeast [46]. The WDD is dispensable for bulk autophagy, but provides a platform for interaction with other proteins for selective autophagy processes, such as xenophagy (a type of selective autophagy for removing invading pathogens), which is impaired by the T300A mutation [47,48]. The T300A mutation causes CASP3/caspase 3-dependent cleavage of ATG16L1, which may lead to separation of the ATG5-12-interacting structure and the WDD [46,49]. T300A affects other ATG16L1-mediated functions, including secretion of antimicrobial proteins from Paneth cells and inflammasome-mediated cytokine production [50,51,52]. ATG16L1 is also involved in repair of plasma membranes following disruption by bacterial pore-forming toxins [53]. Interestingly, T300A is beneficial in other contexts, such as smoking-related COPD (chronic obstructive pulmonary disease) and colorectal cancer [54,55].
ATG5 and systemic lupus erythematosus (SLE) SLE is a common heterogeneous autoimmune disease, characterized by abnormal immune response and production of autoantibodies against double-stranded DNA. GWAS revealed that SNPs in ATG5 and ATG7 are risk factors for SLE [56–59]. Mutations in several trans-acting regulatory loci of ATG5 that elevate the expression level of ATG5 have also been associated with lupus nephritis, an inflammatory kidney disease caused by SLE [60]. ATG5 is required for maintaining normal innate immune function, including cytokine production, xenophagy and antigen processing, and is also indispensable for T and B cell development and activation [7,61]. ATG5 regulates LC3-associated phagocytosis (LAP), a subtype of phagocytosis requiring the LC3 conjugation system. Without LAP, mice develop SLE-like phenotypes and defects in dead cell clearance [62]. ATG5 mutations are also found in neurodegenerative disorders. A homozygous E122D mutation, which attenuates www.sciencedirect.com
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ATG12–ATG5 conjugation and compromises autophagic activity, was identified in patients with ataxia and developmental delay [63]. A variant of ATG5 with lower plasma protein levels is associated with CP [64].
removal of protein aggregates by the autophagic machinery has been extensively studied; however, the properties of protein aggregates that affect their removal remain largely unknown.
The cysteine proteinase Atg4 cleaves Atg8s to expose the C-terminal glycine for conjugation, and also cleaves Atg8PE from closed autophagic structures [2]. Mammals have four Atg4 orthologs, ATG4A/B/C/D [65]. GWASs identified that ATG4C SNPs are associated with Kashin–Beck Disease (KBD), a chronic degenerative disorder of the bones and joints, and ATG4A/B/D SNPs are associated with susceptibility to CD [65–68]. Atg4b knockout mice also show Paneth cell abnormalities and increased sensitivity to experimental colitis [65].
In response to various stresses, stress granules (SGs) are assembled via LLPS to sequester non-polysomal mRNA, translational machinery subunits and signaling molecules for stress adaptation. Autophagy mediates the clearance of SGs to maintain proteostasis [79,80–85]. Accumulation of SGs has been associated with ALS and other neurodegenerative diseases. Consistent with this, autophagy adaptor proteins (p62 and OPTN) and autophagy regulatory protein (C9ORF72, VCP, UBQLN2, CHMP2A, and TBK1) are mutated in ALS and other neurodegenerative diseases [86]. EPG5 knockout mice, which exhibit key features of ALS, also accumulate cytoplasmic TDP-43 aggregates in neurons [40].
Beclin 1 and cancer Autophagy can suppress or promote tumorigenesis, depending on context. Autophagy inhibits tumor initiation, possibly through its role in the antioxidative response. During tumor progression, autophagy enables tumor cell survival through starvation and metabolic stresses. Monoallelic deletion of Beclin 1 (BECN1) is observed in various human carcinomas [6]. Recent studies showed that the apparent tumor-promoting effect of BECN1 deletion in breast and ovarian cancers may be attributed to co-deletion of the nearby tumor suppressor gene BRCA1 [69]. Reduced Beclin 1 levels are also detected in tumor tissues with poor prognosis [6]. In mice, absence of Becn1 is embryonic lethal, while monoallelic deletion of Becn1 causes spontaneous hepatocellular carcinomas [70]. Liver-specific Atg5 and Atg7 knockout mice develop benign hepatoma [71]. The autophagyindependent roles of Beclin 1, including apoptosis, phagocytosis and endocytic trafficking, may contribute to the malignancy. For example, Beclin 1 regulates epidermal growth factor receptor (EGFR) degradation by affecting maturation of early endosomes [72]. The breast cancer tumor protein HER2 suppresses autophagy by interacting with and inhibiting Beclin 1. Induction of autophagy or disruption of HER2-Beclin1 binding reduces HER2-positive tumor growth [73].
Stress granules, autophagy, and neurodegenerative diseases Mutations in RNA-binding proteins such as TDP-43, hnRNPAs, MATR3, and TIA1 have been linked with the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS) and Parkinson’s disease. The disease mutationcontaining proteins are prone to aggregate and accumulate as cytosolic or nuclear inclusions in neurons [74,75]. Protein aggregates can be assembled via liquid–liquid phase separation (LLPS), a process that concentrates proteins into liquid droplets with confined boundaries in a hydrated intracellular environment [76–78]. Liquid protein droplets can transition into gel-like and solid-state aggregates. The www.sciencedirect.com
Mutations in ALS disease proteins such as SOD1, FUS, and hnRNPAs result in abnormal liquid-to-solid phase transition of SGs [82,85,87]. Degradation of the C. elegans P granule components PGL-1 and PGL-3 provides insights into how the biophysical properties of protein aggregates modulate their autophagic degradation. Oocyte-derived PGL-1 and PGL-3 proteins that segregate into somatic cells during early asymmetric cell divisions are removed by autophagy [88], a process requiring the concerted actions of the receptor protein SEPA-1, the scaffold protein EPG-2, and EPG-11-mediated posttranslational arginine methylation [88,89]. SEPA-1 facilitates LLPS of PGL-1/3, ensuring that low levels of PGL1/-3 partition into granules for autophagy [90]. EPG-2 controls the PGL-1/3 droplet size and also makes the droplets transition into gel-like structures, which may serve as a more stable PAS-like structure for autophagosome formation around the granule [90]. Arginine methylation of PGL-1/-3 by EPG-11 inhibits LLPS and also their autophagic degradation [90]. Under heat stress conditions, mTORC1-mediated phosphorylation of PGL-1/3 is increased and LLPS of PGL-1/3 is enhanced. The level of EPG-2 is insufficient to make PGL granules amenable to degradation, and therefore PGL accumulation occurs to confer heat stress adaptation [90]. This provides a framework to understand how the size and biophysical properties of protein aggregates are specified to accommodate autophagosome size and autophagic flux.
Conclusion Numerous disease-linked SNPs and mutations in core autophagy genes involved in autophagosome formation and maturation, and also in genes encoding selective autophagy adaptor, are causatively linked to the pathogenesis of multiple human diseases [4–7]. As terminally differentiated cells, neurons appear to be the most vulnerable type of cell to autophagy dysfunction, and this also explains why neurological disorders are the most Current Opinion in Cell Biology 2019, 61:117–125
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common diseases caused by mutations in autophagy genes [5]. Given the critical role of autophagy in xenophagy and other immunity-related processes, autophagy genes are also frequently linked with inflammatory diseases [7]. The distinct phenotypes caused by different autophagy gene mutations may be a result of their role at distinct steps in the autophagy pathway, and/or the tissuespecific requirement or the autophagy-independent function of specific genes. Genes involved in later autophagosome–lysosome fusion steps, such as CHAMP2B and VCP, which encode endosomal trafficking factors, and EPG5, cause accumulation of nondegradative autophagic structures and selective degeneration of neurons [40]. Blockages at later steps of autophagy and/or piling up of nonfunctional autolysosomes are more toxic to certain types of neuron, resulting in selective neuronal damage. Since mice deficient in key autophagy genes die perinatally [4], it is unlikely that human patients carry complete loss-of-function mutations. The disease-linked mutations are either hypomorphic [37,38,45,56–60,63,64], or the function of the gene may be partially compensated by its homologs, such as WDR45/45B [36]. The neuralspecific defect in Wdr45/45b double knockout mice suggests that the autophagy machinery in different tissues may have unique factors or regulators [36]. From a therapeutic perspective, exploring the molecular mechanism of autophagy gene function in the autophagy pathway will be beneficial for the treatment of autophagy gene-related diseases. For example, induction of autophagosome formation by mTOR inhibitors or AMPK agonists may aggravate the conditions in patients with defects in later autophagic steps. The toxicity of aberrant protein aggregates and damaged organelles in the cytosol or in autophagosomal vesicle-enclosed compartments may be different and require distinct or even opposite treatments. Elucidating the effect of abnormal phase separation and transition on autophagic degradation of protein aggregates will also be of great importance to understand the pathogenesis of various diseases and to explore new therapies.
Conflict of interest statement Nothing declared.
Acknowledgements We are grateful to Dr. Isabel Hanson for editing work. Work in the authors’ lab is supported by the National Natural Science Foundation of China (NSFC) (grant number 31630048, 31421002, 31561143001 to H.Z. and 31671430 to Y. G.Z.), Beijing Municipal Science and Technology Committee (Z181100001318003), the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (grant number XDB19000000), the Key Research Program of Frontier Sciences, CAS (grant number QYZDY-SSW-SMC006) and the Orphan Disease Center’s Million Dollar Bike Ride pilot grant programs (MDBR-18-104-BPAN and MDBR-19-101-BPAN). Current Opinion in Cell Biology 2019, 61:117–125
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