Autophagy

Autophagy is an evolutionarily conserved intracellular degradative process which maintains cell homeostasis and protects cells from stress through degrading or recycling intracellular organelles and protein aggregates under basal and stressed-induced conditions. To date, at least three types of autophagy are characterized, including macroautophagy, microautophagy, and chaperon-mediated autophagy (CMA). Macroautophagy, usually referred to as autophagy, is distinct from the other autophagy types by sequestering substrates into a double-membrane structure termed autophagosome. Followed by fusing with lysosome, the contents of autophagosome are degraded by lysosomal enzymes. Although macroautophagy is generally considered as a non-selective process, there are also specialized types of autophagy, named selective autophagy, which sequester specific cargos by cargo receptors and adaptors (Lamb et al., 2013). Both microautophagy and CMA directly deliver cargo into lysosomes in different ways. Microautophagy imports cargo into the lumen through an inward budding mediated by lateral segregation of lipids and transmembrane-proteins, whereas CMA is conducted by HSP70, which targets proteins by recognizing specific motifs, and LAMP-2A, which combines HSP70 and transfers both chaperon and substrate into the lumen of lysosome (Bejarano and Cuervo, 2010; Li et al., 2012). As the main character of this thesis, macroautophagy will be referred to as autophagy hereafter.

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1.1 Molecular Mechanism

In response to diverse environmental cues, one of the morphological features of autophagy is the formation of autophagosome. Autophagosome originates from extended compartment on endoplasmic reticulum (ER) membrane, named omegasome, and expends with plasma membrane, Golgi, mitochondria and endosomes serving as the reservoir of membrane source (Axe et al., 2008; Hailey et al., 2010; Knaevelsrud et al., 2013; Longatti et al., 2012; Ravikumar et al., 2010). Autophagosome formation can be defined into several stages: initiation, nucleation, expansion, maturation and termination (Lamb et al., 2013) .

In the initiation stage, ULK1 complex, consisting of Ser/Thr kinases ULK1/2 and accessory proteins ATG13, FIP200, and ATG101, plays a major role. Under nutrient-rich condition, ULK1 kinase activity is repressed by mTOR, a negative regulator of autophagy, through phosphorylating ULK1 at Ser757 (Ganley et al., 2009;

Hosokawa et al., 2009). Upon nutrient starvation, mTOR is inactivated to relieve its repression effect on ULK1. In addition, a key energy sensor AMPK is activated in this condition. AMPK contributes to ULK1 activation by several mechanisms. First, it relieves mTOR-mediated ULK1 repression by activating mTOR inhibitor TSC complex, or by inhibiting mTOR activity via directly phosphorylating one of it subunit Raptor (Gwinn et al., 2008; Shaw et al., 2004). Furthermore, AMPK phosphorylates ULK1 complex at Ser317 and Ser777, which is required for ULK1 activation (Kim et al., 2011). Thus, through the combinatory effect of mTOR inactivation and AMPK activation, ULK1 is activated which in turn phosphorylate its partners ATG13L and FIP200 (Dorsey et al., 2009). Activated ULK1 complex translocates from cytosol to certain domains on ER or closely attached membrane structure that would contribute to the autophagosome formation. At these places, ULK1 passes down the signal by

phosphorylating components of the VPS34 complex, composed of class III PI3K VPS34 and its partners VPS15, Beclin1 and ATG14L/UVRAG, thereby enhancing the lipid kinase activity of VPS34 (Egan et al., 2015; Russell et al., 2013; Russell et al., 2014).

Followed by its redistribution to omegasome, VPS34 complex drives the autophagosome nucleation process through producing phosphatidylinositol-3-phosphate (PI3P). Autophagy-specific PI3P binding effectors such as DFCP1 and WIPI family proteins are recruited to the membrane platform where they facilitate the development of isolation membrane (Itakura and Mizushima, 2010; Polson et al., 2010).

During the elongation stage, two ubiquitin-like (UBL) conjugation systems are required. Analogous to the ubiquitination machinery, conjugation of ubiquitin-like molecule ATG12 to ATG5 is catalyzed by E1-like enzyme ATG7 and E2-like enzyme ATG10 (Mizushima et al., 1998). Subsequently, ATG5-ATG12 conjugates associate ATG16L on the isolation membrane, forming ATG5-ATG12-ATG16L complex (Mizushima et al., 2003). In the second UBL system, ubiquitin like protein Atg8 or its mammalian orthologs LC3, GATE16, GABARAP and ATG8L are proteolytically processed by ATG4B, then subjected to ATG7 (E1-like), ATG3 (E2-like), ATG5-ATG12-ATG16L (E3-like) for subsequent phosphotidylethanoamine (PE) conjugation, converting LC3 from unlipidated I form to the lipidated II form (Geng and Klionsky, 2008; Kabeya et al., 2004; Tanida et al., 2004). Among these ATG8 homologs, LC3 has been best characterized and is widely used as a marker of autophagy. The lipidated LC3-II decorates on the membrane of autophagosome and contributes to cargo selection and autophagosome maturation via interacting with LC3-interacting region (LIR) motif-containing proteins (Birgisdottir et al., 2013; Pankiv et al., 2007). Besides the UBL systems, Atg9, a multi-spanning transmembrane protein, also assists in formation and

expansion of autophagosome by shuttling between the membrane sources and autophagosome as a membrane supplier (Yamamoto et al., 2012).

Before the closure of autophagosome, autophagy core proteins such as ATG5-ATG12-ATG16L complex dissociate from the membrane, whereas a portion of LC3-II and cargo receptors remain attached and are retained inside the autophagosome (Lamb et al., 2013). In the maturation process, autophagosome fuses with lysosome to form autolysosome, which is aided by SNAREs and tethering complexes such as STX17-HOPS complex (Jiang et al., 2014). Contents inside the autolysosome are degraded and can be recycled back to the cytosol.

Interestingly, amino acid released from autolysosome can reactivate mTORC1 at the lysosomal membrane, which contributes in part to the termination of autophagic process during prolonged starvation (Yu et al., 2010). A recent work in our lab also demonstrated that starvation-induced autophosphorylation of ULK1 facilitates its interaction with CUL3-KLHL20, which promotes Lys48-linked ubiquitination and subsequent proteasomal degradation. Additionally, CUL3-KLHL20 also governs the turnover of Beclin1, VPS34, ATG14L and ATG13 directly or indirectly, thereby contributing to autophagy termination (Liu et al., 2016).

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1.2 Physiological Role

Basal level of autophagy takes place in all eukaryotic cells, participating in quality control of proteins and organelles and maintaining cell homeostasis. The primordial function of autophagy also includes response to metabolic, genotoxic or hypoxic stress by acting as an adaptive mechanism essential for cell survival.

Autophagy is vital in a range of physiological processes including developmental regulation and aging. Dysregulation of autophagy may lead to a broad range of diseases such as neurodegenerative diseases, infectious or inflammatory diseases, metabolic diseases, tumorigenesis, and myopathy (Choi et al., 2013; Levine and Kroemer, 2008).

Although autophagy has been regarded as a protective process, it also alternatively directs cells to cellular demise through facilitating the activation of apoptosis or necrosis in some cases (Marino et al., 2014). Thus, only by better understanding the fundamental mechanism of the process can we discriminate its character in diseases pathogenesis and eventually provide useful therapies.

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