8 A variety of diﬀerent bacteria, including Mycobacte- ria, Salmonella, Shigella, and Listeria, are recognized by autophagy, yet the speciﬁc signals that mediate recognition of intracellular pathogens by the autophagy machinery have not been deﬁned thus far. It is currently thought that ubiquitin associated with intracellular pathogens promotes targeted autophagosome formation and pathogen destruction. In addition, p62 and NDP52 mediate diﬀerent pathways of selective autophagy for Shigella and Listeria and provide novel insight into the mechanisms by which adaptor proteins target bacteria to autophagy [73]. Infection with Herpes simplex virus type 1 (HSV-1) provoked autophagy under con- ditions that inhibited viral gene expression. The induction of autophagy occurs very early after infection with HSV- 1, and de novo protein synthesis is not required for the response [74]. Autophagy can be induced by the presence of foreign DNA within cells. The interferon-inducible, dsRNA- dependent protein kinase R (PKR) plays an important role in innate immunity against viral infections. PKR activation leads to phosphorylation of eIF2α and a subsequent shut- down of host and viral protein synthesis and viral replication. PKR and eIF2α phosphorylation regulate another funda- mental cellular process, the lysosomal degradation pathway of autophagy. 9.1.10. Proteasome Inhibitors. The ubiquitin-proteasome system and lysosome-dependent autophagy are two major intracellular pathways for protein degradation. The ubiq- uitin-proteasome pathway is a novel therapeutic target for cancer treatment. Inhibition of proteasome function leads to the accumulation of polyubiquitinated proteins and likely causes the accumulation of misfolded proteins in the ER due to the blockage of ER-associated degradation. Thus, proteasome inhibitors activate autophagy via ER stress. Proteasome inhibitors have been potent anticancer agents for various cancers. Proteasome inhibitor MG-132 increased the protein expression of LC3-I and -II in a time-dependent manner. 3-MA signiﬁcantly abolished the formation of LC3+ autophagic vacuoles and the expression of LC3-II, but not LC3-I, induced by MG-132 [75]. A new class of proteasome inhibitors such as glidobactin A treatment also induced autophagy as judged by the presence of the lipidated form of LC3 and autophagosomes [76]. The eukaryotic 20S proteasome contains three catalytic subunits (β1, β2, and β5) conferring caspase-like, trypsin-like, and chymotrypsin-like proteolytic activities, respectively. Glidobactin A blocked the chymotrypsin-like activity irreversibly at low concentrations, whereas the trypsin-like activity was less sensitive and the caspase-like activity was not inhibited at the concentrations tested (up to 20 μM). 9.2. AntiAutophagics (Table 2) 9.2.1. Atg Proteins. The regulation of the autophagic and apoptotic response has been shown to require components that are involved in both processes. Caspases, a family of cystinyl aspartate-requiring proteases, play a central role in apoptosis, a well-studied pathway of programmed cell International Journal of Cell Biology death. Calpain is a calcium-dependent intracellular cysteine protease that plays a crucial role in the regulation of cell spreading, cell migration, programmed cell death, and cell cycle progression. The majority of human Atg proteins can be cleaved by caspases and calpains, which are activated in some apoptotic paradigms [77]. For example, Atg3 is cleaved by caspase-3, -6, and -8, while Atg9, Atg7, and Atg4 homologues can be cleaved by caspase-3. Beclin 1 (Atg6) is a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL. The cleavage of Beclin 1 resulted in abrogation of the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Atg5 is a gene product required for the formation of autophagosomes. Calpains-1 and - 2 activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the antiapoptotic molecule Bcl-xL, and triggered cytochrome c release and caspase activation. Caspase cleavage of autophagy-related proteins can aﬀect the autophagic process. 9.2.2. Autophagosome Formation. Verteporﬁn (visudyne) is a benzoporphyrin derivative used in photodynamic ther- apy, and verteporﬁn inhibited drug- and starvation-induced autophagic degradation and the sequestration of cytoplasmic materials into autophagosomes [78]. Verteporﬁn inhibited autophagy stimulated by serum starvation, a physiological stimulus, and by rapamycin, the chemical inhibitor of mTORC1. Verteporﬁn binds to the membrane of expanding phagophores or to a factor involved in phagophore expansion and prevents expanding phagophores from adopting their characteristic cup shape, so they are unable to capture cytoplasmic cargo and only form empty single-membrane vesicles. 3-Methyladenine (3-MA), as an autophagy inhibitor, was ﬁrst discovered via screening of purine-related sub- stances using isolated hepatocytes from starved rats. 3-MA, which interferes with the formation of autophagosomes in mammalian cells via inhibition of the class III PI3K activity that controls the autophagic pathway [79], has been widely used in studies on autophagy. 3-MA has a dual role in modulation of autophagy; although it is capable of suppressing autophagy induced by starvation, its prolonged treatment in full medium induces autophagy. Under speciﬁc treatment conditions, 3-MA acts similar to rapamycin, a well-established autophagy inducer via suppression of mTOR function. 9.2.3. Bcl-2 Family. Apoptosis and autophagy are both closely regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti- apoptotic protein Bcl-2 functions not only as an antiapop- totic protein but also as an anti-autophagy protein via its inhibitory interaction with Beclin 1 [80]. Beclin 1 functions in the lysosomal degradation pathway of autophagy and induces autophagic cell death in cancer cells. Bcl-2/Bcl-xL can bind Beclin 1 and inhibit Beclin 1-dependent autophagic cell death in cancer cells. The BH3 domain of Beclin 1 also binds