In 1963, the word autophagy was coined by the Belgian cytologist and biochemist Christian de Duve who was rewarded the Nobel Prize in Physiology or Medicine in 1974 for his discovery of lysosomes and peroxisomes. In the 1990s, Japanese biologist Yoshinori Ohsumi identified autophagy-related genes. In 2016, Ohsumi won the Nobel Prize in Physiology or Medicine for his discovery of the molecular mechanisms of autophagy. Ohsumi’s discoveries opened the way to recognize the fundamental significance of autophagy in many physiological processes.

Autophagy, a lysosome-mediated intracellular degradation pathway, is an evolutionarily conserved mechanism in eukaryotes. Autophagy is a Greek word where “auto” means self and “phagy” means eating. This implies that autophagy is a process in which the cell eats its own components, similarly to cellular cannibalism.

Autophagy plays fundamental roles in numerous physiological processes [1]. The physiological roles of autophagy are eliminating unnecessary cargoes, sequestering organelles, recycling cellular components, controlling organelle homeostasis, promoting cell survival, and providing required resources [2]. Autophagy is an intracellular degradation process in which unwanted cargoes, such as old or damaged organelles, and unneeded proteins are sequestrated into double-membrane vesicles called autophagosomes and subsequently delivered to the lysosomes for degradation by lysosomal hydrolases [3]. The macromolecular contents from this digestion are released back into the cytosol in order to be reused for cellular and tissue remodeling [3]. Therefore, the catabolic role of the autophagy pathway allows various cell types to maintain and control cellular homeostasis, renew the cells, and provide energy [4]. Consequently, the dysregulation and dysfunction of autophagy are implicated in various types of diseases, such as neurodegenerative diseases (Alzheimer’s, Huntington’s, and Parkinson’s disease) and tumorigenesis ^[5][6][5,6].

In mammalian species, there are three types of autophagy—macroautophagy, microautophagy, and chaperone-mediated autophagy—and all of them promote the proteolytic degradation of cytosolic components and cargoes in the lysosome and the reuse of synthesized macromolecules by the cell [7]. Different types of autophagy share the common feature of the lysosomal degradation of damaged proteins but differ in their mechanisms of delivering the substrate to the lysosome [8]. Macroautophagy depends on the formation of autophagosomes in order to transport cargo to the lysosome [9], while micro-autophagy involves the direct uptake of cargo via the invagination of the lysosomal membrane [10]. Meanwhile, chaperone-mediated autophagy-targeted proteins are translocated across the lysosomal membrane in a complex with chaperone proteins that are recognized by specific-receptor-lysosomal-associated membrane proteins ^[10][11][10,11]. Neither microautophagy nor chaperone-mediated autophagy involves autophagosome formation but instead depends on the degradation function of lysosomes.

2. Mammalian Autophagy Machinery and Autophagy-Related Genes

Briefly, the process of macroautophagy includes five stages: initiation, elongation, maturation, fusion, and degradation. In mammals, the process of autophagy initiates the formation of intracellular membrane-bounded organelles enriched in phosphatidylinositol 3-phosphate, known as omegasomes, that is dynamically connected to the endoplasmic reticulum ^[12][23]. The small cup-shaped membrane structure termed phagophore is de novo formed from omegasomes. After that, this phagophore undergoes nucleation followed by elongation to engulf cargoes and close to form autophagosomes ^[13][14][24,25]. Autophagy is terminated with the fusion of the autophagosome with the lysosome to form an autolysosome with the subsequent degradation of autolysosomal content by lysosomal hydrolases ^[15][26]. The resulting simple molecules, including free fatty acids, amino acids, and nucleotides, are recycled back to the cytosol by lysosomal permease and reused as an energy source by the cell ^[16][27]. Autophagy is a complex process that is regulated by a series of protein complexes and signaling pathways. In mammals, the core autophagy-related (ATG) genes and their protein products are generally classified into the ULK1 protein kinase complex ^[17][28], Vps34-beclin1 class III PI3-kinase complex ^[18][19][29,30], ATG9A transportation system ^[20][21][31,32], ATG12 conjugation system ^[22][33], and LC3 conjugation system ^[23][34]. Table 12 illustrates the components and role of autophagy complexes participating in the mammalian autophagy machinery. Autophagy is induced by a wide range of stimulating signals such as nutrient deficiency (e.g., amino acids and glucose), growth factors deprivation (insulin and insulin-like growth factors), the depletion of cellular energy levels (ATP), extra- or intracellular stress (endoplasmic reticulum stress, hypoxia, and oxidative stress), and pathogenic infections ^[24][35]. Cells can create mechanisms to adapt their metabolism to the conditions of nutrient availability or metabolic stress in order to maintain cellular homeostasis. The master regulatory complex, the mammalian target of rapamycin (mTOR), is a key player that regulates the rate of anabolic and catabolic processes in response to nutrient availability ^[25][36]. In nutrient-rich conditions, mTOR is activated to enhance the anabolism by stimulating protein synthesis, nucleotide synthesis, glycolysis, lipogenesis, and mitochondrial biogenesis and, at the same time, suppress cellular catabolism via the inhibition of autophagy. On the contrary, the lack of growth factors and amino acids inhibits mTOR and thus stimulates protein breakdown via the catabolic pathway by inducing autophagy ^[25][36]. In addition, when cells are starved, the adenosine-monophosphate-activated protein kinase (AMPK) is activated. Autophagy is promoted by AMPK, which is a key energy sensor that regulates cellular metabolism to maintain energy homeostasis ^[26][37]. Therefore, mTOR and AMPK are considered major negative and positive regulators of autophagy, respectively ^[26][37]. A schematic representation of the mammalian autophagy machinery is shown in Figure 1. The core machinery of the initiation stage in mammalian cells is the Unc-51-like kinase (ULK) complex, which consists of ULK1/2, ATG13, FIP200, and ATG101 ^[27][28][38,39]. The dephosphorylation and autophosphorylation of ULK1, along with the dephosphorylation of ATG13, activate the entire autophagic cascade ^[29][40]. ULK1 post-translational modifications, such as phosphorylation ^[30][41] and ubiquitination ^[31][42], are essential for the induction of autophagy. Unphosphorylated ULK1 also promotes autophagosome–lysosome fusion ^[32][43]. AMPK and mTOR regulate autophagy via the direct phosphorylation of ULK1 ^[33][44]. In the initiation phase, the activation of AMPK by autophagy-stimulating signals inhibits mTORC1, which then dissociates from ULK1, leading to ULK1–AMPK interactions by which AMPK phosphorylates ULK1, activates ULK1 kinase, and dephosphorylates ATG13 in order to eventually initiate autophagy ^[3][33][3,44]. Additionally, the inhibited mTORC1 permits the ULK1 to phosphorylate ATG13, ATG101, and FIP200 ^[34][45], leading to the complete activation of the ULK1 complex. On the contrary, the activation of mTORC1 inhibits autophagy by inactivating ULK1/2 and ATG13 ^[33][35][44,46]. It has been shown that ATG101, the stabilizer of ATG13 ^[28][39], plays a crucial role in bridging the ULK1 and PI3K complex in mammalian autophagy induction ^[36][47]. Moreover, the process of nucleation is initiated when the activated ULK1 complex recruits the class III phosphatidylinositol 3-kinase (PI3K) complex, including VPS34, VPS15, Beclin1, and ATG14L, into phagophore initiation sites ^[37][48]. The activated ULK1 triggers the class III (PI3K) complex by phosphorylating beclin1 (BECN1) and vacuolar protein sorting 34 (VPS34) ^[3][38][39][3,49,50]. In the process of autophagy, the Beclin1-Vps34-Vps15-Atg14L complex is required for autophagosome nucleation and formation ^[3][20][40][3,31,51]. The activation of the Beclin1 complex generates phosphatidylinositol-3-phosphate, which is important for the nucleation of autophagosome ^[38][49]. Beclin1 interacts with VPS34, which activates VPS34 kinase activity to regulate the autophagosome’s size and quantity ^[18][38][29,49]. AMPK and ULK1 mediate the phosphorylation of ATG9A, which is required for proper ATG9A trafficking and autophagic flux ^[41][52]. The ATG9A transportation system consists of ATG9A, WIPI1/2, and ATG2A ^[21][32]. Phagophores require lipids and proteins to mature into autophagosomes. ATG9A is a lipid scramblase that has a key role in lipid mobilization from lipid droplets to autophagosomes for mediating autophagosomal membrane expansion and, hence, the progression of the autophagy process ^[42][43][53,54]. The ATG2 protein also transfers lipids, which are primarily needed for autophagosomal membrane expansion ^[44][55]. Both ATG2 and ATG9 are required for the expansion of the phagophore ^[45][56]. Moreover, an ULK1-independent ATG13-ATG101 complex regulates basal ATG9A trafficking ^[46][57]. During autophagy, WIPI1 and WIPI2 localize to autophagic membranes ^[47][48][58,59]. WIPI1 promotes the fission of endosomal transport carriers and the formation of autophagosomes. In autophagy initiation, WIPI1 binds omegasomes and enables the conjugation of LC3 to phosphatidylethanolamine (PE) in the LC3 lipidation process ^[49][50][60,61]. WIPI2 localizes to omegasome-anchored phagophores and upregulates LC3 lipidation ^[47][58]. Autophagosome elongation and maturation include two ubiquitin-like conjugation systems: the ATG12 conjugation system, the first ubiquitylation-like reaction, is essential for the formation and elongation of the autophagosome. The ATG12 conjugation system consists of ATG7, ATG5, ATG12, ATG10, and ATG16L1 ^[21][32]. ATG7 (E1-like enzyme) activates ATG12, and ATG12 is conjugated to ATG5 by ATG10 (E2-like enzyme) ^[51][62]. The ATG16L conjugates to ATG12-ATG5 to form the ATG12-ATG5-ATG16L complex, which promotes the elongation of the autophagic membrane and helps in the formation of the LC3-conjugated system ^[22][52][33,63]. The second ubiquitin-like conjugation system is the microtubule-associated protein 1 light chain 3 (LC3) conjugation system, and it consists of LC3, ATG7, ATG3, and ATG4. LC3 is widely used as an autophagosome marker in mammalian cells ^[53][64]. LC3 is cleaved by ATG4 to form LC3-I, which is then conjugated to phosphatidylethanolamine (PE) by ATG7 and ATG3 ^[54][65]. This reaction is catalyzed by the ATG12-ATG5-ATG16L complex ^[23][34]. PE-conjugated LC3 changed into a non-soluble form, which is LC3-II, and was steadily inserted into the autophagosome membrane ^[23][34]. LC3-II remains on mature autophagosomes until its fusion with lysosomes ^[55][66]. The number of LC3-II correlates to the number of autophagosomes ^[56][67]. As a result, the conversion of LC3 to LC3-II is considered a marker of autophagy induction. The transport and fusion of autophagosomes with lysosomes are regulated by several molecules, such as Rab7 ^[57][68], EPG5 ^[58][69], SNAREs ^[59][70], LAMPs ^[60][71], FYCO1 ^[61][72], and PLEKHM1 ^[62][73]. Finally, the completion of the autophagy process requires degrading cargoes and transporting synthesized macromolecules back to the cytoplasm in order to be re-used for metabolic mechanisms and/or the synthetization of biomacromolecules ^[63][64][74,75].

Potential Mechanisms of Selective Macroautophagy

The selectivity of autophagy is a prevalent phenomenon in various cells. Current research has proven the presence of various kinds of selective autophagy in eukaryotic cells. According to different cargoes, selective autophagy can be divided into several subcategories, such as mitophagy, proteaphagy, ribophagy, pexophagy, lysophagy, and nucleophagy ^[65][76]. Selective autophagy is principally dependent on both the recognition of the cargo and the coupling of the cargo to the phagophore, which can be carried out by proteins called selective autophagy receptors or cargo receptors ^[66][77]. A representative overview of the mechanisms of selective macroautophagy is illustrated in Figure 2. The first mechanism involves selective autophagy receptors that act as a bridge between the phagophore and cargo to facilitate the recruitment of autophagic machinery, mainly by the binding of LC3 and then the degradation of the cargo ^[67][78]. The second mechanism comprises the selection of cargo that can be achieved by targeted ubiquitination, which is recognized by ubiquitin-dependent selective autophagic cargo receptor proteins such as p62 ^[68][69][79,80], NBR1 ^[70][81], OPTN ^[71][82], and NDP52 ^[72][83], which in turn bind the cargo with ubiquitin to initiate pathways leading to autophagy initiation. Afterward, cargo is directed to the autophagosome by binding LC3/GABARAP proteins via the conserved LC3 interaction region and GABARAP-interacting motifs onto autophagic membranes ^[73][84]. The third mechanism for selective autophagy is that cargo receptors can recruit and bind the autophagy initiation ULK complex to induce autophagy. In mitophagy, the ULK complex is recruited to damaged mitochondria by cargo receptors OPTN and NDP52 ^[74][85]. The ER-phagy receptor CCPG1 (cell cycle progression 1) can bind to FIP200 as well as LC3 ^[75][86]. Moreover, selective cargoes, such as damaged organelles or ubiquitinated proteins, may accumulate at the autophagosome formation site and then are engulfed by autophagosomes even without direct recognition ^[76][87].