Autophagy-gene

The term"autophagy genes", also known as ATG genes, refers to a family of genes involved in the orchestration of autophagy . The lysosomal "disposal" of cytological "waste" (misfolded proteins, portions of cell organelles) involves approximately 20 evolutionarily conserved ATG core genes encoding proteinsof the same name(ATG proteins) (Klionsky et al. 2003).

The ATG genes were originally discovered in yeast genetic screens (Mizushima 2018). With few exceptions, all ATG genes are required for the efficient formation of membrane-enveloped autophagosomes that fuse with lysosomes after autophagosome formation. The proteins encoded by ATG genes are traditionally classified into different biochemical and functional groups that are effective at specific stages of autophagosome assembly.

The process of autophagy is highly conserved in all eukaryotic organisms and is enhanced by various intra- and extracellular stimuli. Autophagy is essential for cellular homeostasis, cellular protein and organelle quality control and disposal, and adaptation of the organism to environmental stress. (Levine and Kroemer, 2008; Mizushima and Komatsu, 2011). In autophagy initiation, the ULK1-serine-threonine kinase complex (involving ULK1, FIP200, ATG13, and ATG101) plays a major role by phosphorylating and thereby activating several downstream proteins. In higher eukaryotes, many autophagy genes have additionally functionally diversified to

• facilitate the transport of extracellular cargo to the lysosome
• promote plasma membrane localization or extracellular release of intracellular cargo
• coordinate intracellular communication with various cell signaling pathways.

These downstream functions are in the true sense, not autophagy. Accordingly, they are referred to as ATG gene-dependent pathways.

• The nuclear ATG proteins are necessary but not sufficient for intact degradative autophagy in human cells. Degradation of autophagosomal cargo can only occur via successful fusion of the autophagosome with a functional lysosome. Prerequisite for these essential processes are: on the one hand an intact lysosomal biogenesis
• an undisturbed autophagolysosomal fusion process per se (Yu et al. 2018)
• an unimpaired lysosomal (enzymatic) function (Shen and Mizushima, 2014)
• and ultimately a restoration of intact lysosomal function (Chen and Yu, 2017).

Mutations in genes required for autophagy and lysosomal function that regulate these processes lead to frequent lines of dysfunction in cellular metabolism and to entirely different, often severe, disease patterns.

Mutations in genes required for autophagy and lysosomal function

• ATG 16L1(Crohn's disease): ATGL16L1 T300A is a major risk allele for CD. The T300A polymorphism has a caspase-3 cleavage site that reduces protein levels. T300A knock-in or hypomorphic or intestinal knockout mice show decreased intestinal bacterial clearance; increased cytokine responses; decreased Paneth cell lysozyme secretion and clearance of IRE la protein aggregates during ER stress; increased enterocytic TNFa-induced necroptosis; dendritic cell defects in regulatory T cell induction and suppression of mucosal inflammation (Chu et al., 2016)
• ATG16L2(systemic lupus erythematosus; Crohn's disease): The ATG16L2 gene encodes a protein of the same name that interacts with Atg5 in a large protein complex consisting of Atg5, Atg12, and Atg16L1. This protein complex has been shown to be essential for stretching isolation membranes (also called phagophores) during autophagy in mammals. To date, however, the precise function and regulation of the Atg12-5-16L1 complex has remained largely unknown. The ATG16L2 protein is an isoform of ATG16 whose function remains unclear (Molineros et al. 2017)
• ATG5 (childhood ataxia, systemic sclerosis, SLE): A loss-of-function mutation reduces autophagy and causes ataxia. Intronic variants are associated with susceptibility to systemic sclerosis. Versch. Polymorphisms are associated with susceptibility to SLE; mouse studies suggest that the mechanism involves deficient LC3-associated phagocytosis (Martinez et al. 2016)
• ATP6AP2(X-linked parkinsonism with spasticity. multisystem disorder): Exon skipping mutations are associated with parkinsonism; missense mutations associated with immunodeficiency, liver disease, and psychomotor impairment result in defective lysosomal acidification due to impaired v-ATPase assembly, leading to defects in autophagy (Rujano et al. 2017)
• BECN1(breast and ovarian cancer): Monoallelic deletion associated with risk and poor prognosis of sporadic breast and ovarian cancer; monoallelic deletion in mice leads to decreased autophagy and increased tumor incidence, including basal breast cancer (Tang et al. 2015)
• CLEC16A(diabetes, multiple sclerosis): CLEC16A variants are associated with several autoimmune diseases. Mice, with Clecl6a deficiencies, suffer from autophagy defects associated with Purkinje degeneration and ataxia, impaired β-cell mitophagy, and autoimmunity (Redmann et al. 2016)
• CTNS gene(cystinosis): Recessive loss-of-function mutations in the CTNS gene, which encodes a transporter that exports cysteine from lysosomes and is associated with renal lysosomal storage disease; gene deletion in mice leads to deficient autophagy, altered lysosomal dynamics, and accumulation of dysfunctional mitochondria that overproduce ROS (Festa et al. 2018)
• EPG5 (Vici syndrome): Recessive mutations in EPG5, a gene required for autophagolysosomal fusion, lead to a neurodevelopmental disorder with multisystem involvement (Hori et al. 2017)
• GBA (Gaucher disease; Parkinson disease): GBA1 encodes the lysosomal enzyme glucocerebrosidase. Homozygous GBA defects cause Gaucher's disease and heterozygous defects predispose to Parkinson's disease (Afiaki et al. 2017).
• GRN(frontotemporal dementia (heterozygous) or neuronal ceroid lipofuscinosis (homozygous): Loss-of-function mutations impair lysosomal function and autophagic flux.
• LAMP2 (Danon's cardiomyopathy; hypertrophic cardiomyopathy): X-linked deletion leads to vacuolar cardiomyopathy and myopathy; Lamp2 deletion in mice leads to autophagosome accumulation and cardiomyopathy.
• PIK3R4 (VPSI 5): Cortical atrophy and epilepsy Mutations in PIK3R4, a component of the Beclin-1 complex required for endosomal-lysosomal trafficking and autophagy, are associated with human neurodevelopmental diseases (Gstrein et al. 2018).
• SNX14(Autosomal recessive spinocerebellar ataxia): SNX14 binds lysosomal phosphatidylinositol(3,5)-bisphosphate and is required for autophagosomal clearance (Akizu et al. 2015;
• SPG11, SPG15 (ZFYVE26), SPG49 (TECPR)(Hereditary Spastic Paraplegia): SPG15 binds phosphatidylinositol-3-phosphate and SPG49 binds LC3 to function in autophagolysosomal trafficking (Ebrahimi-Fakhari et al., 2016).
• WDR45 (WIPI4)(beta-propeller protein-associated neurodegeneration): WDR45 (WIPI4) binds to phosphoinositide-3-phosphate and interacts with ATG2 and ATG9; disease-associated mutations impair autophagy (Ebrahimi-Fakhari et al. 2016).

Mutations in genes regulating autophagy and lysosomal function.

• APP(Alzheimer's disease): Mutated amyloid precursor protein expressed in mouse hippocampal neurons inhibits mitophagy and autophagy.
• AT-1 (SLC33A1)(spastic paraplegia, developmental delay, autism spectrum disorders): AT-1 translocates cytosolic acetyl-CoA into the ER lumen; mutations and duplications are associated with a variety of CNS phenotypes in humans; in mice, overexpression blocks Atg9a-Faml34b-LC3 interactions, leading to defective ER phagia and progeria (Peng et al. 2018).
• C9orf72(amyotrophic lateral sclerosis -ALS; frontotemporal dementia -FTD): Hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTD. Regulates autophagy and lysosomal homeostasis through interactions with SMCR8, ULK1, and Rab GTPases (Corrionero et al. 2018).
• ERBB2(breast cancer): Amplification of ERBB2 and resulting overexpression of ERBB2 (HER2) interacts with Beclin 1 and inhibits autophagy (Vega-Rubin-de-Celis et al., 2018)
• GBA1(Gaucher disease; Parkinson's disease): Mutations in GBA1 decrease glucocerebrosidase activity, resulting in defects in autophagy-lysosomal function and accumulation of a-synuclein aggregates (Afiaki et al. 2017)
• GPR65(Inflammatory bowel disease):GPR65 I231L risk variant of this proton-sensitive G protein-coupled receptor impairs lysosomal acidification, decreases intracellular bacterial clearance, and alters lipid droplet turnover.
• HTT (Huntingtin; Huntington's disease): PolyQ expansion in HTT competitively disrupts the interaction between the deubiquitinase ataxin 3 and Beclin 1, leading to increased proteasomal degradation of Beclin 1 and reduced autophagy (Ashkenazi et al. 2017).
• IRGM (non-alcoholic fatty liver disease (NAFLD); Crohn's disease; tuberculosis): IRGM functions in assembly and activation of the autophagy machinery; a synonymous variant reduces protein expression, leading to reduced autophagy and lipophagy in NAFLD; polymorphisms are associated with risk of Crohn's disease and tuberculosis (Lin et al. 2016)
• LRRK2(Crohn's disease; PD): Risk alleles for CD and PD increase leucine-rich repeat kinase 2 kinase activity and reduce autophagic flux; a protective allele increases flux (Hui et al. 2018)
• MeCP2 (Rett syndrome - X-linked neurodevelopmental disorder-): Deficiency of methyl-CpG-binding protein-2 (MeCP2), a transcriptional regulator, leads to defective autophagy in patient fibroblasts and in the cerebellum of knockout mice and mitochondrial retention in erythrocytes (Sbardella et al. 2017)
• MTMR3(Inflammatory bowel disease): MMTR is a PI3P phosphatase that decreases autophagy. Macrophages from carriers of the risk allele express higher MTMR3 protein levels and have increased pathogen recognition receptor-induced caspase-1 activation and IL-1β secretion (Lahiri et al. 2015)
• PLEKHM1(osteopetrosis): associated mutants impair binding to RAB7A and secretory lysosome trafficking in osteoclasts.
• RAB7A(Charcot-Marie-Tooth disease type 2B): Disease-associated RAB7A mutants reduce autophagic flux in HeLa cells and patient-derived fibroblasts are autophagy-deficient.
• PS1(Alzheimer's disease): Mutations in presenilin 1 that disrupt v-ATPase assembly, lysosomal acidification, and autophagy cause early-onset Alzheimer's disease (Lee et al. 2010).
• PTPN2(type 1 diabetes; juvenile arthritis): Disease-associated SNP in PTPN2, a gene encoding protein tyrosine phosphatase non-receptor type 2, causes impaired autophagosome formation and defective bacterial handling in macrophages and intestinal epithelial cells.
• SMS(Snyder-Robinson syndrome): Loss-of-function mutations in spermine synthase (SMS) cause SRS, an X-linked syndrome with mental retardation; deficiency of SMS produces toxic metabolites that impair lysosomal function and autophagic flux
• TMEM230(Parkinson's disease): Transmembrane protein involved in retromer function; loss reduces autophagic cargo degradation and secretory autophagy (Kim et al. 2017)
• v-ATPase(Autosomal Recessive Osteoporosis): Mutations in the α3-subunit encoded by TCIRG1 impair lysosomal acidification at the grooved border of osteoclasts, leading to defects in bone resorption.
• WASP(Wiskott-Aldrich syndrome) Deficiency of the actin cytoskeleton-regulating WASP protein impairs autophagosome formation, leading to deficient xenophagy and excessive inflammasome activation and pyroptosis.

Mutations in genes required for cargo delivery during selective autophagy.

• ALFY (Primary microcephaly) Dominant mutation in this autophagy scaffold protein causes human microcephaly (Kadir et al. 2016).
• CALC0C02 (NDP52) (Crohn's disease): Missense mutation of this autophagy adaptor reduces its function and enhances NF-kB activation of inflammatory genes (Ellinghaus et al. 2013)
• FAM134B (Hereditary sensory and autonomic neuropathy type II): Mutations disrupt the interaction of this ER protein with LC3 and GABARP and impair ER phagocytosis.
• FANC genes (Fanconi anemia; hereditary breast and ovarian cancer): FA pathway genes are required for removal of damaged mitochondria (mitophagy) and prevention of aberrant inflammasome activation (Sumpter et al. 2016).
• OPTN1 (amyotrophic lateral sclerosis; primary open-angle glaucoma; Paget's disease of bone): Mutations in ALS reduce interaction of this autophagy adaptor with TBK1 and decrease Parkin-dependent mitophagy; mutations in POAG increase interaction with TBK1, activate Bax-dependent apoptosis, and are associated with mitochondrial dysfunction. Truncated protein mutations occur in association with Paget's disease of bone (Silva et al. 2018)
• PARK2/Parkin (Autosomal recessive and sporadic early-onset Parkinson's disease; colon, lung, and brain cancers): Parkin is an E3 ligase that functions in mitophagy and xenophagy; mutations are associated with PD and cancer risk; polymorphisms are associated with increased susceptibility to intracellular bacterial infections.
• PARK6/PINK1 Autosomal recessive and sporadic early-onset Parkinson's disease PINK1 is a serine-threonine kinase that translocates to the outer mitochondrial membrane upon damage and mediates parkin recruitment and mitophagy.
• PEX13 (Zellweger syndrome): Disease-associated mutations impair mitophagy and patients with mutations have accumulation of abnormal mitochondria.
• SQSTM1 (p62) (ALS; FTD; Paget's disease; Distal myopathy) SQSTM1 is an autophagy adaptor that binds ubiquitin and LC3; mutations in the ubiquitin-binding association domain result in a spectrum of multisystem proteinopathies.
• SMURF1 (ulcerative colitis) SMURF1, a susceptibility gene for ulcerative colitis, encodes an E3 ligase that functions in the mitophagy, virophagy, and xenophagy of intracellular bacteria (Franco et al. 2017)
• TBK1 (ALS; Frontotemporal Dementia; Other Neurodegenerative Phenotypes; POAG) TBK1 kinase phosphorylates the autophagy receptor OPTN1 and increases its interaction with ATG8 proteins and polyubiquitinated proteins.
• TRIM20 (Familial Mediterranean Fever): Disease-associated TRIM20 mutants do not interact with inflammasome components and target them for autophagic destruction (Kimura et al.2017).
• VPS13D (ataxia with spasticity) Recessively inherited defects in this ubiquitin-binding protein cause mitophagy failure and mitochondrial dysfunction

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